Edward Mocarski

All Publications

• Programmed Necrosis in Host Defense. Current topics in microbiology and immunology Mocarski, E. S. 2023

  Abstract

  Host control over infectious disease relies on the ability of cells in multicellular organisms to detect and defend against pathogens to prevent disease. Evolution affords mammals with a wide variety of independent immune mechanisms to control or eliminate invading infectious agents. Many pathogens acquire functions to deflect these immune mechanisms and promote infection. Following successful invasion of a host, cell autonomous signaling pathways drive the production of inflammatory cytokines, deployment of restriction factors and induction of cell death. Combined, these innate immune mechanisms attract dendritic cells, neutrophils and macrophages as well as innate lymphoid cells such as natural killer cells that all help control infection. Eventually, the development of adaptive pathogen-specific immunity clears infection and provides immune memory of the encounter. For obligate intracellular pathogens such as viruses, diverse cell death pathways make a pivotal contribution to early control by eliminating host cells before progeny are produced. Pro-apoptotic caspase-8 activity (along with caspase-10 in humans) executes extrinsic apoptosis, a nonlytic form of cell death triggered by TNF family death receptors (DRs). Over the past two decades, alternate extrinsic apoptosis and necroptosis outcomes have been described. Programmed necrosis, or necroptosis, occurs when receptor interacting protein kinase 3 (RIPK3) activates mixed lineage kinase-like (MLKL), causing cell leakage. Thus, activation of DRs, toll-like receptors (TLRs) or pathogen sensor Z-nucleic acid binding protein 1 (ZBP1) initiates apoptosis as well as necroptosis if not blocked by virus-encoded inhibitors. Mammalian cell death pathways are blocked by herpesvirus- and poxvirus-encoded cell death suppressors. Growing evidence has revealed the importance of Z-nucleic acid sensor, ZBP1, in the cell autonomous recognition of both DNA and RNA virus infection. This volume will explore the detente between viruses and cells to manage death machinery and avoid elimination to support dissemination within the host animal.

  View details for DOI 10.1007/82_2023_264

  View details for PubMedID 37563336

• The RIPK3 Scaffold Regulates Lung Inflammation During Pseudomonas Aeruginosa Pneumonia AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY Lyons, J. D., Mandal, P., Otani, S., Chihade, D. B., Easley, K. F., Swift, D. A., Burd, E. M., Liang, Z., Koval, M., Mocarski, E. S., Coopersmith, C. M. 2023; 68 (2): 150-160

  Abstract

  RIPK3 (receptor-interacting protein kinase 3) activity triggers cell death via necroptosis, whereas scaffold function supports protein binding and cytokine production. To determine if RIPK3 kinase or scaffold domains mediate pathology during Pseudomonas aeruginosa infection, control mice and those with deletion or mutation of RIPK3 and associated signaling partners were subjected to Pseudomonas pneumonia and followed for survival or killed for biologic assays. Murine immune cells were studied in vitro for Pseudomonas-induced cytokine production and cell death, and RIPK3 binding interactions were blocked with the viral inhibitor M45. Human tissue effects were assayed by infecting airway epithelial cells with Pseudomonas and measuring cytokine production after siRNA inhibition of RIPK3. Deletion of RIPK3 reduced inflammation and decreased animal mortality after Pseudomonas pneumonia. RIPK3 kinase inactivation did neither. In cell culture, RIPK3 was dispensable for cell killing by Pseudomonas and instead drove cytokine production that required the RIPK3 scaffold domain but not kinase activity. Blocking the RIP homotypic interaction motif (RHIM) with M45 reduced the inflammatory response to infection in vitro. Similarly, siRNA knockdown of RIPK3 decreased infection-triggered inflammation in human airway epithelial cells. Thus, the RIPK3 scaffold drives deleterious pulmonary inflammation and mortality in a relevant clinical model of Pseudomonas pneumonia. This process is distinct from kinase-mediated necroptosis, requiring only the RIPK3 RHIM. Inhibition of RHIM signaling is a potential strategy to reduce lung inflammation during infection.

  View details for DOI 10.1165/rcmb.2021-0474OC

  View details for Web of Science ID 000926612500007

  View details for PubMedID 36178467

  View details for PubMedCentralID PMC9986559

• RIPK3 and caspase 8 collaborate to limit herpes simplex encephalitis. PLoS pathogens Guo, H., Koehler, H. S., Mocarski, E. S., Dix, R. D. 2022; 18 (9): e1010857

  Abstract

  Invasion of the brain by herpes simplex virus 1 (HSV1) can lead to the development of herpes simplex encephalitis (HSE) that is often associated with significant morbidity and mortality regardless of therapeutic intervention. Both virus and host immune factors dictate HSE onset and progression. Because programmed cell death pathways including necroptosis are important antiviral defense mechanisms in HSV1-associated peripheral diseases, they might also play critical roles in HSV1 neuropathogenesis. HSV1-encoded ICP6 prevents receptor-interacting protein kinase 3 (RIPK3)-mediated necroptosis during infection of human cells, but it also acts as a species-dependent inducer of necroptosis in murine cells and thereby restricts virus replication. We therefore used an established mouse model of HSE to investigate RIPK3-mediated necroptosis impact on HSV1 neuropathogenesis. Following corneal HSV1 inoculation, RIPK3 knockout mice showed increased susceptibility to HSE when compared with wildtype mice indicating RIPK3 helps to limit HSE progression. RIPK3-mediated defense against HSE was found to be independent of the kinase domain necessary to drive necroptosis implicating that a death independent function of RIPK3 protects against HSE. Conversely the pro-necroptotic kinase function RIPK3 served to limit viral replication in corneal tissue implicating a tissue-specific RIPK3 function in limiting HSV1. Further evaluation of the kinase-independent mechanism to restrict HSE revealed that the RIPK3 signaling partner, caspase 8, contributes to limiting HSE neuropathogenesis. Increased HSE susceptibility from loss of caspase 8 and RIPK3 correlated with decreased levels of chemokines, cytokines, and antiviral lymphocytes recruitment to the brain. We conclude that RIPK3 contributes toward host control of HSV1 replication in a tissue-specific fashion. Whereas RIPK3-mediated necroptosis restricts virus replication within the cornea, kinase-independent induction of inflammation by RIPK3 in collaboration with caspase 8 restricts virus replication within the brain during HSE neuropathogenesis.

  View details for DOI 10.1371/journal.ppat.1010857

  View details for PubMedID 36121858

  View details for PubMedCentralID PMC9521923

• Multiple Autonomous Cell Death Suppression Strategies Ensure Cytomegalovirus Fitness VIRUSES-BASEL Mandal, P., Nagrani, L. N., Hernandez, L., McCormick, A., Dillon, C. P., Koehler, H. S., Roback, L., Alnemri, E. S., Green, D. R., Mocarski, E. S. 2021; 13 (9)

  Abstract

  Programmed cell death pathways eliminate infected cells and regulate infection-associated inflammation during pathogen invasion. Cytomegaloviruses encode several distinct suppressors that block intrinsic apoptosis, extrinsic apoptosis, and necroptosis, pathways that impact pathogenesis of this ubiquitous herpesvirus. Here, we expanded the understanding of three cell autonomous suppression mechanisms on which murine cytomegalovirus relies: (i) M38.5-encoded viral mitochondrial inhibitor of apoptosis (vMIA), a BAX suppressor that functions in concert with M41.1-encoded viral inhibitor of BAK oligomerization (vIBO), (ii) M36-encoded viral inhibitor of caspase-8 activation (vICA), and (iii) M45-encoded viral inhibitor of RIP/RHIM activation (vIRA). Following infection of bone marrow-derived macrophages, the virus initially deflected receptor-interacting protein kinase (RIPK)3-dependent necroptosis, the most potent of the three cell death pathways. This process remained independent of caspase-8, although suppression of this apoptotic protease enhances necroptosis in most cell types. Second, the virus deflected TNF-mediated extrinsic apoptosis, a pathway dependent on autocrine TNF production by macrophages that proceeds independently of mitochondrial death machinery or RIPK3. Third, cytomegalovirus deflected BCL-2 family protein-dependent mitochondrial cell death through combined TNF-dependent and -independent signaling even in the absence of RIPK1, RIPK3, and caspase-8. Furthermore, each of these cell death pathways dictated a distinct pattern of cytokine and chemokine activation. Therefore, cytomegalovirus employs sequential, non-redundant suppression strategies to specifically modulate the timing and execution of necroptosis, extrinsic apoptosis, and intrinsic apoptosis within infected cells to orchestrate virus control and infection-dependent inflammation. Virus-encoded death suppressors together hold control over an intricate network that upends host defense and supports pathogenesis in the intact mammalian host.

  View details for DOI 10.3390/v13091707

  View details for Web of Science ID 000702076500001

  View details for PubMedID 34578288

  View details for PubMedCentralID PMC8473406

• Vaccinia virus E3 prevents sensing of Z-RNA to block ZBP1-dependent necroptosis CELL HOST & MICROBE Koehler, H., Cotsmire, S., Zhang, T., Balachandran, S., Upton, J. W., Langland, J., Kalman, D., Jacobs, B. L., Mocarski, E. S. 2021; 29 (8): 1266-+

  Abstract

  Necroptosis mediated by Z-nucleic-acid-binding protein (ZBP)1 (also called DAI or DLM1) contributes to innate host defense against viruses by triggering cell death to eliminate infected cells. During infection, vaccinia virus (VACV) protein E3 prevents death signaling by competing for Z-form RNA through an N-terminal Zα domain. In the absence of this E3 domain, Z-form RNA accumulates during the early phase of VACV infection, triggering ZBP1 to recruit receptor interacting protein kinase (RIPK)3 and execute necroptosis. The C-terminal E3 double-strand RNA-binding domain must be retained to observe accumulation of Z-form RNA and induction of necroptosis. Substitutions of Zα from either ZBP1 or the RNA-editing enzyme double-stranded RNA adenosine deaminase (ADAR)1 yields fully functional E3 capable of suppressing virus-induced necroptosis. Overall, our evidence reveals the importance of Z-form RNA generated during VACV infection as a pathogen-associated molecular pattern (PAMP) unleashing ZBP1/RIPK3/MLKL-dependent necroptosis unless suppressed by viral E3.

  View details for DOI 10.1016/j.chom.2021.05.009

  View details for Web of Science ID 000684711700009

  View details for PubMedID 34192517

  View details for PubMedCentralID PMC9333947

• TNF Signaling Dictates Myeloid and Non-Myeloid Cell Crosstalk to Execute MCMV-Induced Extrinsic Apoptosis VIRUSES-BASEL Mandal, P., McCormick, A., Mocarski, E. S. 2020; 12 (11)

  Abstract

  Cytomegaloviruses all encode the viral inhibitor of caspase-8-induced apoptosis (vICA). After binding to this initiator caspase, vICA blocks caspase-8 proteolytic activity and ability to activate caspase-3 and/or caspase-7. In this manner, vICA has long been known to prevent apoptosis triggered via tumor necrosis factor (TNF) family death receptor-dependent extrinsic signaling. Here, we employ fully wild-type murine cytomegalovirus (MCMV) and vICA-deficient MCMV (∆M36) to investigate the contribution of TNF signaling to apoptosis during infection of different cell types. ∆M36 shows the expected ability to kill mouse splenic hematopoietic cells, bone marrow-derived macrophages (BMDM), and dendritic cells (BMDC). Antibody blockade or genetic elimination of TNF protects myeloid cells from death, and caspase-8 activation accompanies cell death. Interferons, necroptosis, and pyroptotic gasdermin D (GSDMD) do not contribute to myeloid cell death. Human and murine fibroblasts or murine endothelial cells (SVEC4-10) normally insensitive to TNF become sensitized to ∆M36-induced apoptosis when treated with TNF or TNF-containing BMDM-conditioned medium. We demonstrate that myeloid cells are the natural source of TNF that triggers apoptosis in either myeloid (autocrine) or non-myeloid cells (paracrine) during ∆M36 infection of mice. Caspase-8 suppression by vICA emerges as key to subverting innate immune elimination of a wide variety of infected cell types.

  View details for DOI 10.3390/v12111221

  View details for Web of Science ID 000593859300001

  View details for PubMedID 33126536

  View details for PubMedCentralID PMC7693317

• Recognizing limits of Z-nucleic acid binding protein (ZBP1/DAI/DLM1) function FEBS JOURNAL Koehler, H. S., Feng, Y., Mandal, P., Mocarski, E. S. 2020; 287 (20): 4362-4369

  Abstract

  Z-nucleic acid binding protein (ZBP)1 (also known as DAI and DLM1) is a pathogen sensor activated by double-strand character RNA to recruit receptor-interacting protein (RIP) kinase via a RIP homotypic interaction motif. The activation of receptor-interacting protein kinase (RIPK)3 and initiation of virus-induced necroptosis were initially reported in a landmark publication Upton et al. (Cell Host Microbe 11: 290, 2012) employing the DNA virus murine cytomegalovirus (MCMV). M45-encoded viral inhibitor of RIP activation prevents virus-induced necroptosis. Additional virus-encoded suppressors of necroptosis were then identified, including herpes simplex virus ICP6 and vaccinia virus E3L. Caspase-8 suppressors encoded by these DNA viruses block apoptosis, unleashing necroptosis mediated through Z-nucleic acid binding protein 1 (ZBP1) recruitment of RIPK3. These studies all utilized ZBP1-deficient mice generated by the Akira Lab (Zbp1-/- AK ) to bring the significance of virus-induced necroptosis to light. C57BL/6 mice were chosen as controls based on the assumption that mutant mice were congenic; however, these mice were recently found to display an unexpected innate immune deficit, lacking C57BL/6-specific NK1.1 and Ly49H natural killer cell subpopulations important in the early control of MCMV infection. Short nucleotide polymorphism analysis of Zbp1-/- AK breeders revealed a mixed genetic background (~ 71% C57BL/6 DNA and ~ 29% 129). Even though this level of 129 strain background does not alter ZBP1 cell-autonomous function as a sensor and mediator of necroptosis, it confounds innate immune response characteristics. In the future, genetic background must be carefully controlled before implicating ZBP1 function in response characteristics that shape immunity, inflammation, metabolism, and pathogenesis.

  View details for DOI 10.1111/febs.15242

  View details for Web of Science ID 000579213600005

  View details for PubMedID 32096322

• Necroptosis-based CRISPR knockout screen reveals Neuropilin-1 as a critical host factor for early stages of murine cytomegalovirus infection. Proceedings of the National Academy of Sciences of the United States of America Lane, R. K., Guo, H., Fisher, A. D., Diep, J., Lai, Z., Chen, Y., Upton, J. W., Carette, J., Mocarski, E. S., Kaiser, W. J. 2020

  Abstract

  Herpesviruses are ubiquitous human pathogens that cause a wide range of health complications. Currently, there is an incomplete understanding of cellular factors that contribute to herpesvirus infection. Here, we report an antiviral necroptosis-based genetic screen to identify novel host cell factors required for infection with the beta-herpesvirus murine cytomegalovirus (MCMV). Our genome-wide CRISPR-based screen harnessed the capacity of herpesvirus mutants that trigger antiviral necroptotic cell death upon early viral gene expression. Vascular endothelial growth factor (VEGF) and semaphorin-binding receptor Neuropilin-1 (Nrp-1) emerge as crucial determinants of MCMV infection. We find that elimination of Nrp-1 impairs early viral gene expression and reduces infection rates in endothelial cells, fibroblasts, and macrophages. Furthermore, preincubation of virus with soluble Nrp-1 dramatically inhibits infection by reducing virus attachment. Thus, Nrp-1 is a key determinant of the initial phase of MCMV infection.

  View details for DOI 10.1073/pnas.1921315117

  View details for PubMedID 32747526

• Squalene-based adjuvants stimulate CD8 T cell, but not antibody responses, through a RIPK3-dependent pathway. eLife Kim, E. H., Woodruff, M. C., Grigoryan, L., Maier, B., Lee, S. H., Mandal, P., Cortese, M., Natrajan, M. S., Ravindran, R., Ma, H., Merad, M., Gitlin, A. D., Mocarski, E. S., Jacob, J., Pulendran, B. 2020; 9

  Abstract

  The squalene-based oil-in-water emulsion (SE) vaccine adjuvant MF59 has been administered to more than 100 million people in more than 30 countries, in both seasonal and pandemic influenza vaccines. Despite its wide use and efficacy, its mechanisms of action remains unclear. In this study we demonstrate that immunization of mice with MF59 or its mimetic AddaVax (AV) plus soluble antigen results in robust antigen-specific antibody and CD8 T cell responses in lymph nodes and non-lymphoid tissues. Immunization triggered rapid RIPK3-kinase dependent necroptosis in the lymph node which peaked at 6 hours, followed by a sequential wave of apoptosis. Immunization with alum plus antigen did not induce RIPK3 kinase-dependent signaling. RIPK3-dependent signaling induced by MF59 or AV was essential for cross-presentation of antigen to CD8 T cells by Batf3-dependent CD8+ DCs. Consistent with this, RIPK3-kinase deficient or Batf3 deficient mice were impaired in their ability to mount adjuvant-enhanced CD8 T cell responses. However, CD8 T cell responses were unaffected in mice deficient in MLKL, a downstream mediator of necroptosis. Surprisingly, antibody responses were unaffected in RIPK3-kinase or Batf3 deficient mice. In contrast, antibody responses were impaired by in vivo administration of the pan-caspase inhibitor Z-VAD-FMK, but normal in caspase-1 deficient mice, suggesting a contribution from apoptotic caspases, in the induction of antibody responses. These results demonstrate that squalene-based vaccine adjuvants induce antigen-specific CD8 T cell and antibody responses, through RIPK3-dependent and-independent pathways, respectively.

  View details for DOI 10.7554/eLife.52687

  View details for PubMedID 32515732

• HIF1α Regulates Early Metabolic Changes due to Activation of Innate Immunity in Nuclear Reprogramming. Stem cell reports Liu, C. n., Ruan, H. n., Himmati, F. n., Zhao, M. T., Chen, C. C., Makar, M. n., Chen, I. Y., Sallam, K. n., Mocarski, E. S., Sayed, D. n., Sayed, N. n. 2020; 14 (2): 192–200

  Abstract

  Innate immune signaling has recently been shown to play an important role in nuclear reprogramming, by altering the epigenetic landscape and thereby facilitating transcription. However, the mechanisms that link innate immune activation and metabolic regulation in pluripotent stem cells remain poorly defined, particularly with regard to key molecular components. In this study, we show that hypoxia-inducible factor 1α (HIF1α), a central regulator of adaptation to limiting oxygen tension, is an unexpected but crucial regulator of innate immune-mediated nuclear reprogramming. HIF1α is dramatically upregulated as a consequence of Toll-like receptor 3 (TLR3) signaling and is necessary for efficient induction of pluripotency and transdifferentiation. Bioenergetics studies reveal that HIF1α regulates the reconfiguration of innate immune-mediated reprogramming through its well-established role in throwing a glycolytic switch. We believe that results from these studies can help us better understand the influence of immune signaling in tissue regeneration and lead to new therapeutic strategies.

  View details for DOI 10.1016/j.stemcr.2020.01.006

  View details for PubMedID 32048999

• Caspase-8 restricts natural killer cell accumulation during MCMV Infection MEDICAL MICROBIOLOGY AND IMMUNOLOGY Feng, Y., Daley-Bauer, L. P., Roback, L., Potempa, M., Lanier, L. L., Mocarski, E. S. 2019; 208 (3-4): 543-554

  Abstract

  Natural killer (NK) cells provide important host defense against herpesvirus infections and influence subsequent T cell control of replication and maintenance of latency. NK cells exhibit phases of expansion, contraction and memory formation in response to the natural mouse pathogen murine cytomegalovirus (MCMV). Innate and adaptive immune responses are tightly regulated in mammals to avoid excess tissue damage while preventing acute and chronic viral disease and assuring resistance to reinfection. Caspase (CASP)8 is an autoactivating aspartate-specific cysteine protease that initiates extrinsic apoptosis and prevents receptor interacting protein (RIP) kinase (RIPK)1-RIPK3-driven necroptosis. CASP8 also promotes death-independent signal transduction. All of these activities make contributions to inflammation. Here, we demonstrate that CASP8 restricts NK cell expansion during MCMV infection but does not influence NK memory. Casp8-/-Ripk3-/- mice mount higher NK response levels than Casp8+/-Ripk3-/- littermate controls or WT C57BL/6 J mice, indicating that RIPK3 deficiency alone does not contribute to NK response patterns. MCMV m157-responsive Ly49H+ NK cells support increased expansion of both Ly49H- NK cells and CD8 T cells in Casp8-/-Ripk3-/- mice. Surprisingly, hyperaccumulation of NK cells depends on the pronecrotic kinase RIPK1. Ripk1-/-Casp8-/-Ripk3-/- mice fail to show the enhanced expansion of lymphocytes observed in Casp8-/-Ripk3-/- mice even though development and homeostasis are preserved in uninfected Ripk1-/-Casp8-/-Ripk3-/- mice. Thus, CASP8 naturally regulates the magnitude of NK cell responses in response to infection where strong activation signals depend on another key regulator of death signaling, RIPK1. In addition, the strong NK cell response promotes survival of effector CD8 T cells during their expansion. Thus, hyperaccumulation of NK cells and crosstalk with T cells becomes amplified in the absence of extrinsic cell death machinery.

  View details for DOI 10.1007/s00430-019-00617-6

  View details for Web of Science ID 000475697400027

  View details for PubMedID 31115653

  View details for PubMedCentralID PMC6635065

• Caspase-8 restricts antiviral CD8 T cell hyperaccumulation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Feng, Y., Daley-Bauer, L. P., Roback, L., Guo, H., Koehler, H. S., Potempa, M., Lanier, L. L., Mocarski, E. S. 2019; 116 (30): 15170-15177

  Abstract

  The magnitude of CD8 T cell responses against viruses is checked by the balance of proliferation and death. Caspase-8 (CASP8) has the potential to influence response characteristics through initiation of apoptosis, suppression of necroptosis, and modulation of cell death-independent signal transduction. Mice deficient in CASP8 and RIPK3 (Casp8-/-Ripk3-/- ) mount enhanced peak CD8 T cell levels against the natural mouse pathogen murine cytomegalovirus (MCMV) or the human pathogen herpes simplex virus-1 compared with littermate control RIPK3-deficient or WT C57BL/6 mice, suggesting an impact of CASP8 on the magnitude of antiviral CD8 T cell expansion and not on contraction. The higher peak response to MCMV in Casp8-/-Ripk3-/- mice resulted from accumulation of greater numbers of terminally differentiated KLRG1hi effector CD8 T cell subsets. Antiviral Casp8-/-Ripk3-/- T cells exhibited enhanced proliferation when splenocytes were transferred into WT recipient mice. Thus, cell-autonomous CASP8 normally restricts CD8 T cell proliferation following T cell receptor activation in response to foreign antigen. Memory inflation is a hallmark quality of the T cell response to cytomegalovirus infection. Surprisingly, MCMV-specific memory inflation was not sustained long-term in Casp8-/-Ripk3-/- mice even though these mice retained immunity to secondary challenge. In addition, the accumulation of abnormal B220+CD3+ T cells in these viable CASP8-deficient mice was reduced by chronic MCMV infection. Combined, these data brings to light the cell death-independent role of CASP8 during CD8 T cell expansion in mice lacking the confounding impact of RIPK3-mediated necroptosis.

  View details for DOI 10.1073/pnas.1904319116

  View details for Web of Science ID 000476715500060

  View details for PubMedID 31285326

  View details for PubMedCentralID PMC6660791

• Herpes simplex virus 1 ICP6 impedes TNF receptor 1-induced necrosome assembly during compartmentalization to detergent-resistant membrane vesicles JOURNAL OF BIOLOGICAL CHEMISTRY Ali, M., Roback, L., Mocarski, E. S. 2019; 294 (3): 991-1004

  Abstract

  Receptor-interacting protein (RIP) kinase 3 (RIPK3)-dependent necroptosis directs inflammation and tissue injury, as well as anti-viral host defense. In human cells, herpes simplex virus 1 (HSV1) UL39-encoded ICP6 blocks RIP homotypic interacting motif (RHIM) signal transduction, preventing this leakage form of cell death and sustaining viral infection. TNF receptor 1 (TNFR1)-induced necroptosis is known to require the formation of a RIPK1-RIPK3-mixed lineage kinase domain-like pseudokinase (MLKL) signaling complex (necrosome) that we find compartmentalizes exclusively to caveolin-1-associated detergent-resistant membrane (DRM) vesicles in HT-29 cells. Translocation proceeds in the presence of RIPK3 kinase inhibitor GSK'840 or MLKL inhibitor necrosulfonomide but requires the kinase activity, as well as RHIM signaling of RIPK1. ICP6 impedes the translocation of RIPK1, RIPK3, and MLKL to caveolin-1-containing DRM vesicles without fully blocking the activation of RIPK3 or phosphorylation of MLKL. Consistent with the important contribution of RIPK1 RHIM-dependent recruitment of RIPK3, overexpression of RHIM-deficient RIPK3 results in phosphorylation of MLKL, but this does not lead to either translocation or necroptosis. Combined, these data reveal a critical role of RHIM signaling in the recruitment of the MLKL-containing necrosome to membrane vesicle-associated sites of aggregation. A similar mechanism is predicted for other RHIM-containing signaling adaptors, Z-nucleic acid-binding protein 1 (ZBP1) (also called DAI and DLM1), and TIR domain-containing adapter-inducing interferon-β (TRIF).

  View details for DOI 10.1074/jbc.RA118.004651

  View details for Web of Science ID 000458670200023

  View details for PubMedID 30504227

  View details for PubMedCentralID PMC6341382

• Remarkably Robust Antiviral Immune Response despite Combined Deficiency in Caspase-8 and RIPK3 JOURNAL OF IMMUNOLOGY Feng, Y., Livingston-Rosanoff, D., Roback, L., Sundararajan, A., Speck, S. H., Mocarski, E. S., Daley-Bauer, L. P. 2018; 201 (8): 2244-2255

  Abstract

  Caspase-8 (Casp8)-mediated signaling triggers extrinsic apoptosis while suppressing receptor-interacting protein kinase (RIPK) 3-dependent necroptosis. Although Casp8 is dispensable for the development of innate and adaptive immune compartments in mice, the importance of this proapoptotic protease in the orchestration of immune response to pathogens remains to be fully explored. In this study, Casp8-/-Ripk3-/- C57BL/6 mice show robust innate and adaptive immune responses to the natural mouse pathogen, murine CMV. When young, these mice lack lpr-like lymphoid hyperplasia and accumulation of either B220 + CD3+ or B220-CD3+CD4+ and CD8+ T cells with increased numbers of immature myeloid cells that are evident in older mice. Dendritic cell activation and cytokine production drive both NK and T cell responses to control viral infection in these mice, suggesting that Casp8 is dispensable to the generation of antiviral host defense. Curiously, NK and T cell expansion is amplified, with greater numbers observed by 7 d postinfection compared with either Casp8+/-Ripk3-/- or wild type (Casp8+/+Ripk3+/+ ) littermate controls. Casp8 and RIPK3 are natural targets of virus-encoded cell death suppressors that prevent infected cell apoptosis and necroptosis, respectively. It is clear from the current studies that the initiation of innate immunity and the execution of cytotoxic lymphocyte functions are all preserved despite the absence of Casp8 in responding cells. Thus, Casp8 and RIPK3 signaling is completely dispensable to the generation of immunity against this natural herpesvirus infection, although the pathways driven by these initiators serve as a crucial first line for host defense within virus-infected cells.

  View details for DOI 10.4049/jimmunol.1800110

  View details for Web of Science ID 000447646000007

  View details for PubMedID 30194111

  View details for PubMedCentralID PMC6211196

• Species-independent contribution of ZBP1/DAI/DLM-1-triggered necroptosis in host defense against HSV1 CELL DEATH & DISEASE Guo, H., Gilley, R. P., Fisher, A., Lane, R., Landsteiner, V. J., Ragan, K. B., Dovey, C. M., Carette, J. E., Upton, J. W., Mocarski, E. S., Kaiser, W. J. 2018; 9: 816

  Abstract

  Necroptosis complements apoptosis as a host defense pathway to stop virus infection. Herpes simplex virus shows a propensity to trigger necroptosis of mouse cells and mice even though cell death is blocked in human cells through UL39-encoded ICP6. This ribonucleotide reductase large subunit (R1) nucleates RHIM-dependent oligomerization of RIP3 kinase (RIPK3, also known as RIP3) in mouse cells but inhibits activation in cells from the natural human host. By interrogating the comparative behavior of ICP6-deficient viruses in mouse and human cells, here we unveil virus-induced necroptosis mediated by Z-DNA-binding protein 1 (ZBP1, also known as DAI). ZBP1 acts as a pathogen sensor to detect nascent RNA transcripts rather than input viral DNA or viral DNA generated through replication. Consistent with the implicated role of virus-induced necroptosis in restricting infection, viral pathogenesis is restored in Zbp1-/-, Ripk3-/- and Mlkl-/- mice. Thus, in addition to direct activation of RIPK3 via ICP6, HSV1 infection in mice and mouse cells triggers virus-induced necroptosis through ZBP1. Importantly, virus-induced necroptosis is also induced in human HT-29 cells by ICP6 mutant viruses; however, ZBP1 levels must be elevated for this pathway to be active. Thus, our studies reveal a common, species-independent role of this nucleic acid sensor to detect the presence of this virus. HSV1 ICP6 functions as a bona fide RHIM signaling inhibitor to block virus-induced necroptosis in its natural host. Altogether, ZBP1-dependent restriction of herpesvirus infection emerges as a potent antiviral armament of the innate immune system.

  View details for PubMedID 30050136

• Caspase-8 Collaborates with Caspase-11 to Drive Tissue Damage and Execution of Endotoxic Shock IMMUNITY Manda, P., Feng, Y., Lyons, J. D., Berger, S. B., Otani, S., DeLaney, A., Tharp, G. K., Maner-Smith, K., Burd, E. M., Schaeffer, M., Hoffman, S., Capriotti, C., Roback, L., Young, C. B., Liang, Z., Ortlund, E. A., DiPaolo, N. C., Bosinger, S., Bertin, J., Gough, P. J., Brodsky, I. E., Coopersmith, C. M., Shayakhmetov, D. M., Mocarski, E. S. 2018; 49 (1): 42-+

  Abstract

  The execution of shock following high dose E. coli lipopolysaccharide (LPS) or bacterial sepsis in mice required pro-apoptotic caspase-8 in addition to pro-pyroptotic caspase-11 and gasdermin D. Hematopoietic cells produced MyD88- and TRIF-dependent inflammatory cytokines sufficient to initiate shock without any contribution from caspase-8 or caspase-11. Both proteases had to be present to support tumor necrosis factor- and interferon-β-dependent tissue injury first observed in the small intestine and later in spleen and thymus. Caspase-11 enhanced the activation of caspase-8 and extrinsic cell death machinery within the lower small intestine. Neither caspase-8 nor caspase-11 was individually sufficient for shock. Both caspases collaborated to amplify inflammatory signals associated with tissue damage. Therefore, combined pyroptotic and apoptotic signaling mediated endotoxemia independently of RIPK1 kinase activity and RIPK3 function. These observations bring to light the relevance of tissue compartmentalization to disease processes in vivo where cytokines act in parallel to execute diverse cell death pathways.

  View details for DOI 10.1016/j.immuni.2018.06.011

  View details for Web of Science ID 000438949100010

  View details for PubMedID 30021146

  View details for PubMedCentralID PMC6064639

• MLKL Requires the Inositol Phosphate Code to Execute Necroptosis. Molecular cell Dovey, C. M., Diep, J. n., Clarke, B. P., Hale, A. T., McNamara, D. E., Guo, H. n., Brown, N. W., Cao, J. Y., Grace, C. R., Gough, P. J., Bertin, J. n., Dixon, S. J., Fiedler, D. n., Mocarski, E. S., Kaiser, W. J., Moldoveanu, T. n., York, J. D., Carette, J. E. 2018; 70 (5): 936–48.e7

  Abstract

  Necroptosis is an important form of lytic cell death triggered by injury and infection, but whether mixed lineage kinase domain-like (MLKL) is sufficient to execute this pathway is unknown. In a genetic selection for human cell mutants defective for MLKL-dependent necroptosis, we identified mutations in IPMK and ITPK1, which encode inositol phosphate (IP) kinases that regulate the IP code of soluble molecules. We show that IP kinases are essential for necroptosis triggered by death receptor activation, herpesvirus infection, or a pro-necrotic MLKL mutant. In IP kinase mutant cells, MLKL failed to oligomerize and localize to membranes despite proper receptor-interacting protein kinase-3 (RIPK3)-dependent phosphorylation. We demonstrate that necroptosis requires IP-specific kinase activity and that a highly phosphorylated product, but not a lowly phosphorylated precursor, potently displaces the MLKL auto-inhibitory brace region. These observations reveal control of MLKL-mediated necroptosis by a metabolite and identify a key molecular mechanism underlying regulated cell death.

  View details for PubMedID 29883610

• Retinoic Acid Inducible Gene 1 Protein (RIG1)-Like Receptor Pathway Is Required for Efficient Nuclear Reprogramming STEM CELLS Sayed, N., Ospino, F., Himmati, F., Lee, J., Chanda, P., Mocarski, E. S., Cooke, J. P. 2017; 35 (5): 1197-1207

  Abstract

  We have revealed a critical role for innate immune signaling in nuclear reprogramming to pluripotency, and in the nuclear reprogramming required for somatic cell transdifferentiation. Activation of innate immune signaling causes global changes in the expression and activity of epigenetic modifiers to promote epigenetic plasticity. In our previous papers, we focused on the role of toll-like receptor 3 (TLR3) in this signaling pathway. Here we define the role of another innate immunity pathway known to participate in the response to viral RNA, the retinoic acid-inducible gene 1 receptor (RIG-1)-like receptor (RLR) pathway. This pathway is represented by the sensors of viral RNA, RIG-1, LGP2 and MDA5. We first found that TLR3 deficiency only causes a partial inhibition of nuclear reprogramming to pluripotency in mouse tail-tip fibroblasts, which motivated us to determine the contribution of RLR. We found that knockdown of iPS-1, the common adaptor protein for the RLR family, substantially reduced nuclear reprogramming induced by retroviral or by mmRNA expression of Oct 4, Sox2, KLF4 and cMYC (OSKM). Importantly a double knockdown of both RLR and TLR3 pathway led to a further decrease in iPSC colonies suggesting an additive effect of both these pathways on nuclear reprogramming. Furthermore, in murine embryonic fibroblasts expressing a dox-inducible cassette of the genes encoding OSKM, an RLR agonist increased the yield of iPSCs. Similarly, the RLR agonist enhanced nuclear reprogramming by cell permeant peptides of the Yamanaka factors. Finally, in the dox-inducible system, RLR activation promotes activating histone marks in the promoter region of pluripotency genes. To conclude, innate immune signaling mediated by RLR plays a critical role in nuclear reprogramming. Manipulation of innate immune signaling may facilitate nuclear reprogramming to achieve pluripotency. This article is protected by copyright. All rights reserved.

  View details for DOI 10.1002/stem.2607

  View details for Web of Science ID 000400017200008

  View details for PubMedID 28276156

• Mouse cytomegalovirus M36 and M45 death suppressors cooperate to prevent inflammation resulting from antiviral programmed cell death pathways PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Daley-Bauer, L. P., Roback, L., Crosby, L. N., McCormick, A. L., Feng, Y., Kaiser, W. J., Mocarski, E. S. 2017; 114 (13): E2786-E2795

  Abstract

  The complex interplay between caspase-8 and receptor-interacting protein (RIP) kinase RIP 3 (RIPK3) driving extrinsic apoptosis and necroptosis is not fully understood. Murine cytomegalovirus triggers both apoptosis and necroptosis in infected cells; however, encoded inhibitors of caspase-8 activity (M36) and RIP3 signaling (M45) suppress these antiviral responses. Here, we report that this virus activates caspase-8 in macrophages to trigger apoptosis that gives rise to secondary necroptosis. Infection with double-mutant ΔM36/M45mutRHIM virus reveals a signaling pattern in which caspase-8 activates caspase-3 to drive apoptosis with subsequent RIP3-dependent activation of mixed lineage kinase domain-like (MLKL) leading to necroptosis. This combined cell death signaling is highly inflammatory, greater than either apoptosis induced by ΔM36 or necroptosis induced by M45mutRHIM virus. IL-6 production by macrophages is dramatically increased during double-mutant virus infection and correlates with faster antiviral responses in the host. Collaboratively, M36 and M45 target caspase-8 and RIP3 pathways together to suppress this proinflammatory cell death. This study reveals the effect of antiviral programmed cell death pathways on inflammation, shows that caspase-8 activation may go hand-in-hand with necroptosis in macrophages, and revises current understanding of independent and collaborative functions of M36 and M45 in blocking apoptotic and necroptotic cell death responses.

  View details for DOI 10.1073/pnas.1616829114

  View details for Web of Science ID 000397607300029

  View details for PubMedID 28292903

• A Phase 1 Study of 4 Live, Recombinant Human Cytomegalovirus Towne/Toledo Chimera Vaccines in Cytomegalovirus-Seronegative Men JOURNAL OF INFECTIOUS DISEASES Adler, S. P., Manganello, A., Lee, R., McVoy, M. A., Nixon, D. E., Plotkin, S., Mocarski, E., Cox, J. H., Fast, P. E., Nesterenko, P. A., Murray, S. E., Hill, A. B., Kemble, G. 2016; 214 (9): 1341-1348

  Abstract

   Human cytomegalovirus (HCMV) infection causes disease in newborns and transplant recipients. A HCMV vaccine (Towne) protects transplant recipients. The genomes of Towne and the nonattenuated Toledo strain were recombined, yielding 4 Towne/Toledo chimera vaccines. Each of 36 HCMV-seronegative men received 1 subcutaneous dose of 10, 100, or 1000 plaque-forming units (PFU) in cohorts of 3. Safety and immunogenicity were evaluated over 12 weeks after immunization and for 52 weeks for those who seroconverted. There were no serious local or systemic reactions. No subject had HCMV in urine or saliva. For chimera 3, none of 9 subjects seroconverted. For chimera 1, 1 of 9 seroconverted (the seroconverter received 100 PFU). For chimera 2, 3 subjects seroconverted (1 received 100 PFU, and 2 received 1000 PFU). For chimera 4, 7 subjects seroconverted (1 received 10 PFU, 3 received 100 PFU, and 3 received 1000 PFU). All 11 seroconverters developed low but detectable levels of neutralizing activity. CD4(+) T-cell responses were detectable in 1 subject (who received 100 PFU of chimera 4). Seven subjects receiving chimera 2 or 4 had detectable CD8(+) T-cell responses to IE1; 3 responded to 1-2 additional antigens. The Towne/Toledo chimera vaccine candidates were well tolerated and were not excreted. Additional human trials of chimeras 2 and 4 are appropriate. NCT01195571.

  View details for DOI 10.1093/infdis/jiw365

  View details for PubMedID 27521362

• RIPK3 Activates Parallel Pathways of MLKL-Driven Necroptosis and FADD-Mediated Apoptosis to Protect against Influenza A Virus CELL HOST & MICROBE Nogusa, S., Thapa, R. J., Dillon, C. P., Liedmann, S., Oguin, T. H., Ingram, J. P., Rodriguez, D. A., Kosoff, R., Sharma, S., Sturm, O., Verbist, K., Gough, P. J., Bertin, J., Hartmann, B. M., Sealfon, S. C., Kaiser, W. J., Mocarski, E. S., Lopez, C. B., Thomas, P. G., Oberst, A., Green, D. R., Balachandran, S. 2016; 20 (1): 13-24

  Abstract

  Influenza A virus (IAV) is a lytic virus in primary cultures of many cell types and in vivo. We report that the kinase RIPK3 is essential for IAV-induced lysis of mammalian fibroblasts and lung epithelial cells. Replicating IAV drives assembly of a RIPK3-containing complex that includes the kinase RIPK1, the pseudokinase MLKL, and the adaptor protein FADD, and forms independently of signaling by RNA-sensing innate immune receptors (RLRs, TLRs, PKR), or the cytokines type I interferons and TNF-α. Downstream of RIPK3, IAV activates parallel pathways of MLKL-driven necroptosis and FADD-mediated apoptosis, with the former reliant on RIPK3 kinase activity and neither on RIPK1 activity. Mice deficient in RIPK3 or doubly deficient in MLKL and FADD, but not MLKL alone, are more susceptible to IAV than their wild-type counterparts, revealing an important role for RIPK3-mediated apoptosis in antiviral immunity. Collectively, these results outline RIPK3-activated cytolytic mechanisms essential for controlling respiratory IAV infection.

  View details for DOI 10.1016/j.chom.2016.05.011

  View details for Web of Science ID 000380707200006

  View details for PubMedID 27321907

• T cell-intrinsic ASC critically promotes T(H)17-mediated experimental autoimmune encephalomyelitis NATURE IMMUNOLOGY Martin, B. N., Wang, C., Zhang, C., Kang, Z., Gulen, M. F., Zepp, J. A., Zhao, J., Bian, G., Do, J., Min, B., Pavicic, P. G., El-Sanadi, C., Fox, P. L., Akitsu, A., Iwakura, Y., Sarkar, A., Wewers, M. D., Kaiser, W. J., Mocarski, E. S., Rothenberg, M. E., Hise, A. G., Dubyak, G. R., Ransohoff, R. M., Li, X. 2016; 17 (5): 583-?

  Abstract

  Interleukin 1β (IL-1β) is critical for the in vivo survival, expansion and effector function of IL-17-producing helper T (T(H)17) cells during autoimmune responses, including experimental autoimmune encephalomyelitis (EAE). However, the spatiotemporal role and cellular source of IL-1β during EAE pathogenesis are poorly defined. In the present study, we uncovered a T cell-intrinsic inflammasome that drives IL-1β production during T(H)17-mediated EAE pathogenesis. Activation of T cell antigen receptors induced expression of pro-IL-1β, whereas ATP stimulation triggered T cell production of IL-1β via ASC-NLRP3-dependent caspase-8 activation. IL-1R was detected on T(H)17 cells but not on type 1 helper T (T(H)1) cells, and ATP-treated T(H)17 cells showed enhanced survival compared with ATP-treated T(H)1 cells, suggesting autocrine action of T(H)17-derived IL-1β. Together these data reveal a critical role for IL-1β produced by a T(H)17 cell-intrinsic ASC-NLRP3-caspase-8 inflammasome during inflammation of the central nervous system.

  View details for DOI 10.1038/ni.3389

  View details for Web of Science ID 000374323100018

  View details for PubMedID 26998763

• Caspase-8 as an Effector and Regulator of NLRP3 Inflammasome Signaling JOURNAL OF BIOLOGICAL CHEMISTRY Antonopoulos, C., Russo, H. M., El Sanadi, C., Martin, B. N., Li, X., Kaiser, W. J., Mocarski, E. S., Dubyak, G. R. 2015; 290 (33): 20167-20184

  Abstract

  We recently described the induction of noncanonical IL-1β processing via caspase-8 recruited to ripoptosome signaling platforms in myeloid leukocytes. Here, we demonstrate that activated NLRP3·ASC inflammasomes recruit caspase-8 to drive IL-1β processing in murine bone marrow-derived dendritic cells (BMDC) independent of caspase-1 and -11. Sustained stimulation (>2 h) of LPS-primed caspase-1-deficient (Casp1/11(-/-)) BMDC with the canonical NLRP3 inflammasome agonist nigericin results in release of bioactive IL-1β in conjunction with robust caspase-8 activation. This IL-1β processing and caspase-8 activation do not proceed in Nlrp3(-/-) or Asc(-/-) BMDC and are suppressed by pharmacological inhibition of caspase-8, indicating that caspase-8 can act as a direct IL-1β-converting enzyme during NLRP3 inflammasome activation. In contrast to the rapid caspase-1-mediated death of wild type (WT) BMDC via NLRP3-dependent pyroptosis, nigericin-stimulated Casp1/11(-/-) BMDC exhibit markedly delayed cell death via NLRP3-dependent apoptosis. Biochemical analyses of WT and Casp1/11(-/-) BMDC indicated that caspase-8 is proteolytically processed within detergent-insoluble ASC-enriched protein complexes prior to extracellular export during nigericin treatment. Although nigericin-stimulated caspase-1 activation and activity are only modestly attenuated in caspase-8-deficient (Casp8(-/-)Rip3(-/-)) BMDC, these cells do not exhibit the rapid loss of viability of WT cells. These results support a contribution of caspase-8 to both IL-1β production and regulated death signaling via NLRP3 inflammasomes. In the absence of caspase-1, NLRP3 inflammasomes directly utilize caspase-8 as both a pro-apoptotic initiator and major IL-1β-converting protease. In the presence of caspase-1, caspase-8 acts as a positive modulator of the NLRP3-dependent caspase-1 signaling cascades that drive both IL-1β production and pyroptotic death.

  View details for DOI 10.1074/jbc.M115.652321

  View details for Web of Science ID 000359608900019

  View details for PubMedID 26100631

  View details for PubMedCentralID PMC4536427

• Manipulation of apoptosis and necroptosis signaling by herpesviruses MEDICAL MICROBIOLOGY AND IMMUNOLOGY Guo, H., Kaiser, W. J., Mocarski, E. S. 2015; 204 (3): 439-448

  Abstract

  Like apoptosis, necroptosis is an innate immune mechanism that eliminates pathogen-infected cells. Receptor-interacting protein kinase (RIP)3 (also called RIPK3) mediates necrotic death by phosphorylating an executioner protein, MLKL, leading to plasma membrane leakage. The pathway is triggered against viruses that block caspase 8. In murine CMV, the viral inhibitor of caspase 8 activation prevents extrinsic apoptosis but also has the potential to unleash necroptosis. This virus encodes the viral inhibitor of RIP activation to prevent RIP homotypic interaction motif (RHIM)-dependent signal transduction and necroptosis. Recent investigations reveal a similar mechanism at play in the human alpha-herpesviruses, herpes simplex virus (HSV)1 and HSV2, where RHIM competitor function and caspase 8 suppression are carried out by the virus-encoded large subunit of ribonucleotide reductase (R1). In human cells, R1 inhibition of caspase 8 prevents TNF-induced apoptosis, but sensitizes to TNF-induced necroptosis. The RHIM and caspase 8 interaction domains of R1 collaborate to prevent RIP3-dependent steps and enable both herpesviruses to deflect host cell death machinery that would cut short infection. In mouse cells, HSV1 infection by itself triggers necroptosis by driving RIP3 protein kinase activity. HSV1 R1 contributes to the activation of RIP3 adaptor function in mice, a popular host animal for experimental infection. Based on these studies, infection of RIP3-kinase inactive mice should be explored in models of pathogenesis and latency. The necrotic death pathway that is suppressed during infection in the natural host becomes a cross-species barrier to infection in a non-natural host.

  View details for DOI 10.1007/s00430-015-0410-5

  View details for Web of Science ID 000354897600021

  View details for PubMedID 25828583

• Caspase-8 scaffolding function and MLKL regulate NLRP3 inflammasome activation downstream of TLR3 NATURE COMMUNICATIONS Kang, S., Fernandes-Alnemri, T., Rogers, C., Mayes, L., Wang, Y., Dillon, C., Roback, L., Kaiser, W., Oberst, A., Sagara, J., Fitzgerald, K. A., Green, D. R., Zhang, J., Mocarski, E. S., Alnemri, E. S. 2015; 6

  Abstract

  TLR2 promotes NLRP3 inflammasome activation via an early MyD88-IRAK1-dependent pathway that provides a priming signal (signal 1) necessary for activation of the inflammasome by a second potassium-depleting signal (signal 2). Here we show that TLR3 binding to dsRNA promotes post-translational inflammasome activation through intermediate and late TRIF/RIPK1/FADD-dependent pathways. Both pathways require the scaffolding but not the catalytic function of caspase-8 or RIPK1. Only the late pathway requires kinase competent RIPK3 and MLKL function. Mechanistically, FADD/caspase-8 scaffolding function provides a post-translational signal 1 in the intermediate pathway, whereas in the late pathway it helps the oligomerization of RIPK3, which together with MLKL provides both signal 1 and 2 for inflammasome assembly. Cytoplasmic dsRNA activates NLRP3 independent of TRIF, RIPK1, RIPK3 or mitochondrial DRP1, but requires FADD/caspase-8 in wildtype macrophages to remove RIPK3 inhibition. Our study provides a comprehensive analysis of pathways that lead to NLRP3 inflammasome activation in response to dsRNA.

  View details for DOI 10.1038/ncomms8515

  View details for Web of Science ID 000357178900001

  View details for PubMedID 26104484

  View details for PubMedCentralID PMC4480782

• Necroptosis: The Trojan horse in cell autonomous antiviral host defense VIROLOGY Mocarski, E. S., Guo, H., Kaiser, W. J. 2015; 479: 160-166

  View details for DOI 10.1016/j.virol.2015.03.016

  View details for Web of Science ID 000354909500015

• Suppression of RIP3-dependent Necroptosis by Human Cytomegalovirus JOURNAL OF BIOLOGICAL CHEMISTRY Omoto, S., Guo, H., Talekar, G. R., Roback, L., Kaiser, W. J., Mocarski, E. S. 2015; 290 (18): 11635-11648

  Abstract

  Necroptosis is an alternate programmed cell death pathway that is unleashed by caspase-8 compromise and mediated by receptor-interacting protein kinase 3 (RIP3). Murine cytomegalovirus (CMV) and herpes simplex virus (HSV) encode caspase-8 inhibitors that prevent apoptosis together with competitors of RIP homotypic interaction motif (RHIM)-dependent signal transduction to interrupt the necroptosis. Here, we show that pro-necrotic murine CMV M45 mutant virus drives virus-induced necroptosis during nonproductive infection of RIP3-expressing human fibroblasts, whereas WT virus does not. Thus, M45-encoded RHIM competitor, viral inhibitor of RIP activation, sustains viability of human cells like it is known to function in infected mouse cells. Importantly, human CMV is shown to block necroptosis induced by either TNF or M45 mutant murine CMV in RIP3-expressing human cells. Human CMV blocks TNF-induced necroptosis after RIP3 activation and phosphorylation of the mixed lineage kinase domain-like (MLKL) pseudokinase. An early, IE1-regulated viral gene product acts on a necroptosis step that follows MLKL phosphorylation prior to membrane leakage. This suppression strategy is distinct from RHIM signaling competition by murine CMV or HSV and interrupts an execution process that has not yet been fully elaborated.

  View details for DOI 10.1074/jbc.M115.646042

  View details for Web of Science ID 000353719400038

  View details for PubMedID 25778401

• The immunological underpinnings of vaccinations to prevent cytomegalovirus disease CELLULAR & MOLECULAR IMMUNOLOGY McCormick, A. L., Mocarski, E. S. 2015; 12 (2): 170-179

  Abstract

  A universal cytomegalovirus (CMV) vaccination promises to reduce the burden of the developmental damage that afflicts up to 0.5% of live births worldwide. An effective vaccination that prevents transplacental transmission would reduce CMV congenital disease and CMV-associated still births and leave populations less susceptible to opportunistic CMV disease. Thus, a vaccination against this virus has long been recognized for the potential of enormous health-care savings because congenital damage is life-long and existing anti-viral options are limited. Vaccine researchers, industry leaders, and regulatory representatives have discussed the challenges posed by clinical efficacy trials that would lead to a universal CMV vaccine, reviewing the links between infection and disease, and identifying settings where disrupting viral transmission might provide a surrogate endpoint for disease prevention. Reducing the complexity of such trials would facilitate vaccine development. Children and adolescents are the targets for universal vaccination, with the expectation of protecting the offspring of immunized women. Given that a majority of females worldwide experience CMV infection during childhood, a universal vaccine must boost natural immunity and reduce transmission due to reactivation and re-infection as well as primary infection during pregnancy. Although current vaccine strategies recognize the value of humoral and cellular immunity, the precise mechanisms that act at the placental interface remain elusive. Immunity resulting from natural infection appears to limit rather than prevent reactivation of latent viruses and susceptibility to re-infection, leaving a challenge for universal vaccination to improve upon natural immunity levels. Despite these hurdles, early phase clinical trials have achieved primary end points in CMV seronegative subjects. Efficacy studies must be expanded to mixed populations of CMV-naive and naturally infected subjects to understand the overall efficacy and potential. Together with CMV vaccine candidates currently in clinical development, additional promising preclinical strategies continue to come forward; however, these face limitations due to the insufficient understanding of host defense mechanisms that prevent transmission, as well as the age-old challenges of reaching the appropriate threshold of immunogenicity, efficacy, durability and potency. This review focuses on the current understanding of natural and CMV vaccine-induced protective immunity.

  View details for DOI 10.1038/cmi.2014.120

  View details for Web of Science ID 000350571100005

  View details for PubMedID 25544503

• The A, B, Cs of Herpesvirus Capsids VIRUSES-BASEL Tandon, R., Mocarski, E. S., Conway, J. F. 2015; 7 (3): 899-914

  Abstract

  Assembly of herpesvirus nucleocapsids shares significant similarities with the assembly of tailed dsDNA bacteriophages; however, important differences exist. A unique feature of herpesviruses is the presence of different mature capsid forms in the host cell nucleus during infection. These capsid forms, referred to as A-, B-, and C-capsids, represent empty capsids, scaffold containing capsids and viral DNA containing capsids, respectively. The C-capsids are the closest in form to those encapsidated into mature virions and are considered precursors to infectious virus. The evidence supporting A- and B-capsids as either abortive forms or assembly intermediates has been lacking. Interaction of specific capsid forms with viral tegument proteins has been proposed to be a mechanism for quality control at the point of nuclear egress of mature particles. Here, we will review the available literature on these capsid forms and present data to debate whether A- and B-capsids play an important or an extraneous role in the herpesvirus life cycle.

  View details for DOI 10.3390/v7030899

  View details for Web of Science ID 000351943000004

  View details for PubMedID 25730559

  View details for PubMedCentralID PMC4379554

• Herpes Simplex Virus Suppresses Necroptosis in Human Cells CELL HOST & MICROBE Guo, H., Omoto, S., Harris, P. A., Finger, J. N., Bertin, J., Gough, P. J., Kaiser, W. J., Mocarski, E. S. 2015; 17 (2): 243-251

  Abstract

  Herpes simplex virus (HSV)-1 and HSV-2 are significant human pathogens causing recurrent disease. During infection, HSV modulates cell death pathways using the large subunit (R1) of ribonucleotide reductase (RR) to suppress apoptosis by binding to and blocking caspase-8. Here, we demonstrate that HSV-1 and HSV-2 R1 proteins (ICP6 and ICP10, respectively) also prevent necroptosis in human cells by inhibiting the interaction between receptor-interacting protein kinase 1 (RIP1) and RIP3, a key step in tumor necrosis factor (TNF)-induced necroptosis. We show that suppression of this cell death pathway requires an N-terminal RIP homotypic interaction motif (RHIM) within R1, acting in concert with the caspase-8-binding domain, which unleashes necroptosis independent of RHIM function. Thus, necroptosis is a human host defense pathway against two important viral pathogens that naturally subvert multiple death pathways via a single evolutionarily conserved gene product.

  View details for DOI 10.1016/j.chom.2015.01.003

  View details for Web of Science ID 000349761700013

  View details for PubMedID 25674983

• MicroRNA miR-21 Attenuates Human Cytomegalovirus Replication in Neural Cells by Targeting Cdc25a JOURNAL OF VIROLOGY Fu, Y., Liu, X., Li, X., Shen, Z., Yang, B., Wu, C., Li, J., Miao, L., Ye, H., Qiao, G., Rayner, S., Chavanas, S., Davrinche, C., Britt, W. J., Tang, Q., McVoy, M., Mocarski, E., Luo, M. 2015; 89 (2): 1070-1082

  Abstract

  Congenital human cytomegalovirus (HCMV) infection is a leading cause of birth defects, primarily manifesting as neurological disorders. HCMV infection alters expression of cellular microRNAs (miRs) and induces cell cycle arrest, which in turn modifies the cellular environment to favor virus replication. Previous observations found that HCMV infection reduces miR-21 expression in neural progenitor/stem cells (NPCs). Here, we show that infection of NPCs and U-251MG cells represses miR-21 while increasing the levels of Cdc25a, a cell cycle regulator and known target of miR-21. These opposing responses to infection prompted an investigation of the relationship between miR-21, Cdc25a, and viral replication. Overexpression of miR-21 in NPCs and U-251MG cells inhibited viral gene expression, genome replication, and production of infectious progeny, while shRNA-knockdown of miR-21 in U-251MG cells increased viral gene expression. In contrast, overexpression of Cdc25a in U-251MG cells increased viral gene expression and production of infectious progeny and overcame the inhibitory effects of miR-21 overexpression. Three viral gene products-IE1, pp71, and UL26-were shown to inhibit miR-21 expression at the transcriptional level. These results suggest that Cdc25a promotes HCMV replication and elevation of Cdc25a levels after HCMV infection are due in part to HCMV-mediated repression of miR-21. Thus, miR-21 is an intrinsic antiviral factor that is modulated by HCMV infection. This suggests a role for miR-21 downregulation in the neuropathogenesis of HCMV infection of the developing CNS.Human cytomegalovirus (HCMV) is a ubiquitous pathogen and has very high prevalence among population, especially in China, and congenital HCMV infection is a major cause for birth defects. Elucidating virus-host interactions that govern HCMV replication in neuronal cells is critical to understanding the neuropathogenesis of birth defects resulting from congenital infection. In this study, we confirm that HCMV infection downregulates miR-21 but upregulates Cdc25a. Further determined the negative effects of cellular miRNA miR-21 on HCMV replication in neural progenitor/stem cells and U-251MG glioblastoma/astrocytoma cells. More importantly, our results provide the first evidence that miR-21 negatively regulates HCMV replication by targeting Cdc25a, a vital cell cycle regulator. We further found that viral gene products of IE1, pp71, and UL26 play roles in inhibiting miR-21 expression, which in turn causes increases in Cdc25a and benefits HCMV replication. Thus, miR-21 appears to be an intrinsic antiviral factor that represents a potential target for therapeutic intervention.

  View details for DOI 10.1128/JVI01740-14

  View details for Web of Science ID 000347178900016

  View details for PubMedID 25378484

• TNFR1-dependent cell death drives inflammation in Sharpin-deficient mice ELIFE Rickard, J. A., Anderton, H., Etemadi, N., Nachbur, U., Darding, M., Peltzer, N., Lalaoui, N., Lawlor, K. E., Vanyai, H., Hall, C., Bankovacki, A., Gangoda, L., Wong, W. W., Corbin, J., Huang, C., Mocarski, E. S., Murphy, J. M., Alexander, W. S., Voss, A. K., Vaux, D. L., Kaiser, W. J., Walczak, H., Silke, J. 2014; 3

  Abstract

  SHARPIN regulates immune signaling and contributes to full transcriptional activity and prevention of cell death in response to TNF in vitro. The inactivating mouse Sharpin cpdm mutation causes TNF-dependent multi-organ inflammation, characterized by dermatitis, liver inflammation, splenomegaly, and loss of Peyer's patches. TNF-dependent cell death has been proposed to cause the inflammatory phenotype and consistent with this we show Tnfr1, but not Tnfr2, deficiency suppresses the phenotype (and it does so more efficiently than Il1r1 loss). TNFR1-induced apoptosis can proceed through caspase-8 and BID, but reduction in or loss of these players generally did not suppress inflammation, although Casp8 heterozygosity significantly delayed dermatitis. Ripk3 or Mlkl deficiency partially ameliorated the multi-organ phenotype, and combined Ripk3 deletion and Casp8 heterozygosity almost completely suppressed it, even restoring Peyer's patches. Unexpectedly, Sharpin, Ripk3 and Casp8 triple deficiency caused perinatal lethality. These results provide unexpected insights into the developmental importance of SHARPIN.

  View details for DOI 10.7554/eLife.03464

  View details for Web of Science ID 000345643700006

  View details for PubMedID 25443632

  View details for PubMedCentralID PMC4270099

• RIP3 Induces Apoptosis Independent of Pronecrotic Kinase Activity MOLECULAR CELL Mandal, P., Berger, S. B., Pillay, S., Moriwaki, K., Huang, C., Guo, H., Lich, J. D., Finger, J., Kasparcova, V., Votta, B., Ouellette, M., King, B. W., Wisnoski, D., Lakdawala, A. S., DeMartino, M. P., Casillas, L. N., Haile, P. A., Sehon, C. A., Marquis, R. W., Upton, J., Daley-Bauer, L. P., Roback, L., Ramia, N., Dovey, C. M., Carette, J. E., Chan, F. K., Bertin, J., Gough, P. J., Mocarski, E. S., Kaiser, W. J. 2014; 56 (4): 481-495

  View details for DOI 10.1016/j.molcel.2014.10.021

  View details for Web of Science ID 000345502200005

• Caspase-8 Modulates Dectin-1 and Complement Receptor 3-Driven IL-1 beta Production in Response to beta-Glucans and the Fungal Pathogen, Candida albicans JOURNAL OF IMMUNOLOGY Ganesan, S., Rathinam, V. A., Bossaller, L., Army, K., Kaiser, W. J., Mocarski, E. S., Dillon, C. P., Green, D. R., Mayadas, T. N., Levitz, S. M., Hise, A. G., Silverman, N., Fitzgerald, K. A. 2014; 193 (5): 2519-2530

  Abstract

  Inflammasomes are central mediators of host defense to a wide range of microbial pathogens. The nucleotide-binding domain and leucine-rich repeat containing family (NLR), pyrin domain-containing 3 (NLRP3) inflammasome plays a key role in triggering caspase-1-dependent IL-1β maturation and resistance to fungal dissemination in Candida albicans infection. β-Glucans are major components of fungal cell walls that trigger IL-1β secretion in both murine and human immune cells. In this study, we sought to determine the contribution of β-glucans to C. albicans-induced inflammasome responses in mouse dendritic cells. We show that the NLRP3-apoptosis-associated speck-like protein containing caspase recruitment domain protein-caspase-1 inflammasome is absolutely critical for IL-1β production in response to β-glucans. Interestingly, we also found that both complement receptor 3 (CR3) and dectin-1 play a crucial role in coordinating β-glucan-induced IL-1β processing as well as a cell death response. In addition to the essential role of caspase-1, we identify an important role for the proapoptotic protease caspase-8 in promoting β-glucan-induced cell death and NLRP3 inflammasome-dependent IL-1β maturation. A strong requirement for CR3 and caspase-8 also was found for NLRP3-dependent IL-1β production in response to heat-killed C. albicans. Taken together, these results define the importance of dectin-1, CR3, and caspase-8, in addition to the canonical NLRP3 inflammasome, in mediating β-glucan- and C. albicans-induced innate responses in dendritic cells. Collectively, these findings establish a novel link between β-glucan recognition receptors and the inflammatory proteases caspase-8 and caspase-1 in coordinating cytokine secretion and cell death in response to immunostimulatory fungal components.

  View details for DOI 10.4049/jimmunol.1400276

  View details for Web of Science ID 000341140600052

  View details for PubMedID 25063877

  View details for PubMedCentralID PMC4134963

• Cutting Edge: RIP1 Kinase Activity Is Dispensable for Normal Development but Is a Key Regulator of Inflammation in SHARPIN-Deficient Mice JOURNAL OF IMMUNOLOGY Berger, S. B., Kasparcova, V., Hoffman, S., Swift, B., Dare, L., Schaeffer, M., Capriotti, C., Cook, M., Finger, J., Hughes-Earle, A., Harris, P. A., Kaiser, W. J., Mocarski, E. S., Bertin, J., Gough, P. J. 2014; 192 (12): 5476-5480

  Abstract

  RIP1 (RIPK1) kinase is a key regulator of TNF-induced NF-κB activation, apoptosis, and necroptosis through its kinase and scaffolding activities. Dissecting the balance of RIP1 kinase activity and scaffolding function in vivo during development and TNF-dependent inflammation has been hampered by the perinatal lethality of RIP1-deficient mice. In this study, we generated RIP1 kinase-dead (Ripk1(K45A)) mice and showed they are viable and healthy, indicating that the kinase activity of RIP1, but not its scaffolding function, is dispensable for viability and homeostasis. After validating that the Ripk1(K45A) mice were specifically protected against necroptotic stimuli in vitro and in vivo, we crossed them with SHARPIN-deficient cpdm mice, which develop severe skin and multiorgan inflammation that has been hypothesized to be mediated by TNF-dependent apoptosis and/or necroptosis. Remarkably, crossing Ripk1(K45A) mice with the cpdm strain protected against all cpdm-related pathology. Together, these data suggest that RIP1 kinase represents an attractive therapeutic target for TNF-driven inflammatory diseases.

  View details for DOI 10.4049/jimmunol.1400499

  View details for Web of Science ID 000337172100006

  View details for PubMedID 24821972

• RIP1 suppresses innate immune necrotic as well as apoptotic cell death during mammalian parturition PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kaiser, W. J., Daley-Bauer, L. P., Thapa, R. J., Mandal, P., Berger, S. B., Huang, C., Sundararajan, A., Guo, H., Roback, L., Speck, S. H., Bertin, J., Gough, P. J., Balachandran, S., Mocarski, E. S. 2014; 111 (21): 7753-7758

  Abstract

  The pronecrotic kinase, receptor interacting protein (RIP1, also called RIPK1) mediates programmed necrosis and, together with its partner, RIP3 (RIPK3), drives midgestational death of caspase 8 (Casp8)-deficient embryos. RIP1 controls a second vital step in mammalian development immediately after birth, the mechanism of which remains unresolved. Rip1(-/-) mice display perinatal lethality, accompanied by gross immune system abnormalities. Here we show that RIP1 K45A (kinase dead) knockin mice develop normally into adulthood, indicating that development does not require RIP1 kinase activity. In the face of complete RIP1 deficiency, cells develop sensitivity to RIP3-mixed lineage kinase domain-like-mediated necroptosis as well as to Casp8-mediated apoptosis activated by diverse innate immune stimuli (e.g., TNF, IFN, double-stranded RNA). When either RIP3 or Casp8 is disrupted in combination with RIP1, the resulting double knockout mice exhibit slightly prolonged survival over RIP1-deficient animals. Surprisingly, triple knockout mice with combined RIP1, RIP3, and Casp8 deficiency develop into viable and fertile adults, with the capacity to produce normal levels of myeloid and lymphoid lineage cells. Despite the combined deficiency, these mice sustain a functional immune system that responds robustly to viral challenge. A single allele of Rip3 is tolerated in Rip1(-/-)Casp8(-/-)Rip3(+/-) mice, contrasting the need to eliminate both alleles of either Rip1 or Rip3 to rescue midgestational death of Casp8-deficient mice. These observations reveal a vital kinase-independent role for RIP1 in preventing pronecrotic as well as proapoptotic signaling events associated with life-threatening innate immune activation at the time of mammalian parturition.

  View details for DOI 10.1073/pnas.1401857111

  View details for Web of Science ID 000336411300059

  View details for PubMedID 24821786

• Caspase-8 and RIP kinases regulate bacteria-induced innate immune responses and cell death PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Weng, D., Marty-Roix, R., Ganesan, S., Proulx, M. K., Vladimer, G. I., Kaiser, W. J., Mocarski, E. S., Pouliot, K., Chan, F. K., Kelliher, M. A., Harris, P. A., Bertin, J., Gough, P. J., Shayakhmetov, D. M., Goguen, J. D., Fitzgerald, K. A., Silverman, N., Lien, E. 2014; 111 (20): 7391-7396

  Abstract

  A number of pathogens cause host cell death upon infection, and Yersinia pestis, infamous for its role in large pandemics such as the "Black Death" in medieval Europe, induces considerable cytotoxicity. The rapid killing of macrophages induced by Y. pestis, dependent upon type III secretion system effector Yersinia outer protein J (YopJ), is minimally affected by the absence of caspase-1, caspase-11, Fas ligand, and TNF. Caspase-8 is known to mediate apoptotic death in response to infection with several viruses and to regulate programmed necrosis (necroptosis), but its role in bacterially induced cell death is poorly understood. Here we provide genetic evidence for a receptor-interacting protein (RIP) kinase-caspase-8-dependent macrophage apoptotic death pathway after infection with Y. pestis, influenced by Toll-like receptor 4-TIR-domain-containing adapter-inducing interferon-β (TLR4-TRIF). Interestingly, macrophages lacking either RIP1, or caspase-8 and RIP3, also had reduced infection-induced production of IL-1β, IL-18, TNF, and IL-6; impaired activation of the transcription factor NF-κB; and greatly compromised caspase-1 processing. Cleavage of the proform of caspase-1 is associated with triggering inflammasome activity, which leads to the maturation of IL-1β and IL-18, cytokines important to host responses against Y. pestis and many other infectious agents. Our results identify a RIP1-caspase-8/RIP3-dependent caspase-1 activation pathway after Y. pestis challenge. Mice defective in caspase-8 and RIP3 were also highly susceptible to infection and displayed reduced proinflammatory cytokines and myeloid cell death. We propose that caspase-8 and the RIP kinases are key regulators of macrophage cell death, NF-κB and inflammasome activation, and host resistance after Y. pestis infection.

  View details for DOI 10.1073/pnas.1403477111

  View details for Web of Science ID 000336168100053

  View details for PubMedID 24799678

  View details for PubMedCentralID PMC4034196

• Highly Acidic C-Terminal Region of Cytomegalovirus pUL96 Determines Its Functions during Virus Maturation Independently of a Direct pp150 Interaction JOURNAL OF VIROLOGY Brechtel, T. M., Mocarski, E. S., Tandon, R. 2014; 88 (8): 4493-4503

  Abstract

  Tegument proteins pp150 and pUL96 function at a late step in cytomegalovirus (CMV) maturation. Here, we show that pp150 interacts directly with pUL96; however, the N-terminal region of pp150 and the C-terminal region of pUL96, which are critical for these proteins to function, are not required for this interaction. Moreover, the largely dispensable C-terminal region of pp150 is critical for pp150-pUL96 interaction. To further study the role of pUL96, several point and clustered mutations were engineered into the CMV Towne bacterial artificial chromosome (Towne-BAC) genome, replacing the conserved negatively charged C-terminal residues of pUL96. Although individual point mutations (E122A, D124A, and D125A) reduced virus growth slightly, the clustered mutations of 122EVDDAV127 significantly reduced virus growth, produced small syncytial plaque phenotypes, and impacted a late stage of virus maturation. When the UL96 C-terminal alanine conversion mutant (B6-BAC) virus was serially passaged in cell culture, it gained a plaque size comparable to that of Towne-BAC, displayed an altered restriction fragment length pattern, and replicated with increased growth kinetics. Whole-genome sequencing of this passaged virus (UL96P10) and the similarly passaged Towne-BAC virus revealed major differences only in the RNA4.9 and UL96 regions. When one of the mutations in the UL96 coding region was engineered into the B6-BAC virus, it significantly increased the plaque size and rescued the virus growth rate. Thus, accumulation of compensatory mutations only in UL96 in this revertant and the specific involvement of functionally dispensable regions of pp150 in the pUL96-pp150 interaction point toward a role for pUL96 in virus maturation that does not depend upon pp150.Human cytomegalovirus causes significant medical problems in newborns, as well as in people with low immunity. In this study, we investigated the functions of two essential virus proteins, pp150 and pUL96, and determined the impact of their mutual interaction on virus replication. These studies provide valuable information that is critical for the development of targeted antiviral therapies.

  View details for DOI 10.1128/JVI.03784-13

  View details for Web of Science ID 000333676400048

  View details for PubMedID 24501413

  View details for PubMedCentralID PMC3993780

• Cytomegalovirus Hijacks CX3CR1(hi) Patrolling Monocytes as Immune-Privileged Vehicles for Dissemination in Mice. Cell host & microbe Daley-Bauer, L. P., Roback, L. J., Wynn, G. M., Mocarski, E. S. 2014; 15 (3): 351-362

  Abstract

  Peripheral blood myelomonocytic cells are important for cytomegalovirus dissemination to distal organs such as salivary glands where persistent replication and shedding dictates transmission patterns. We find that this process is markedly enhanced by the murine cytomegalovirus (MCMV)-encoded CC chemokine, MCK2, which promotes recruitment of CX3CR1(hi) patrolling monocytes to initial infection sites in the mouse. There, these cells become infected and traffic via the bloodstream to distal sites. In contrast, inflammatory monocytes, the other major myelomonocytic subset, remain virus negative. CX3CR1 deficiency prevents patrolling monocyte migration on the vascular endothelium and interrupts MCMV dissemination to the salivary glands independent of antiviral NK and T cell immune control. In this manner, CX3CR1(hi) patrolling monocytes serve as immune-privileged vehicles to transport MCMV via the bloodstream to distal organs. MCMV commandeers patrolling monocytes to mediate systemic infection and seed a persistent reservoir essential for horizontal transmission.

  View details for DOI 10.1016/j.chom.2014.02.002

  View details for PubMedID 24629341

• True grit: programmed necrosis in antiviral host defense, inflammation, and immunogenicity. Journal of immunology Mocarski, E. S., Kaiser, W. J., Livingston-Rosanoff, D., Upton, J. W., Daley-Bauer, L. P. 2014; 192 (5): 2019-2026

  Abstract

  Programmed necrosis mediated by receptor interacting protein kinase (RIP)3 (also called RIPK3) has emerged as an alternate death pathway triggered by TNF family death receptors, pathogen sensors, IFNRs, Ag-specific TCR activation, and genotoxic stress. Necrosis leads to cell leakage and acts as a "trap door," eliminating cells that cannot die by apoptosis because of the elaboration of pathogen-encoded caspase inhibitors. Necrotic signaling requires RIP3 binding to one of three partners-RIP1, DAI, or TRIF-via a common RIP homotypic interaction motif. Once activated, RIP3 kinase targets the pseudokinase mixed lineage kinase domain-like to drive cell lysis. Although necrotic and apoptotic death can enhance T cell cross-priming during infection, mice that lack these extrinsic programmed cell death pathways are able to produce Ag-specific T cells and control viral infection. The entwined relationship of apoptosis and necrosis evolved in response to pathogen-encoded suppressors to support host defense and contribute to inflammation.

  View details for DOI 10.4049/jimmunol.1302426

  View details for PubMedID 24563506

• Transcription of True Late (gamma 2) Cytomegalovirus Genes Requires UL92 Function That Is Conserved among Beta- and Gammaherpesviruses JOURNAL OF VIROLOGY Omoto, S., Mocarski, E. S. 2014; 88 (1): 120-130

  Abstract

  Human cytomegalovirus-encoded UL92 plays an essential role in viral replication that has not been resolved. We show here that this gene controls the accumulation of true late (γ2) viral transcripts, a property shared with several other recently evaluated genes (UL79, UL87, UL91, and UL95) conserved among beta- and gammaherpesviruses. When the UL92 mutant virus was evaluated, function was fully complemented by either the natural protein or the homologous Rh127 protein from rhesus cytomegalovirus. N-terminal epitope-tagged UL92 protein is functional, follows complex early-late expression kinetics, and localizes in the nucleus within viral replication compartments. UL92 severely impacts the late (72-h postinfection) expression of nine genes encoding virion proteins (UL32, UL55, UL73, UL75, UL80, UL86, UL99, and UL115), as well as UL91 and itself, but does not influence the levels of UL44, UL82, or UL83 accumulation. Although viral DNA is made at normal levels, viral capsid accumulation in the nucleus is severely compromised in UL92 mutant virus-infected cells, and mature virions are not observed in the cytoplasm. Taken together, UL92 is a key regulator of late viral gene expression, apparently functioning with four other beta- or gammaherpesvirus gene products in a pattern that appears reminiscent of gene regulation in T4 DNA bacteriophage.

  View details for DOI 10.1128/JVI.02983-13

  View details for Web of Science ID 000329194600011

  View details for PubMedID 24131715

• Natural antisense transcripts of UL123 packaged in human cytomegalovirus virions ARCHIVES OF VIROLOGY Yang, C., Miao, L., Pan, X., Wu, C., Rayner, S., Mocarski, E. S., Ye, H., Luo, M. 2014; 159 (1): 147-151

  Abstract

  In this study, we demonstrated that antisense transcripts of human cytomegalovirus (HCMV) UL123, UL21.5 and cellular GAPDH genes were present in highly purified virions. These virion RNAs were delivered into the host cells upon infection, and de novo synthesized ones appeared in the infected cell at the immediate early stage. Although the sequence of UL123 antisense transcripts in virions is uncertain, we found that these transcripts in Towne-infected human fibroblasts had novel transcriptional start sites (TSSs) with various 5'-terminal deletions of open reading frame (ORF) 59. These findings not only provide new insight into the composition of HCMV virions but also reveal a possible viral strategy for initiating latent infection and switching between latent and productive infections.

  View details for DOI 10.1007/s00705-013-1793-5

  View details for Web of Science ID 000330584800014

  View details for PubMedID 23884634

• Priorities for CMV vaccine development VACCINE Krause, P. R., Bialek, S. R., Boppana, S. B., Griffiths, P. D., Laughlin, C. A., Ljungman, P., Mocarski, E. S., Pass, R. F., Read, J. S., Schleiss, M. R., Plotkin, S. A. 2013; 32 (1): 4-10

  Abstract

  A multidisciplinary meeting addressed priorities related to development of vaccines against cytomegalovirus (CMV), the cause of congenital CMV (cCMV) disease and of serious disease in the immunocompromised. Participants discussed optimal uses of a CMV vaccine, aspects of clinical study design, and the value of additional research. A universal childhood CMV vaccine could potentially rapidly reduce cCMV disease, as infected children are sources of viral transmission to seronegative and seropositive mothers. A vaccine administered to adolescents or adult women could also reduce cCMV disease by making them immune prior to pregnancy. Clinical trials of CMV vaccines in women should evaluate protection against cCMV infection, an essential precursor of cCMV disease, which is a more practical and acceptable endpoint for assessing vaccine effects on maternal-fetal transmission. Clinical trials of vaccines to evaluate prevention of CMV disease in stem cell transplant recipients could use CMV viremia at a level triggering pre-emptive antiviral therapy as an endpoint, because widespread use of pre-emptive and prophylactic antivirals has rendered CMV-induced disease too rare to be a practical endpoint for clinical trials. In solid organ transplant patients, CMV-associated disease is sufficiently common for use as a primary endpoint. Additional research to advance CMV vaccine development should include identifying factors that predict fetal loss due to CMV, determining age-specific incidence and transmission rates, defining the mechanism and relative contributions of maternal reactivation and re-infection to cCMV disease, developing assays that can distinguish between reactivation and re-infection in seropositive vaccinees, further defining predictors of sequelae from cCMV infection, and identifying clinically relevant immune response parameters to CMV (including developing validated assays that could assess CMV antibody avidity) that could lead to the establishment of immune correlates of protection.

  View details for DOI 10.1016/j.vaccine.2013.09.042

  View details for Web of Science ID 000329684700003

  View details for PubMedID 24129123

• Apaf1 apoptotic function critically limits Sonic hedgehog signaling during craniofacial development CELL DEATH AND DIFFERENTIATION Long, A. B., Kaiser, W. J., Mocarski, E. S., Caspary, T. 2013; 20 (11): 1510-1520

  Abstract

  Apaf1 is an evolutionarily conserved component of the apoptosome. In mammals, the apoptosome assembles when cytochrome c is released from mitochondria, binding Apaf1 in an ATP-dependent manner and activating caspase 9 to execute apoptosis. Here we identify and characterize a novel mouse mutant, yautja, and find it results from a leucine-to-proline substitution in the winged-helix domain of Apaf1. We show that this allele of Apaf1 is unique, as the yautja mutant Apaf1 protein is stable, yet does not possess apoptotic function in cell culture or in vivo assays. Mutant embryos die perinatally with defects in craniofacial and nervous system development, as well as reduced levels of apoptosis. We further investigated the defects in craniofacial development in the yautja mutation and found altered Sonic hedgehog (Shh) signaling between the prechordal plate and the frontonasal ectoderm, leading to increased mesenchymal proliferation in the face and delayed or absent ossification of the skull base. Taken together, our data highlight the time-sensitive link between Shh signaling and the regulation of apoptosis function in craniofacial development to sculpt the face. We propose that decreased apoptosis in the developing nervous system allows Shh-producing cells to persist and direct a lateral outgrowth of the upper jaw, resulting in the craniofacial defects we see. Finally, the novel yautja Apaf1 allele offers the first in vivo understanding of a stable Apaf1 protein that lacks a function, which should make a useful tool with which to explore the regulation of programmed cell death in mammals.

  View details for DOI 10.1038/cdd.2013.97

  View details for Web of Science ID 000325548900011

  View details for PubMedID 23892366

• Proapoptotic Chemotherapeutic Drugs Induce Noncanonical Processing and Release of IL-1 beta via Caspase-8 in Dendritic Cells JOURNAL OF IMMUNOLOGY Antonopoulos, C., El Sanadi, C., Kaiser, W. J., Mocarski, E. S., Dubyak, G. R. 2013; 191 (9): 4789-4803

  Abstract

  The identification of noncanonical (caspase-1-independent) pathways for IL-1β production has unveiled an intricate interplay between inflammatory and death-inducing signaling platforms. We found a heretofore unappreciated role for caspase-8 as a major pathway for IL-1β processing and release in murine bone marrow-derived dendritic cells (BMDC) costimulated with TLR4 agonists and proapoptotic chemotherapeutic agents such as doxorubicin (Dox) or staurosporine (STS). The ability of Dox to stimulate release of mature (17-kDa) IL-1β was nearly equivalent in wild-type (WT) BMDC, Casp1(-/-)Casp11(-/-) BMDC, WT BMDC treated with the caspase-1 inhibitor YVAD, and BMDC lacking the inflammasome regulators ASC, NLRP3, or NLRC4. Notably, Dox-induced production of mature IL-1β was temporally correlated with caspase-8 activation in WT cells and greatly suppressed in Casp8(-/-)Rip3(-/-) or Trif(-/-) BMDC, as well as in WT BMDC treated with the caspase-8 inhibitor, IETD. Similarly, STS stimulated robust IL-1β processing and release in Casp1(-/-)Casp11(-/-) BMDC that was IETD sensitive. These data suggest that TLR4 induces assembly of caspase-8-based signaling complexes that become licensed as IL-1β-converting enzymes in response to Dox and STS. The responses were temporally correlated with downregulation of cellular inhibitor of apoptosis protein 1, suggesting suppressive roles for this and likely other inhibitor of apoptosis proteins on the stability and/or proteolytic activity of the caspase-8 platforms. Thus, proapoptotic chemotherapeutic agents stimulate the caspase-8-mediated processing and release of IL-1β, implicating direct effects of such drugs on a noncanonical inflammatory cascade that may modulate immune responses in tumor microenvironments.

  View details for DOI 10.4049/jimmunol.1300645

  View details for Web of Science ID 000325929000036

  View details for PubMedID 24078693

  View details for PubMedCentralID PMC3870469

• Toll-like Receptor 3-mediated Necrosis via TRIF, RIP3, and MLKL JOURNAL OF BIOLOGICAL CHEMISTRY Kaiser, W. J., Sridharan, H., Huang, C., Mandal, P., Upton, J. W., Gough, P. J., Sehon, C. A., Marquis, R. W., Bertin, J., Mocarski, E. S. 2013; 288 (43): 31268-31279

  Abstract

  Toll-like receptor (TLR) signaling is triggered by pathogen-associated molecular patterns that mediate well established cytokine-driven pathways, activating NF-κB together with IRF3/IRF7. In addition, TLR3 drives caspase 8-regulated programmed cell death pathways reminiscent of TNF family death receptor signaling. We find that inhibition or elimination of caspase 8 during stimulation of TLR2, TLR3, TLR4, TLR5, or TLR9 results in receptor interacting protein (RIP) 3 kinase-dependent programmed necrosis that occurs through either TIR domain-containing adapter-inducing interferon-β (TRIF) or MyD88 signal transduction. TLR3 or TLR4 directly activates programmed necrosis through a RIP homotypic interaction motif-dependent association of TRIF with RIP3 kinase (also called RIPK3). In fibroblasts, this pathway proceeds independent of RIP1 or its kinase activity, but it remains dependent on mixed lineage kinase domain-like protein (MLKL) downstream of RIP3 kinase. Here, we describe two small molecule RIP3 kinase inhibitors and employ them to demonstrate the common requirement for RIP3 kinase in programmed necrosis induced by RIP1-RIP3, DAI-RIP3, and TRIF-RIP3 complexes. Cell fate decisions following TLR signaling parallel death receptor signaling and rely on caspase 8 to suppress RIP3-dependent programmed necrosis whether initiated directly by a TRIF-RIP3-MLKL pathway or indirectly via TNF activation and the RIP1-RIP3-MLKL necroptosis pathway.

  View details for DOI 10.1074/jbc.M113.462341

  View details for Web of Science ID 000330778900043

  View details for PubMedID 24019532

• Cytomegalovirus UL91 Is Essential for Transcription of Viral True Late (gamma 2) Genes JOURNAL OF VIROLOGY Omoto, S., Mocarski, E. S. 2013; 87 (15): 8651-8664

  Abstract

  Human cytomegalovirus-encoded UL91 is a betagamma gene that is essential for viral replication. Here we show that the 111-amino-acid (aa) UL91 protein controls accumulation of true-late (γ2) viral transcripts. The primate betaherpesvirus conserved N-terminal region from aa 1 to 71 is sufficient to fully reconstitute function. Evaluation of viral DNA, RNA, and antigen revealed that UL91 protein is expressed with leaky-late (γ1) kinetics, localizes in the nucleus without influencing viral DNA synthesis, and must be present from 48 h postinfection to support full expression of late viral transcripts and proteins. In the absence of UL91, viral capsid assembly in the nucleus of infected cells is significantly reduced, and mature, cytoplasmic virions fail to form. Taken together, the evidence shows that UL91 regulates late viral gene expression by a mechanism that is apparently conserved in betaherpesviruses and gammaherpesviruses.

  View details for DOI 10.1128/JVI.01052-13

  View details for Web of Science ID 000321590200034

  View details for PubMedID 23720731

• Viral modulation of programmed necrosis CURRENT OPINION IN VIROLOGY Kaiser, W. J., Upton, J. W., Mocarski, E. S. 2013; 3 (3): 296-306

  Abstract

  Apoptosis and programmed necrosis balance each other as alternate first line host defense pathways against which viruses have evolved countermeasures. Intrinsic apoptosis, the critical programmed cell death pathway that removes excess cells during embryonic development and tissue homeostasis, follows a caspase cascade triggered at mitochondria and modulated by virus-encoded anti-apoptotic B cell leukemia (BCL)2-like suppressors. Extrinsic apoptosis controlled by caspase 8 arose during evolution to trigger executioner caspases directly, circumventing viral suppressors of intrinsic (mitochondrial) apoptosis and providing the selective pressure for viruses to acquire caspase 8 suppressors. Programmed necrosis likely evolved most recently as a 'trap door' adaptation to extrinsic apoptosis. Receptor interacting protein (RIP)3 kinase (also called RIPK3) becomes active when either caspase 8 activity or polyubiquitylation of RIP1 is compromised. This evolutionary dialog implicates caspase 8 as a 'supersensor' alternatively activating and suppressing cell death pathways.

  View details for DOI 10.1016/j.coviro.2013.05.019

  View details for Web of Science ID 000322416300011

  View details for PubMedID 23773332

• Desirability and feasibility of a vaccine against cytomegalovirus VACCINE Griffiths, P., Plotkin, S., Mocarski, E., Pass, R., Schleiss, M., Krause, P., Bialek, S. 2013; 31: B197-B203

  Abstract

  Publication of a report from the Institute of Medicine in 2000 showing that a vaccine against cytomegalovirus (CMV) would likely be cost saving was very influential and encouraged the clinical evaluation of candidate vaccines. The major objective of a CMV vaccination program would be to reduce disease caused by congenital CMV infection, which is the leading viral cause of sensorineural hearing loss and neurodevelopmental delay. CMV has challenges as a vaccine target because it is a herpesvirus, it persists lifelong despite host immunity, infected individuals can be reinfected with new strains, overt disease occurs in those with immature or impaired immune systems and persons with this infection do not usually report symptoms. Nevertheless, natural immunity against CMV provides some protection against infection and disease, natural history studies have defined the serological and molecular biological techniques needed for endpoints in future clinical trials of vaccines and CMV is not highly communicable, suggesting that it may not be necessary to achieve very high levels of population immunity through vaccination in order to affect transmission. Three phase 2 CMV vaccine studies have been completed in the last 3 years and all report encouraging outcomes. A key international meeting was organized by the Food and Drug Administration in January 2012 at which interested parties from regulatory bodies, industry and academia discussed and prioritised designs for phase 2 and phase 3 clinical trials. Vaccines able to prevent primary infection with CMV and to boost the immune response of those already infected are desirable. The major target populations for a CMV vaccine include women of childbearing age and adolescents. Toddlers represent another potential population, since an effect of vaccine in this age group could potentially decrease transmission to adults. In addition, prospective recipients of transplants and patients with AIDS would be expected to benefit.

  View details for DOI 10.1016/j.vaccine.2012.10.074

  View details for Web of Science ID 000318462000023

  View details for PubMedID 23598482

• Gene products of the embedded m41/m41.1 locus of murine cytomegalovirus differentially influence replication and pathogenesis VIROLOGY Crosby, L. N., McCormick, A. L., Mocarski, E. S. 2013; 436 (2): 274-283

  Abstract

  Cytomegaloviruses utilize overlapping and embedded reading frames as a way to efficiently package and express all genes necessary to carry out a complex lifecycle. Murine cytomegalovirus encodes a mitochondrial-localized inhibitor of Bak oligomerization (vIBO) from m41.1, a reading frame that is embedded within the m41 gene. The m41.1-encoded mitochondrial protein and m41-encoded Golgi-localized protein have both been implicated in cell death suppression; however, their contribution to viral infection within the host has not been investigated. Here, we report that mitochondrial-localized m41.1 (vIBO) is required for optimal viral replication in macrophages and has a modest impact on dissemination in infected mice. In contrast, Golgi-localized m41 protein is dispensable during acute infection and dissemination as well as for latency. All together, these data indicate that the primary evolutionary focus of this locus is to maintain mitochondrial function through inhibition of Bak-mediated death pathways in support of viral pathogenesis.

  View details for DOI 10.1016/j.virol.2012.12.002

  View details for Web of Science ID 000314446600004

  View details for PubMedID 23295021

• Multiplicity-dependent activation of a serine protease-dependent cytomegalovirus-associated programmed cell death pathway VIROLOGY McCormick, A. L., Roback, L., Wynn, G., Mocarski, E. S. 2013; 435 (2): 250-257

  Abstract

  At a low MOI (≤0.01), cytomegalovirus-associated programmed cell death terminates productive infection via a pathway triggered by the mitochondrial serine protease HtrA2/Omi. This infected cell death is associated with late phase replication events naturally suppressed by the viral mitochondrial inhibitor of apoptosis (vMIA). Here, higher MOI (ranging from 0.1-3.0) triggers cell death earlier during infection independent of viral DNA synthesis. Thus, MOI-dependent activating signals early, at high MOI, or late, at low MOI, during replication promote serine protease-dependent death that is suppressed by vMIA. Treatment with an antioxidant targeting reactive oxygen species (ROS) or the serine protease inhibitor N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) delays cell death, and the combination has an additive impact. These studies identify serine proteases and ROS as important factors triggering programmed cell death induced by vMIA-deficient virus, and show that this death pathway occurs earlier and reduces viral yields as the MOI is increased.

  View details for DOI 10.1016/j.viro1.2012.08.042

  View details for Web of Science ID 000313459200007

  View details for PubMedID 23159167

• Cutting Edge: FAS (CD95) Mediates Noncanonical IL-1 beta and IL-18 Maturation via Caspase-8 in an RIP3-Independent Manner JOURNAL OF IMMUNOLOGY Bossaller, L., Chiang, P., Schmidt-Lauber, C., Ganesan, S., Kaiser, W. J., Rathinam, V. A., Mocarski, E. S., Subramanian, D., Green, D. R., Silverman, N., Fitzgerald, K. A., Marshak-Rothstein, A., Latz, E. 2012; 189 (12): 5508-5512

  Abstract

  Fas, a TNF family receptor, is activated by the membrane protein Fas ligand expressed on various immune cells. Fas signaling triggers apoptosis and induces inflammatory cytokine production. Among the Fas-induced cytokines, the IL-1β family cytokines require proteolysis to gain biological activity. Inflammasomes, which respond to pathogens and danger signals, cleave IL-1β cytokines via caspase-1. However, the mechanisms by which Fas regulates IL-1β activation remain unresolved. In this article, we demonstrate that macrophages exposed to TLR ligands upregulate Fas, which renders them responsive to receptor engagement by Fas ligand. Fas signaling activates caspase-8 in macrophages and dendritic cells, leading to the maturation of IL-1β and IL-18 independently of inflammasomes or RIP3. Hence, Fas controls a novel noncanonical IL-1β activation pathway in myeloid cells, which could play an essential role in inflammatory processes, tumor surveillance, and control of infectious diseases.

  View details for DOI 10.4049/jimmunol.1202121

  View details for Web of Science ID 000311995800005

  View details for PubMedID 23144495

• Antiviral T Cell Response Triggers Cytomegalovirus Hepatitis in Mice JOURNAL OF VIROLOGY Livingston-Rosanoff, D., Daley-Bauer, L. P., Garcia, A., McCormick, A. L., Huang, J., Mocarski, E. S. 2012; 86 (23): 12879-12890

  Abstract

  One common sign of human cytomegalovirus infection is altered liver function. Murine cytomegalovirus strain v70 induces a rapid and severe hepatitis in immunocompetent mice that requires the presence of T cells in order to develop. v70 exhibits approximately 10-fold-greater virulence than the commonly used strain K181, resulting in a more severe, sustained, and lethal hepatitis but not dramatically higher viral replication levels. Hepatitis and death are markedly delayed in immunodeficient SCID compared to immunocompetent BALB/c mice. Transfer of BALB/c splenocytes to SCID mice conferred rapid disease following infection, and depletion of either CD4 or CD8 T cells in BALB/c mice reduced virus-induced hepatitis. The frequency of CD8 T cells producing gamma interferon and tumor necrosis factor in response to viral antigen was higher in settings where more severe disease occurred. Thus, virus-specific effector CD8 T cells appear to contribute to lethal virus-induced hepatitis, contrasting their protective role during sublethal infection. This study reveals how protection and disease during cytomegalovirus infection depend on viral strain and dose, as well as the quality of the T cell response.

  View details for DOI 10.1128/JVI.01752-12

  View details for Web of Science ID 000310585300038

  View details for PubMedID 22993151

  View details for PubMedCentralID PMC3497643

• Activation of Innate Immunity Is Required for Efficient Nuclear Reprogramming CELL Lee, J., Sayed, N., Hunter, A., Au, K. F., Wong, W. H., Mocarski, E. S., Pera, R. R., Yakubov, E., Cooke, J. P. 2012; 151 (3): 547-558

  Abstract

  Retroviral overexpression of reprogramming factors (Oct4, Sox2, Klf4, c-Myc) generates induced pluripotent stem cells (iPSCs). However, the integration of foreign DNA could induce genomic dysregulation. Cell-permeant proteins (CPPs) could overcome this limitation. To date, this approach has proved exceedingly inefficient. We discovered a striking difference in the pattern of gene expression induced by viral versus CPP-based delivery of the reprogramming factors, suggesting that a signaling pathway required for efficient nuclear reprogramming was activated by the retroviral, but not CPP approach. In gain- and loss-of-function studies, we find that the toll-like receptor 3 (TLR3) pathway enables efficient induction of pluripotency by viral or mmRNA approaches. Stimulation of TLR3 causes rapid and global changes in the expression of epigenetic modifiers to enhance chromatin remodeling and nuclear reprogramming. Activation of inflammatory pathways are required for efficient nuclear reprogramming in the induction of pluripotency.

  View details for DOI 10.1016/j.cell.2012.09.034

  View details for PubMedID 23101625

• Mechanisms modulating immune clearance during human cytomegalovirus latency PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Slobedman, B., Mocarski, E. S. 2012; 109 (36): 14291-14292

  View details for DOI 10.1073/pnas.1212245109

  View details for Web of Science ID 000308912600010

  View details for PubMedID 22949568

• Toll-Like Receptor 3 Activation Promotes Efficient Nuclear Reprogramming and Endothelial Differentiation Basic Cardiovascular Sciences Scientific Session Sayed, N., Lee, J., Hunter, A., Au, K. F., Wong, W., Mocarski, E., Pera, R. R., Cooke, J. P. LIPPINCOTT WILLIAMS & WILKINS. 2012

  View details for Web of Science ID 000312506400344

• Viral and host control of cytomegalovirus maturation TRENDS IN MICROBIOLOGY Tandon, R., Mocarski, E. S. 2012; 20 (8): 392-401

  Abstract

  Maturation in herpesviruses initiates in the nucleus of the infected cell, with encapsidation of viral DNA to form nucleocapsids, and concludes with envelopment in the cytoplasm to form infectious virions that egress the cell. The entire process of virus maturation is orchestrated by protein-protein interactions and enzymatic activities of viral and host origin. Viral tegument proteins play important roles in maintaining the structural stability of capsids and directing the acquisition of virus envelope. Envelopment occurs at modified host membranes and exploits host vesicular trafficking. In this review, we summarize current knowledge of and concepts in human cytomegalovirus (HCMV) maturation and their parallels in other herpesviruses, with an emphasis on viral and host factors that regulate this process.

  View details for DOI 10.1016/j.tim.2012.04.008

  View details for Web of Science ID 000307802700007

  View details for PubMedID 22633075

• Cytomegalovirus Impairs Antiviral CD8(+) T Cell Immunity by Recruiting Inflammatory Monocytes IMMUNITY Daley-Bauer, L. P., Wynn, G. M., Mocarski, E. S. 2012; 37 (1): 122-133

  Abstract

  Inflammatory monocytes are key early responders to infection that contribute to pathogen-host interactions in diverse ways. Here, we report that the murine cytomegalovirus-encoded CC chemokine, MCK2, enhanced CCR2-dependent recruitment of these cells to modulate antiviral immunity, impairing virus-specific CD8(+) T cell expansion and differentiation into effector cytotoxic T lymphocytes, thus reducing the capacity to eliminate viral antigen-bearing cells and slowing viral clearance. Adoptive transfer of inflammatory monocytes into Ccr2(-/-)Ccl2(-/-) mice impaired virus antigen-specific clearance. Cytomegalovirus therefore enhances a natural CCR2-dependent immune regulatory network to modulate adaptive immunity via nitric oxide production, reminiscent of the monocytic subtype of myeloid-derived suppressor cells primarily implicated in cancer immunomodulation.

  View details for DOI 10.1016/j.immuni.2012.04.014

  View details for Web of Science ID 000307133200015

  View details for PubMedID 22840843

• DAI/ZBP1/DLM-1 Complexes with RIP3 to Mediate Virus-Induced Programmed Necrosis that Is Targeted by Murine Cytomegalovirus vIRA CELL HOST & MICROBE Upton, J. W., Kaiser, W. J., Mocarski, E. S. 2012; 11 (3): 290-297

  Abstract

  Programmed necrosis, like apoptosis, eliminates pathogen-infected cells as a component of host defense. Receptor-interacting protein kinase (RIP) 3 (also called RIPK3) mediates RIP homotypic interaction motif (RHIM)-dependent programmed necrosis induced by murine cytomegalovirus (MCMV) infection or death receptor activation and suppressed by the MCMV-encoded viral inhibitor of RIP activation (vIRA). We find that interferon-independent expression of DNA-dependent activator of interferon regulatory factors (DAI, also known as ZBP1 or DLM-1) sensitizes cells to virus-induced necrosis and that DAI knockdown or knockout cells are resistant to this death pathway. Importantly, as with RIP3(-/-) mice, vIRA mutant MCMV pathogenesis is restored in DAI(-/-) mice, consistent with a DAI-RIP3 complex being the natural target of vIRA. Thus, DAI interacts with RIP3 to mediate virus-induced necrosis analogous to the RIP1-RIP3 complex controlling death receptor-induced necroptosis. These studies unveil a role for DAI as the RIP3 partner mediating virus-induced necrosis.

  View details for DOI 10.1016/j.chom.2012.01.016

  View details for Web of Science ID 000302050700009

  View details for PubMedID 22423968

• Viral infection and the evolution of caspase 8-regulated apoptotic and necrotic death pathways. Nature reviews. Immunology Mocarski, E. S., Upton, J. W., Kaiser, W. J. 2012; 12 (2): 79-88

  Abstract

  Pathogens specifically target both the caspase 8-dependent apoptotic cell death pathway and the necrotic cell death pathway that is dependent on receptor-interacting protein 1 (RIP1; also known as RIPK1) and RIP3 (also known as RIPK3). The fundamental co-regulation of these two cell death pathways emerged when the midgestational death of mice deficient in FAS-associated death domain protein (FADD) or caspase 8 was reversed by elimination of RIP1 or RIP3, indicating a far more entwined relationship than previously appreciated. Thus, mammals require caspase 8 activity during embryogenesis to suppress the kinases RIP1 and RIP3 as part of the dialogue between two distinct cell death processes that together fulfil reinforcing roles in the host defence against intracellular pathogens such as herpesviruses.

  View details for DOI 10.1038/nri3131

  View details for PubMedID 22193709

• Viral infection and the evolution of caspase 8-regulated apoptotic and necrotic death pathways NATURE REVIEWS IMMUNOLOGY Mocarski, E. S., Upton, J. W., Kaiser, W. J. 2012; 12 (2): 79-88

  View details for DOI 10.1038/nri3131

  View details for Web of Science ID 000300296300008

• Cytomegalovirus pUL96 Is Critical for the Stability of pp150-Associated Nucleocapsids JOURNAL OF VIROLOGY Tandon, R., Mocarski, E. S. 2011; 85 (14): 7129-7141

  Abstract

  Maturation of human cytomegalovirus (HCMV) initiates with nucleocapsids that egress from the nucleus and associate with a juxtanuclear cytoplasmic assembly compartment, where virion envelopment and release are orchestrated. Betaherpesvirus conserved proteins pp150 (encoded by UL32) and pUL96 are critical for HCMV growth in cell culture. pp150 is a capsid-proximal tegument protein that preserves the integrity of nucleocapsids during maturation. pUL96, although expressed as an early protein, acts late during virus maturation, similar to pp150, based on the comparable antigen distribution in UL96, UL32, or UL96/UL32 dual mutant virus-infected cells. pp150 associates with nuclear capsids prior to DNA encapsidation, whereas both pp150 and pUL96 associate with extracellular virus, suggesting that pUL96 is added after pp150. In the absence of pUL96, capsid egress from the nucleus continues; however, unlike wild-type virus infection, pp150 accumulates in the nuclear, as well as in the cytoplasmic, compartment. Ultrastructural evaluation of a UL96 conditional mutant revealed intact nuclear stages but aberrant nucleocapsids accumulating in the cytoplasm comparable to the known phenotype of UL32 mutant virus. In summary, pUL96 preserves the integrity of pp150-associated nucleocapsids during translocation from the nucleus to the cytoplasm.

  View details for DOI 10.1128/JVI.02549-10

  View details for Web of Science ID 000291932400028

  View details for PubMedID 21593167

• Inhibition of 2 ',5 '-Oligoadenylate Synthetase Expression and Function by the Human Cytomegalovirus ORF94 Gene Product JOURNAL OF VIROLOGY Tan, J. C., Avdic, S., Cao, J. Z., Mocarski, E. S., White, K. L., Abendroth, A., Slobedman, B. 2011; 85 (11): 5696-5700

  Abstract

  The human cytomegalovirus (HCMV) ORF94 gene product has been reported to be expressed during both productive and latent phases of infection, although its function is unknown. We report that expression of pORF94 leads to decreased 2',5'-oligoadenylate synthetase (OAS) expression in transfected cells with and without interferon stimulation. Furthermore, the functional activity of OAS was inhibited by pORF94. Finally, we present evidence of OAS modulation by pORF94 during productive HCMV infection of human fibroblasts. This study provides the first identification of a function for pORF94 and identifies an additional means by which HCMV may limit a critical host cell antiviral response.

  View details for DOI 10.1128/JVI.02463-10

  View details for Web of Science ID 000290298700048

  View details for PubMedID 21450824

• Identification and Classification of Acute Cardiac Rejection by Intragraft Transcriptional Profiling CIRCULATION Holweg, C. T., Potena, L., Luikart, H., Yu, T., Berry, G. J., Cooke, J. P., Valantine, H. A., Mocarski, E. S. 2011; 123 (20): 2236-U154

  Abstract

  Treatment of acute rejection (AR) in heart transplantation relies on histopathological grading of endomyocardial biopsies according to International Society for Heart and Lung Transplantation guidelines. Intragraft gene expression profiling may be a way to complement histological evaluation.Transcriptional profiling was performed on 26 endomyocardial biopsies, and expression patterns were compared with the 1990 International Society for Heart and Lung Transplantation AR grades. Importantly, transcriptional profiles from settings with an equivalent AR grade appeared the same. In addition, grade 0 profiles could not be distinguished from 1A profiles, and grade 3A profiles could not be distinguished from 3B profiles. Comparing the AR groupings (0+1A, 1B, and 3A+3B), 0+1A showed more striking differences from 1B than from 3A+3B. When these findings were extrapolated to the 2005 revised guidelines, the combination of 1A and 1B into a single category (1R) appears to have brought together endomyocardial biopsies with different underlying processes that are not evident from histological evaluation. Grade 1B was associated with upregulated immune response genes, as 1 categorical distinction from grade 1A. Although grade 1B was distinct from the clinically relevant AR grades 3A and 3B, all of these grades shared a small number of overlapping pathways consistent with common physiological underpinnings.The gene expression similarities and differences identified here in different AR settings have the potential to revise the clinical perspective on acute graft rejection, pending the results of larger studies.

  View details for DOI 10.1161/CIRCULATIONAHA.109.913921

  View details for Web of Science ID 000290852200018

  View details for PubMedID 21555702

  View details for PubMedCentralID PMC3115694

• Cytomegalovirus UL103 Controls Virion and Dense Body Egress JOURNAL OF VIROLOGY Ahlqvist, J., Mocarski, E. 2011; 85 (10): 5125-5135

  Abstract

  Human cytomegalovirus UL103 encodes a tegument protein that is conserved across herpesvirus subgroups. Mutant viruses lacking this gene product exhibit dramatically reduced accumulation of cell-free virus progeny and poor cell-to-cell spread. Given that viral proteins and viral DNA accumulate with normal kinetics in cells infected with mutant virus, UL103 appears to function during the late phase of replication, playing a critical role in egress of capsidless dense bodies and virions. Few dense bodies were observed in the extracellular space in mutant virus-infected cells in the presence or absence of the DNA encapsidation inhibitor 2-bromo-5,6-dichloro-1-(β-d-ribofuranosyl)benzimidazole. Upon reversal of encapsidation inhibition, UL103 had a striking impact on accumulation of cell-free virus, but not on accumulation of cell-associated virus. Thus, UL103 plays a novel and important role during maturation, regulating virus particle and dense body egress from infected cells.

  View details for DOI 10.1128/JVI.01682-10

  View details for Web of Science ID 000289787300047

  View details for PubMedID 21345947

• RIP3 mediates the embryonic lethality of caspase-8-deficient mice NATURE Kaiser, W. J., Upton, J. W., Long, A. B., Livingston-Rosanoff, D., Daley-Bauer, L. P., Hakem, R., Caspary, T., Mocarski, E. S. 2011; 471 (7338): 368-?

  Abstract

  Apoptosis and necroptosis are complementary pathways controlled by common signalling adaptors, kinases and proteases; among these, caspase-8 (Casp8) is critical for death receptor-induced apoptosis. This caspase has also been implicated in non-apoptotic pathways that regulate Fas-associated via death domain (FADD)-dependent signalling and other less defined biological processes as diverse as innate immune signalling and myeloid or lymphoid differentiation patterns. Casp8 suppresses RIP3-RIP1 (also known as RIPK3-RIPK1) kinase complex-dependent necroptosis that follows death receptor activation as well as a RIP3-dependent, RIP1-independent necrotic pathway that has emerged as a host defence mechanism against murine cytomegalovirus. Disruption of Casp8 expression leads to embryonic lethality in mice between embryonic days 10.5 and 11.5 (ref. 7). Thus, Casp8 may naturally hold alternative RIP3-dependent death pathways in check in addition to promoting apoptosis. We find that RIP3 is responsible for the mid-gestational death of Casp8-deficient embryos. Remarkably, Casp8(-/-)Rip3(-/-) double mutant mice are viable and mature into fertile adults with a full immune complement of myeloid and lymphoid cell types. These mice seem immunocompetent but develop lymphadenopathy by four months of age marked by accumulation of abnormal T cells in the periphery, a phenotype reminiscent of mice with Fas-deficiency (lpr/lpr; also known as Fas). Thus, Casp8 contributes to homeostatic control in the adult immune system; however, RIP3 and Casp8 are together completely dispensable for mammalian development.

  View details for DOI 10.1038/nature09857

  View details for Web of Science ID 000288444000043

  View details for PubMedID 21368762

• Virus Inhibition of RIP3-Dependent Necrosis CELL HOST & MICROBE Upton, J. W., Kaiser, W. J., Mocarski, E. S. 2010; 7 (4): 302-313

  Abstract

  Viral infection activates cytokine expression and triggers cell death, the modulation of which is important for successful pathogenesis. Necroptosis is a form of programmed necrosis dependent on two related RIP homotypic interaction motif (RHIM)-containing signaling adaptors, receptor-interacting protein kinases (RIP) 1 and 3. We find that murine cytomegalovirus infection induces RIP3-dependent necrosis. Whereas RIP3 kinase activity and RHIM-dependent interactions control virus-associated necrosis, virus-induced death proceeds independently of RIP1 and is therefore distinct from TNFalpha-dependent necroptosis. Viral M45-encoded inhibitor of RIP activation (vIRA) targets RIP3 during infection and disrupts RIP3-RIP1 interactions characteristic of TNFalpha-induced necroptosis, thereby suppressing both death pathways. Importantly, attenuation of vIRA mutant virus in wild-type mice is normalized in RIP3-deficient mice. Thus, vIRA function validates necrosis as central to host defense against viral infections and highlights the benefit of multiple virus-encoded cell-death suppressors that inhibit not only apoptotic, but also necrotic mechanisms of virus clearance.

  View details for DOI 10.1016/j.chom.2010.03.006

  View details for Web of Science ID 000277111400007

  View details for PubMedID 20413098

• Human Cytomegalovirus Exploits ESCRT Machinery in the Process of Virion Maturation JOURNAL OF VIROLOGY Tandon, R., AuCoin, D. P., Mocarski, E. S. 2009; 83 (20): 10797-10807

  Abstract

  The endosomal sorting complex required for transport (ESCRT) machinery controls the incorporation of cargo into intraluminal vesicles of multivesicular bodies. This machinery is used during envelopment of many RNA viruses and some DNA viruses, including herpes simplex virus type 1. Other viruses mature independent of ESCRT components, instead relying on the intrinsic behavior of viral matrix and envelope proteins to drive envelopment. Human cytomegalovirus (HCMV) maturation has been reported to proceed independent of ESCRT components (A. Fraile-Ramos et al. Cell. Microbiol. 9:2955-2967, 2007). A virus complementation assay was used to evaluate the role of dominant-negative (DN) form of a key ESCRT ATPase, vacuolar protein sorting-4 (Vps4DN) in HCMV replication. Vps4DN specifically inhibited viral replication, whereas wild-type-Vps4 had no effect. In addition, a DN form of charged multivesicular body protein 1 (CHMP1DN) was found to inhibit HCMV. In contrast, DN tumor susceptibility gene-101 (Tsg101DN) did not impact viral replication despite the presence of a PTAP motif within pp150/ppUL32, an essential tegument protein involved in the last steps of viral maturation and release. Either Vps4DN or CHMP1DN blocked viral replication at a step after the accumulation of late viral proteins, suggesting that both are involved in maturation. Both Vps4A and CHMP1A localized in the vicinity of viral cytoplasmic assembly compartments, sites of viral maturation that develop in CMV-infected cells. Thus, ESCRT machinery is involved in the final steps of HCMV replication.

  View details for DOI 10.1128/JVI.01093-09

  View details for Web of Science ID 000270121600045

  View details for PubMedID 19640981

• The spleen plays a central role in primary humoral alloimmunization to transfused mHEL red blood cells TRANSFUSION Hendrickson, J. E., Saakadze, N., Cadwell, C. M., Upton, J. W., Mocarski, E. S., Hillyer, C. D., Zimring, J. C. 2009; 49 (8): 1678-1684

  Abstract

  Several differences exist between antigens on transfused red blood cells (RBCs) and other immunogens, including anatomical compartmentalization. Whereas antigens from microbial pathogens and solid organ transplants drain into local lymph nodes, circulating RBCs remain segregated in the peripheral circulation, where they are consumed by antigen-presenting cells (APCs) in the spleen and liver. Accordingly, it was hypothesized that the splenic APCs play a central role in primary alloimmunization to transfused RBCs.Recipient mice were splenectomized and transfused with transgenic RBCs expressing the membrane-bound hen egg lysozyme (mHEL) model RBC antigen. In some experiments, mHEL-specific CD4+ T cells were adoptively transferred into recipient mice to allow investigation of helper T-cell responses. Unmanipulated or sham-splenectomized mice served as controls. Recombinant murine cytomegalovirus expressing mHEL (mHEL-MCMV) was used as a control non-RBC immunogen. Humoral responses were measured by mHEL-specific enzyme-linked immunosorbent assay and flow cytometric–based RBC cross-match.Control animals synthesized detectable anti-HEL immunoglobulin (Ig)G after a single mHEL RBC transfusion. mHEL-specific CD4+ T cells underwent robust expansion, and adoptive transfer of CD4+ T cells resulted in a 1000-fold increase in anti-HEL IgG. In contrast, minimal anti-HEL IgG was detectable in splenectomized mice, mHEL-specific CD4+ T cells did not proliferate, and adoptive transfer did not increase anti-HEL IgG. However, anti-HEL IgG response after exposure to mHEL-MCMV was equivalent in control and splenectomized mice.Together, these findings illustrate the distinct properties of transfused RBCs as immunologic stimuli, with the spleen playing a critical role in primary RBC alloimmunization at the level of CD4+ T-cell activation.

  View details for DOI 10.1111/j.1537-2995.2009.02200.x

  View details for Web of Science ID 000268590400022

  View details for PubMedID 19413728

• Human Herpesvirus Replication and Abnormal CD8+T Cell Activation and Low CD4+T Cell Counts in Antiretroviral-Suppressed HIV-Infected Patients PLOS ONE Jacobson, M. A., Ditmer, D. P., Sinclair, E., Martin, J. N., Deeks, S. G., Hunt, P., Mocarski, E. S., Shiboski, C. 2009; 4 (4)

  Abstract

  Most HIV-infected patients receiving virologically suppressive antiretroviral therapy continue to have abnormal, generalized T cell activation. We explored whether the degree of ongoing cytomegalovirus (CMV), Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus (KSHV) replication was associated with higher virus-specific T cell activation and the failure to achieve normal absolute CD4+ T cell counts in the face of long-term suppressive antiretroviral therapy.Longitudinally collected PBMC and saliva specimens obtained from HIV-infected patients on effective antiretroviral therapy for at least one year (plasma HIV RNA <75 copies/mL) were examined using a multiplex CMV, EBV and KSHV DNA PCR assay. Eleven cases were chosen who had CD8+ T cell CD38+HLA-DR+ expression >10% and plateau absolute CD4+ T cell counts <500 cells/microL. Five controls from the same study had CD8+ T cell CD38 expression <10% and plateau absolute CD4+ T cell counts >500 cells/microL.Among all subjects combined, 18% of PMBC samples were positive for CMV DNA, and 27%, 73% and 24% of saliva samples were positive for CMV, EBV and KSHV DNA, respectively. No significant differences or trends were observed between cases and controls in proportions of all CMV, EBV or KSHV DNA positive specimens, proportions of subjects in each group that intermittently or continuously shed CMV, EBV or KSHV DNA in saliva, or the median number of genome copies of CMV, EBV and KSHV DNA in saliva. Overall, number of genome copies in saliva were lower for KSHV than for CMV and lower for CMV than for EBV. Although replication of CMV, EBV and KSHV persists in many antiretroviral-suppressed, HIV-infected patients, we observed no evidence in this pilot case-control study that the magnitude of such human herpesvirus replication is associated with abnormally increased CD8+ T cell activation and sub-normal plateau absolute CD4+ T cell counts following virologically suppressive antiretroviral therapy.

  View details for DOI 10.1371/journal.pone.0005277

  View details for Web of Science ID 000265510800013

  View details for PubMedID 19381272

• Frequent Occult Infection with Cytomegalovirus in Cardiac Transplant Recipients despite Antiviral Prophylaxis (vol 45, pg 1804, 2007) JOURNAL OF CLINICAL MICROBIOLOGY Potena, L., Holweg, C. J., Vana, M. L., Bashyam, L., Rajamani, J., McCormick, A., Cooke, J. P., Valantine, H. A., Mocarski, E. S. 2008; 46 (12): 4121

  View details for DOI 10.1128/JCM.01837-08

  View details for Web of Science ID 000261247900054

• Receptor-Interacting Protein Homotypic Interaction Motif-Dependent Control of NF-kappa B Activation via the DNA-Dependent Activator of IFN Regulatory Factors JOURNAL OF IMMUNOLOGY Kaiser, W. J., Upton, J. W., Mocarski, E. S. 2008; 181 (9): 6427-6434

  Abstract

  DNA-dependent activator of IFN regulatory factors (IRF; DAI, also known as ZBP1 or DLM-1) is a cytosolic DNA sensor that initiates IRF3 and NF-kappaB pathways leading to activation of type I IFNs (IFNalpha, IFNbeta) and other cytokines. In this study, induction of NF-kappaB is shown to depend on the adaptor receptor-interacting protein kinase (RIP)1, acting via a RIP homotypic interaction motif (RHIM)-dependent interaction with DAI. DAI binds to and colocalizes with endogenous RIP1 at characteristic cytoplasmic granules. Suppression of RIP1 expression by RNAi abrogates NF-kappaB activation as well as IFNbeta induction by immunostimulatory DNA. DAI also interacts with RIP3 and this interaction potentiates DAI-mediated activation of NF-kappaB, implicating RIP3 in regulating this RHIM-dependent pathway. The role of DAI in activation of NF-kappaB in response to immunostimulatory DNA appears to be analogous to sensing of dsRNA by TLR3 in that both pathways involve RHIM-dependent signaling that is mediated via RIP1, reinforcing a central role for this adaptor in innate sensing of intracellular microbes.

  View details for Web of Science ID 000260659000068

  View details for PubMedID 18941233

• Repression of human cytomegalovirus major immediate early gene expression by the cellular transcription factor CCAAT displacement protein VIROLOGY Stern, J. L., Cao, J. Z., Xu, J., Mocarski, E. S., Slobedman, B. 2008; 378 (2): 214-225

  Abstract

  Initiation of human cytomegalovirus (HCMV) productive infection is dependent on the major immediate early (MIE) genes ie1 and ie2. Several putative binding sites for CCAAT displacement protein (CDP or CUX1) were identified within the MIE promoter/regulatory region. Binding assays demonstrated binding of CUX1 to MIE-region oligonucleotides containing the CUX1 core binding sequence ATCGAT and mutagenesis of this sequence abrogated CUX1 binding. Furthermore, CUX1 repressed expression of a luciferase reporter construct controlled by the MIE promoter, and mutation of CUX1 binding sites within the promoter diminished this repressive function of CUX1. In the context of virus infection of HEK293 cells transfected with the CUX1 expression vector, CUX1 showed evidence of association with the HCMV MIE regulatory region and inhibited the capacity of the virus to express ie1 and ie2 transcripts, suggesting that this cellular factor regulates MIE gene expression following virus entry. These data identify a role for CUX1 in repressing HCMV gene expression essential for initiation of the replicative cycle.

  View details for DOI 10.1016/j.virol.2008.05.024

  View details for Web of Science ID 000258631600003

  View details for PubMedID 18614194

• Cytomegalovirus m45 cell death suppression requires receptor-interacting protein (RIP) homotypic interaction motif (RHIM)-dependent interaction with RIP1 JOURNAL OF BIOLOGICAL CHEMISTRY Upton, J. W., Kaiser, W. J., Mocarski, E. S. 2008; 283 (25): 16966-16970

  Abstract

  Herpesviruses such as cytomegaloviruses encode functions that modulate the innate response in diverse ways to counteract host sensing and delay host clearance during infection. The murine cytomegalovirus M45 protein interacts with receptor-interacting protein (RIP) 1 and RIP3 via a RIP homotypic interaction motif. Cell death suppression by M45 requires RIP homotypic interaction motif-dependent interaction with RIP1. This interaction also underlies the cell tropism role of M45 in preventing premature death of endothelial cells during murine cytomegalovirus infection. Thus, M45 is a viral inhibitor of RIP activation that provides a direct cell type-dependent replication benefit to the virus while modulating other biological processes signaling via the RIP1 adaptor such as activation of Toll-like receptor (TLR)3 as well as other mediators of cell death.

  View details for DOI 10.1074/jbc.C800051200

  View details for Web of Science ID 000256720600002

  View details for PubMedID 18442983

• HtrA2/Omi terminates cytomegalovirus infection and is controlled by the viral mitochondrial inhibitor of apoptosis (vMIA) PLOS PATHOGENS McCormick, A. L., Roback, L., Mocarski, E. S. 2008; 4 (5)

  Abstract

  Viruses encode suppressors of cell death to block intrinsic and extrinsic host-initiated death pathways that reduce viral yield as well as control the termination of infection. Cytomegalovirus (CMV) infection terminates by a caspase-independent cell fragmentation process after an extended period of continuous virus production. The viral mitochondria-localized inhibitor of apoptosis (vMIA; a product of the UL37x1 gene) controls this fragmentation process. UL37x1 mutant virus-infected cells fragment three to four days earlier than cells infected with wt virus. Here, we demonstrate that infected cell death is dependent on serine proteases. We identify mitochondrial serine protease HtrA2/Omi as the initiator of this caspase-independent death pathway. Infected fibroblasts develop susceptibility to death as levels of mitochondria-resident HtrA2/Omi protease increase. Cell death is suppressed by the serine protease inhibitor TLCK as well as by the HtrA2-specific inhibitor UCF-101. Experimental overexpression of HtrA2/Omi, but not a catalytic site mutant of the enzyme, sensitizes infected cells to death that can be blocked by vMIA or protease inhibitors. Uninfected cells are completely resistant to HtrA2/Omi induced death. Thus, in addition to suppression of apoptosis and autophagy, vMIA naturally controls a novel serine protease-dependent CMV-infected cell-specific programmed cell death (cmvPCD) pathway that terminates the CMV replication cycle.

  View details for DOI 10.1371/journal.ppat.1000063

  View details for Web of Science ID 000256668900021

  View details for PubMedID 18769594

  View details for PubMedCentralID PMC2528007

• RNA analysis by biosynthetic tagging using 4-thiouracil and uracil phosphoribosyltransferase. Methods in molecular biology (Clifton, N.J.) Zeiner, G. M., Cleary, M. D., Fouts, A. E., Meiring, C. D., Mocarski, E. S., Boothroyd, J. C. 2008; 419: 135-146

  Abstract

  RNA analysis by biosynthetic tagging (RABT) enables sensitive and specific queries of (a) how gene expression is regulated on a genome-wide scale and (b) transcriptional profiling of a single cell or tissue type in vivo. RABT can be achieved by exploiting unique properties of Toxoplasma gondii uracil phosphoribosyltransferase (TgUPRT), a pyrimidine salvage enzyme that couples ribose-5-phosphate to the N1 nitrogen of uracil to yield uridine monophosphate (UMP). When 4-thiouracil is provided as a TgUPRT substrate, the resultant product is 4-thiouridine monophosphate which can, ultimately, be incorporated into RNA. Thio-substituted nucleotides are not a natural component of nucleic acids and are readily tagged, detected, and purified with commercially available reagents. Thus, one can do pulse/chase experiments to measure synthesis and decay rates and/or use cell-specific expression of the TgUPRT to tag only RNA synthesized in a given cell type. This chapter updates the original RABT protocol (1) and addresses methodological details associated with RABT that were beyond the scope or space allotment of the initial report.

  View details for DOI 10.1007/978-1-59745-033-1_9

  View details for PubMedID 18369980

• Pathogen subversion of cell-intrinsic innate immunity NATURE IMMUNOLOGY Roy, C. R., Mocarski, E. S. 2007; 8 (11): 1179-1187

  Abstract

  The mammalian immune system has evolved under continuous selective pressure from a wide range of microorganisms that colonize and replicate in animal hosts. A complex set of signaling networks initiate both innate and adaptive immunity in response to the diverse pathogens that mammalian hosts encounter. In response, viral and microbial pathogens have developed or acquired sophisticated mechanisms to avoid, counteract and subvert sensors, signaling networks and a range of effector functions that constitute the host immune response. This balance of host response and pathogen countermeasures contributes to chronic infection in highly adapted pathogens that have coevolved with their host. In this review we outline some of the themes that are beginning to emerge in the mechanisms by which pathogens subvert the early innate immune response.

  View details for DOI 10.1038/ni1528

  View details for Web of Science ID 000250338900008

  View details for PubMedID 17952043

• Cyclosporine inhibits mouse cytomegalovirus infection via a cyclophilin-dependent pathway specifically in neural stem/progenitor cells JOURNAL OF VIROLOGY Kawasaki, H., Mocarski, E. S., Kosugi, I., Tsutsui, Y. 2007; 81 (17): 9013-9023

  Abstract

  The potential of neural stem and progenitor cell (NSPC) transplantation in neurodegenerative disease raises a concern about immunosuppressive agents and opportunistic neurotropic pathogens that may interfere with engraftment. Cytomegalovirus (CMV) is an important opportunistic pathogen infecting the central nervous system, where it may remain latent for life, following transplacental transmission. Cyclosporine (Cs), an immunosuppressive drug used in organ transplantation, where its use is associated with CMV reactivation, suppressed murine CMV (MCMV) infection in cultured NSPCs but not in fibroblasts. This activity of Cs appears to be mediated via cyclophilin (CyP) rather than via calcineurin. First, the calcineurin-specific inhibitor FK506 failed to suppress replication. Second, the CyP-specific inhibitor NIM811 strongly suppressed replication in NSPC. NSPCs maintained in the presence of NIM811 retained viral genomes for several weeks without detectable viral gene expression or obvious deleterious effects. The withdrawal of NIM811 reactivated viral replication, suggesting that the inhibitory mechanism was reversible. Finally, inhibition of endogenous CyP A (CyPA) by small interfering RNA also inhibited replication in NSPCs. These results show that MCMV replication depends upon cellular CyPA pathways in NSPCs (in a specific cell type-dependent fashion), that CyPA plays an important role in viral infection in this cell type, and that inhibition of viral replication via CyP leads to persistence of the viral genome without cell damage. Further, the calcineurin-signaling pathway conferring immunosuppression in T cells does not influence viral replication in a detectable fashion.

  View details for DOI 10.1128/JVI.00261-07

  View details for Web of Science ID 000248923700015

  View details for PubMedID 17553872

• Frequent occult infection with cytomegalovirus in cardiac transplant recipients despite antiviral prophylaxis JOURNAL OF CLINICAL MICROBIOLOGY Potena, L., Holweg, C. T., Vana, M. L., Bashyam, L., Rajamani, J., McCormick, A. L., Cooke, J. P., Valantine, H. A., Mocarski, E. S. 2007; 45 (6): 1804-1810

  Abstract

  Despite antiviral prophylaxis, a high percentage (over 90%) of heart transplant patients experience active cytomegalovirus (CMV) infection, diagnosed by detection of viral DNA in peripheral blood polymorphonuclear leukocytes within the first few months posttransplantation. Viral DNA was detected in mononuclear cells prior to detection in granulocytes from CMV-seropositive recipients (R+) receiving a heart from a CMV-seropositive donor (D+). Based on assessment of systemic infection in leukocyte populations, both R+ subgroups (R+/D- and R+/D+) experienced a greater infection burden than the R-/D+ subgroup, which was aggressively treated because of a higher risk of acute CMV disease. Despite widespread systemic infection in all at-risk patient subgroups, CMV DNA was rarely (< 3% of patients) detected in transplanted heart biopsy specimens. The R+ patients more frequently exceeded the 75th percentile of the CMV DNA copy number distribution in leukocytes (110 copies/10(5) polymorphonuclear leukocytes) than the R-/D+ subgroup. Therefore, active systemic CMV infection involving leukocytes is common in heart transplant recipients receiving prophylaxis to reduce acute disease. Infection of the transplanted organ is rare, suggesting that chronic vascular disease attributed to CMV may be driven by the consequences of systemic infection.

  View details for DOI 10.1128/JCM.01362-06

  View details for Web of Science ID 000247286500022

  View details for PubMedID 17409205

  View details for PubMedCentralID PMC1933112

• Interplay between systemic inflammation and markers of insulin resistance in cardiovascular prognosis after heart transplantation JOURNAL OF HEART AND LUNG TRANSPLANTATION Biadi, O., Potena, L., Fearon, W. F., Luikart, H. I., Yeung, A., Ferrara, R., Hunt, S. A., Mocarski, E. S., Valantine, H. A. 2007; 26 (4): 324-330

  Abstract

  Metabolic and immuno-inflammatory risk factors contribute to cardiac allograft vasculopathy (CAV) pathogenesis. Although systemic inflammation, as detected by C-reactive protein (CRP), predicts CAV development, the relationship between CRP and markers of metabolic abnormalities remains unexplored.CRP and the entire metabolic panel were evaluated in 98 consecutive heart transplant recipients at the time of annual coronary angiography, 5.8 years after transplant (range, 1-12 years). A ratio of triglycerides (TG) to high-density lipoproteins (HDL) of 3.0 or more was considered a marker of insulin resistance. CAV prevalence was defined by angiography, and subsequent prognosis was evaluated as incidence of major cardiac adverse events.CRP was higher in the 34 patients with angiographic CAV than in those without CAV (1.10 +/- 0.20 vs 0.50 +/- 0.05 mg/dl, p < 0.001). Patients with insulin resistance had higher CRP concentrations (p = 0.023) and higher CAV prevalence (p = 0.005). High CRP and a TG/HDL of 3.0 or more were independently associated with an increased likelihood of CAV (odds ratio, > or = 3.9; p = 0.02) and predicted an increased risk of major cardiac adverse events. The combination of high CRP and a TG/HDL of 3.0 or more identified a subgroup of patients having a 4-fold increased risk for CAV and a 3-fold increased risk for major cardiac adverse events compared with patients with low CRP and normal values for metabolic indicators.Both CRP and insulin resistance, as estimated by TG/HDL, appear to be strong, synergic risk factors for CAV and for major cardiac adverse events. These findings support the hypothesis that in heart transplant recipients, systemic inflammation may be an important mediator of graft vascular injury associated with metabolic syndrome.

  View details for DOI 10.1016/j.healun.2007.01.020

  View details for PubMedID 17403472

• Changes in coronary arterial dimensions early after cardiac transplantation TRANSPLANTATION Fearon, W. F., Potena, L., Hirohata, A., Sakurai, R., Yamasaki, M., Luikart, H., Lee, J., Vana, M. L., Cooke, J. P., Mocarski, E. S., Yeung, A. C., Valantine, H. A. 2007; 83 (6): 700-705

  Abstract

  Significant changes in coronary artery structure, including intimal thickening and vessel remodeling, occur early after cardiac transplantation. The degree to which these changes compromise coronary lumen dimensions, and the clinical factors that affect these changes, remain controversial.Thirty-eight adult cardiac transplant recipients underwent coronary angiography and volumetric intravascular ultrasound (IVUS) evaluation of the left anterior descending artery within 8 weeks of transplantation and at 1 year. Clinical parameters including donor and recipient characteristics, rejection episodes, and serology were prospectively recorded. Two-dimensional IVUS measurements and vessel, lumen and plaque volume were calculated at both time points and compared. Multivariate regression analysis was performed to reveal clinical predictors of change in coronary dimensions.During the first year after transplantation, significant decreases in vessel size (negative remodeling) and lumen size were observed with significant increases in plaque burden based on IVUS analyses. Loss of lumen volume correlated significantly with the degree of negative remodeling (R=0.82, P<0.0001), but not with changes in plaque burden (R=0.08, P=0.64). Patients with the greatest increase in plaque volume had significantly less negative remodeling (R=0.53, P=0.0006). Transplant recipient cytomegalovirus (CMV) antibody seropositivity and lack of aggressive prophylaxis against CMV infection/reactivation were significant independent predictors of greater negative remodeling (P<0.01 and P=0.03, respectively) and greater lumen loss (P=0.02 and P=0.03, respectively).Negative remodeling is primarily responsible for coronary artery lumen loss during the first year after cardiac transplantation. CMV seropositivity and lack of aggressive CMV prophylaxis correlate with increased negative remodeling, resulting in greater lumen loss.

  View details for DOI 10.1097/01.tp.0000256335.84363.9b

  View details for PubMedID 17414701

• [Coronary allograft vasculopathy: pathophysiological interaction between the immune system, infections and metabolic syndrome]. Giornale italiano di cardiologia (2006) Potena, L., Ferrara, R., Mocarski, E. S., Lewis, D. B., Cooke, J. P., Grigioni, F., Coccolo, F., Magnani, G., Fallani, F., Magelli, C., Valantine, H. A., Branzi, A. 2007; 8 (2): 73-82

  Abstract

  Cardiac allograft vasculopathy is still the main cause of long-term graft loss after heart transplantation. Indeed, recent advances in immunosuppression management led to a significant improvement in short-term survival, while long-term death rate did not change significantly in the last 20 years. In this paper, we will review the latest advances in the understanding of this peculiar form of atherosclerosis, focusing on the mechanisms that can be potentially targeted by specific therapeutic interventions.

  View details for PubMedID 17402351

• Viral and cell cycle-regulated kinases in cytomegalovirus-induced pseudomitosis and replication PLOS PATHOGENS Hertel, L., Chou, S., Mocarski, E. S. 2007; 3 (1): 14-22

  Abstract

  A process of pseudomitosis occurs during human cytomegalovirus infection that appears similar to cellular mitosis but involves the formation of multiple spindle poles, abnormal condensation, and mislocalization of chromosomal DNA. The relationship of this process to viral replication and cell cycle regulation during infection has been poorly understood. Pseudomitosis consistently peaks at late times of infection in all viral strains examined but at overall highest frequencies (30% to 35% of cells) using one common laboratory strain variant (AD169varATCC). Cyclin-dependent kinase 1 (Cdk1) plays a crucial role in pseudomitosis, mirroring its role in conventional mitosis. Dominant negative Cdk1 inhibits and wild-type Cdk1 stimulates this process; however, viral yields remain the same regardless of pseudomitosis levels. Broad inhibition of cell cycle-regulated kinases (Cdk1/Cdk2/Cdk5/Cdk9) with indirubin-3'-monoxime substantially decreases viral yields and synergizes with the viral UL97 kinase inhibitor, maribavir. Thus, Cdk1 is necessary and sufficient to drive pseudomitosis, whereas a combination of viral and cell cycle-regulated kinases is important during viral replication.

  View details for DOI 10.1371/journal.ppat.0030006

  View details for Web of Science ID 000248492500002

  View details for PubMedID 17206862

• Viral modulation of the host response to infection HUMAN HERPESVIRUSES: BIOLOGY, THERAPY, AND IMMUNOPROPHYLAXIS McCormick, A., Mocarski, E. S., Arvin, A., CampadelliFiume, G., Mocarski, E., Moore, P. S., Roizman, B., Whitley, R., Yamanishi, K. 2007: 324–37

  View details for Web of Science ID 000296850300022

• Betaherpes viral genes and their functions HUMAN HERPESVIRUSES: BIOLOGY, THERAPY, AND IMMUNOPROPHYLAXIS Mocarski, E. S., Arvin, A., CampadelliFiume, G., Mocarski, E., Moore, P. S., Roizman, B., Whitley, R., Yamanishi, K. 2007: 204–30

  View details for Web of Science ID 000296850300016

• Comparative analysis of herpesvirus-common proteins HUMAN HERPESVIRUSES: BIOLOGY, THERAPY, AND IMMUNOPROPHYLAXIS Mocarski, E. S., Arvin, A., CampadelliFiume, G., Mocarski, E., Moore, P. S., Roizman, B., Whitley, R., Yamanishi, K. 2007: 44–58

  View details for Web of Science ID 000296850300005

• T-cell immunity to subclinical cytomegalovirus infection reduces cardiac allograft disease CIRCULATION Tu, W., Potena, L., Stepick-Biek, P., Liu, L., Dionis, K. Y., Luikart, H., Fearon, W. F., Holmes, T. H., Chin, C., Cooke, J. P., Valantine, H. A., Mocarski, E. S., Lewis, D. B. 2006; 114 (15): 1608-1615

  Abstract

  Asymptomatic cytomegalovirus (CMV) replication is frequent after cardiac transplantation in recipients with pretransplantation CMV infection. How subclinical viral replication influences cardiac allograft disease remains poorly understood, as does the importance of T-cell immunity in controlling such replication.Thirty-nine cardiac recipients who were pretransplantation CMV antibody positive were longitudinally studied for circulating CMV-specific CD4 and CD8 T-cell responses, CMV viral load in blood neutrophils, and allograft rejection during the first posttransplantation year. Nineteen of these recipients were also analyzed for changes of coronary artery intimal, lumen, and whole-vessel area. All recipients received early prophylactic therapy with ganciclovir. No recipients developed overt CMV disease. Those with detectable levels of CMV-specific CD4 T cells in the first month after transplantation were significantly protected from high mean and peak posttransplantation viral load (P<0.05), acute rejection (P<0.005), and loss of allograft coronary artery lumen (P<0.05) and of whole-vessel area (P<0.05) compared with those who lacked this immune response. The losses of lumen and vessel area were both significantly correlated with the time after transplantation at which a CD4 T-cell response was first detected (P<0.05) and with the cumulative graft rejection score (P<0.05).The early control of subclinical CMV replication after transplantation by T-cell immunity may limit cardiac allograft rejection and vascular disease. Interventions to increase T-cell immunity might be clinically useful in limiting these adverse viral effects.

  View details for DOI 10.1161/CIRCULATIONAHA.105.607549

  View details for PubMedID 17015794

• Human cytomegalovirus alters localization of MHC class II and dendrite morphology in mature Langerhans cells JOURNAL OF IMMUNOLOGY Lee, A. W., Hertel, L., Louie, R. K., Burster, T., Lacaille, V., Pashine, A., Abate, D. A., Mocarski, E. S., Mellins, E. D. 2006; 177 (6): 3960-3971

  Abstract

  Hemopoietic stem cell-derived mature Langerhans-type dendritic cells (LC) are susceptible to productive infection by human CMV (HCMV). To investigate the impact of infection on this cell type, we examined HLA-DR biosynthesis and trafficking in mature LC cultures exposed to HCMV. We found decreased surface HLA-DR levels in viral Ag-positive as well as in Ag-negative mature LC. Inhibition of HLA-DR was independent of expression of unique short US2-US11 region gene products by HCMV. Indeed, exposure to UV-inactivated virus, but not to conditioned medium from infected cells, was sufficient to reduce HLA-DR on mature LC, implicating particle binding/penetration in this effect. Reduced surface levels reflected an altered distribution of HLA-DR because total cellular HLA-DR was not diminished. Accumulation of HLA-DR was not explained by altered cathepsin S activity. Mature, peptide-loaded HLA-DR molecules were retained within cells, as assessed by the proportion of SDS-stable HLA-DR dimers. A block in egress was implicated, as endocytosis of surface HLA-DR was not increased. Immunofluorescence microscopy corroborated the intracellular retention of HLA-DR and revealed markedly fewer HLA-DR-positive dendritic projections in infected mature LC. Unexpectedly, light microscopic analyses showed a dramatic loss of the dendrites themselves and immunofluorescence revealed that cytoskeletal elements crucial for the formation and maintenance of dendrites are disrupted in viral Ag-positive cells. Consistent with these dendrite effects, HCMV-infected mature LC exhibit markedly reduced chemotaxis in response to lymphoid chemokines. Thus, HCMV impedes MHC class II molecule trafficking, dendritic projections, and migration of mature LC. These changes likely contribute to the reduced activation of CD4+ T cells by HCMV-infected mature LC.

  View details for Web of Science ID 000240475300052

  View details for PubMedID 16951359

• Cytomegalovirus-induced embryopathology: mouse submandibular salivary gland epithelial-mesenchymal ontogeny as a model BMC DEVELOPMENTAL BIOLOGY Melnick, M., Mocarski, E. S., Abichaker, G., Huang, J., Jaskoll, T. 2006; 6

  Abstract

  Human studies suggest, and mouse models clearly demonstrate, that cytomegalovirus (CMV) is dysmorphic to early organ and tissue development. CMV has a particular tropism for embryonic salivary gland and other head mesenchyme. CMV has evolved to co-opt cell signaling networks so to optimize replication and survival, to the detriment of infected tissues. It has been postulated that mesenchymal infection is the critical step in disrupting organogenesis. If so, organogenesis dependent on epithelial-mesenchymal interactions would be particularly vulnerable. In this study, we chose to model the vulnerability by investigating the cell and molecular pathogenesis of CMV infected mouse embryonic submandibular salivary glands (SMGs).We infected E15 SMG explants with mouse CMV (mCMV). Active infection for up to 12 days in vitro results in a remarkable cell and molecular pathology characterized by atypical ductal epithelial hyperplasia, apparent epitheliomesenchymal transformation, oncocytic-like stromal metaplasia, beta-catenin nuclear localization, and upregulation of Nfkb2, Relb, Il6, Stat3, and Cox2. Rescue with an antiviral nucleoside analogue indicates that mCMV replication is necessary to initiate and maintain SMG dysmorphogenesis.mCMV infection of embryonic mouse explants results in dysplasia, metaplasia, and, possibly, anaplasia. The molecular pathogenesis appears to center around the activation of canonical and, perhaps more importantly, noncanonical NFkappaB. Further, COX-2 and IL-6 are important downstream effectors of embryopathology. At the cellular level, there appears to be a consequential interplay between the transformed SMG cells and the surrounding extracellular matrix, resulting in the nuclear translocation of beta-catenin. From these studies, a tentative framework has emerged within which additional studies may be planned and performed.

  View details for DOI 10.1186/1471-213X-6-42

  View details for Web of Science ID 000241284000001

  View details for PubMedID 16959038

  View details for PubMedCentralID PMC1601957

• Acute rejection and cardiac allograft vascular disease is reduced by suppression of subclinical cytomegalovirus infection TRANSPLANTATION Potena, L., Holweg, C. T., Chin, C., Luikart, H., Weisshaar, D., Narasimhan, B., Fearon, W. F., Lewis, D. B., Cooke, J. P., Mocarski, E. S., Valantine, H. A. 2006; 82 (3): 398-405

  Abstract

  Anticytomegalovirus (CMV) prophylaxis prevents the acute disease but its impact on subclinical infection and allograft outcome is unknown. We sought to determine whether CMV prophylaxis administered for three months after heart transplant would improve patient outcomes.This prospective cohort study of 66 heart transplant recipients compared aggressive CMV prophylaxis (n = 21, CMV hyperimmune globulin [CMVIG] plus four weeks of intravenous ganciclovir followed by two months of valganciclovir); with standard prophylaxis (n = 45, intravenous ganciclovir for four weeks). Prophylaxis was based on pretransplant donor (D) and recipient (R) CMV serology: R-/D+ received aggressive prophylaxis; R+ received standard prophylaxis. Outcome measures were: CMV infection assessed by DNA-polymerase chain reaction on peripheral blood polymorphonuclear leukocytes, acute rejection, and cardiac allograft vascular disease (CAV) assessed by intravascular ultrasound. All patients completed one year of follow-up. RESULTS.: CMV infection was subclinical in all but four patients (two in each group). Aggressively treated patients had a lower incidence of CMV infection (73 +/- 10% vs. 94 +/- 4%; P = 0.038), and an independent reduced relative risk for acute rejection graded > or =3A (relative risk [95% CI] = 0.55 [0.26-0.96]; P = 0.03), as compared with the standard prophylaxis group. Aggressively prophylaxed patients also showed a slower progression of CAV, in terms of coronary artery lumen loss (lumen volume change=-21 +/- 13% vs. -10+/-14%; P = 0.05); and vessel shrinkage (vessel volume change = -15 +/- 11% vs. -3 +/- 18%; P = 0.03).Prolonged (val)ganciclovir plus CMVIG reduces viral levels, acute rejection, and allograft vascular disease, suggesting a role for chronic subclinical infection in the pathophysiology of the most common diseases affecting heart transplant recipients.

  View details for DOI 10.1097/01.tp.0000229039.87735.76

  View details for PubMedID 16906040

• Betaherpesvirus-conserved cytomegalovirus tegument protein ppUL32 (pp150) controls cytoplasmic events during virion maturation JOURNAL OF VIROLOGY AuCoin, D. P., Smith, G. B., Meiering, C. D., Mocarski, E. S. 2006; 80 (16): 8199-8210

  Abstract

  The UL32 gene of human cytomegalovirus (CMV) encodes a prominent betaherpesvirus-conserved virion tegument protein, called pp150 (basic phosphoprotein/ppUL32), that accumulates within a cytoplasmic inclusion adjacent to the nucleus at late times during infection. Using a UL32 deletion mutant (DeltaUL32-BAC) (where BAC is bacterial artificial chromosome), we demonstrate that pp150 is critical for virion maturation in the cytoplasmic compartment. Cotransfection of a pp150 expression plasmid with DeltaUL32-BAC DNA led to complementation of the replication defect with focus formation due to secondary spread. Deletion of the amino terminus of pp150 or disruption of the betaherpesvirus conserved regions, CR1 and CR2, revealed these regions to be critical for replication. In contrast, deletion of the carboxyl terminus only partially compromised maturation while disruption of glycosylation sites had no effect. An African green monkey CMV UL32 homolog complemented DeltaUL32-BAC replication but murine CMV M32 failed to complement, consistent with evolutionary divergence of rodent and primate cytomegaloviruses. Infection with DeltaUL32-BAC showed normal expression of all kinetic classes of viral genes and replication of viral DNA, with accumulation of viral DNA-containing particles in the cytoplasm; however, mutant virus did not spread to adjacent cells. In contrast to this block in virion infectivity, cell-to-cell transfer of pp65-containing particles was observed, suggesting that release of dense bodies continued in the absence of pp150. These observations demonstrate that pp150 is critical for virion egress, possibly at the stage of final envelopment.

  View details for DOI 10.1128/JVI.00457-06

  View details for Web of Science ID 000239557700041

  View details for PubMedID 16873276

  View details for PubMedCentralID PMC1563810

• Prevention of subclinical CMV infection reduces cardiac allograft disease progression by positively affecting coronary remodeling Potena, L., Fearon, W., Holweg, C., Luikart, H., Chin, C., Cooke, J., Lewis, D., Mocarski, E., Valantine, H. ELSEVIER SCIENCE INC. 2006: S138–S139

  View details for Web of Science ID 000203407400273

• Increased sensitivity of two-step cytomegalovirus PCR analysis on peripheral blood leukocytes permits detailed analysis of reactivation and immune reconstitution 32nd Annual Meeting of the American-Society-for-Blood-and-Marrow-Transplantation Vana, M. L., Formankova, D., Cha, S., Sharma, A., Potena, L., Brown, J. M., Mocarski, E. S. ELSEVIER SCIENCE INC. 2006: 30–30

  View details for Web of Science ID 000235344100076

• Inhibition of cardiac allograft vasculopathy by sirolimus and mycofenolate: Asymmetric dimethyl arginine as a potential therapeutic target Potena, L., Fearon, W., Sydow, K., Holweg, C., Luikart, H., Mocarski, E., Valantine, H., Cooke, J. ELSEVIER SCIENCE INC. 2006: S100–S100

  View details for Web of Science ID 000203407400162

• Cytomegalovirus MCK-2 controls mobilization and recruitment of myeloid progenitor cells to facilitate dissemination BLOOD Noda, S., Aguirre, S. A., Bitmansour, A., Brown, J. M., Sparer, T. E., Huang, J., Mocarski, E. S. 2006; 107 (1): 30-38

  Abstract

  Murine cytomegalovirus encodes a secreted, pro-inflammatory chemokine-like protein, MCK-2, that recruits leukocytes and facilitates viral dissemination. We have shown that MCK-2-enhanced recruitment of myelomonocytic leukocytes with an immature phenotype occurs early during infection and is associated with efficient viral dissemination. Expression of MCK-2 drives the mobilization of a population of leukocytes from bone marrow that express myeloid marker Mac-1 (CD11b), intermediate levels of Gr-1 (Ly6 G/C), platelet-endothelial-cell adhesion molecule-1 (PECAM-1, CD31), together with heterogeneous levels of stem-cell antigen-1 (Sca-1, Ly-6 A /E). Recombinant MCK-2 mediates recruitment of this population even in the absence of viral infection. Recruitment of this cell population and viral dissemination via the bloodstream to salivary glands proceeds normally in mice that lack CCR2 and MCP-1 (CCL2), suggesting that recruitment of macrophages is not a requisite component of pathogenesis. Thus, a systemic impact of MCK-2 enhances the normal host response and causes a marked increase in myelomonocytic recruitment with an immature phenotype to initial sites of infection. Mobilization influences levels of virus dissemination via the bloodstream to salivary glands and is dependent on a myelomonocytic cell type other than mature macrophages.

  View details for Web of Science ID 000234235200012

  View details for PubMedID 16046529

• Human cytomegalovirus alters localization of MHC class II and dendrite morphology in mature Langerhans cells. 6th Annual Meeting of the Federation-of-Clinical-Immunology-Societies Lee, A., Hertel, L., Louie, R., Burster, T., Lacaille, V., Pashine, A., Abate, D., Mocarski, E., Mellins, E. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2006: S22–S23

  View details for DOI 10.1016/j.clim.2006.04.206

  View details for Web of Science ID 000237924300051

• Contribution of GADD45 family members to cell death suppression by cellular Bcl-x(L) and cytomegalovirus vMIA JOURNAL OF VIROLOGY Smith, G. B., Mocarski, E. S. 2005; 79 (23): 14923-14932

  Abstract

  Mammalian cells and viruses encode inhibitors of programmed cell death that localize to mitochondria and suppress apoptosis initiated by a wide variety of inducers. Mutagenesis was used to probe the role of a predicted alpha-helical region within the hydrophobic antiapoptotic domain (AAD) of cytomegalovirus vMIA, the UL37x1 gene product. This region was found to be essential for cell death suppression activity. A screen for proteins that interacted with the AAD of functional vMIA but that failed to interact with mutants identified growth arrest and DNA damage 45 (GADD45alpha), a cell cycle regulatory protein activated by genotoxic stress, as a candidate cellular binding partner. GADD45alpha interaction required the AAD alpha-helical character that also dictated GADD45alpha-mediated enhancement of death suppression. vMIA mutants that failed to interact with GADD45alpha were completely nonfunctional in cell death suppression, and any of the three GADD45 family members (GADD45alpha, GADD45beta/MyD118, or GADD45gamma/OIG37/CR6/GRP17) was able to cooperate with vMIA; however, none influenced cell death when introduced into cells alone. GADD45alpha was found to increase vMIA protein levels comparably to treatment with protease inhibitors MG132 and ALLN. Targeted short interfering RNA knockdown of all three GADD45 family members maximally reduced vMIA activity, and this reduction was abrogated by additional GADD45alpha. Interestingly, GADD45 family members were also able to bind and enhance cell death suppression by Bcl-xL, a member of the Bcl-2 family of cell death suppressors, suggesting a direct cooperative link between apoptosis and the proteins that regulate the DNA damage response.

  View details for DOI 10.1128/JVI.79.23.14923-14932.2005

  View details for Web of Science ID 000233279300046

  View details for PubMedID 16282491

  View details for PubMedCentralID PMC1287561

• Mitochondrial cell death suppressors carried by human and murine cytomegalovirus confer resistance to proteasome inhibitor-induced apoptosis JOURNAL OF VIROLOGY McCormick, A. L., Meiering, C. D., Smith, G. B., Mocarski, E. S. 2005; 79 (19): 12205-12217

  Abstract

  Human cytomegalovirus carries a mitochondria-localized inhibitor of apoptosis (vMIA) that is conserved in primate cytomegaloviruses. We find that inactivating mutations within UL37x1, which encodes vMIA, do not substantially affect replication in TownevarATCC (Towne-BAC), a virus that carries a functional copy of the betaherpesvirus-conserved viral inhibitor of caspase 8 activation, the UL36 gene product. In Towne-BAC infection, vMIA reduces susceptibility of infected cells to intrinsic death induced by proteasome inhibition. vMIA is sufficient to confer resistance to proteasome inhibition when expressed independent of viral infection. Murine cytomegalovirus m38.5, whose position in the viral genome is analogous to UL37x1, exhibits mitochondrial association and functions in much the same manner as vMIA in inhibiting intrinsic cell death. This work suggests a common role for vMIA in rodent and primate cytomegaloviruses, modulating the threshold of virus-infected cells to intrinsic cell death.

  View details for DOI 10.1128/JVI.79.19.12205-12217.2005

  View details for Web of Science ID 000231992500008

  View details for PubMedID 16160147

  View details for PubMedCentralID PMC1211555

• Predicting coding potential from genome sequence: Application to betaherpesviruses infecting rats and mice JOURNAL OF VIROLOGY Brocchieri, L., Kledal, T. N., Karlin, S., Mocarski, E. S. 2005; 79 (12): 7570-7596

  Abstract

  Prediction of protein-coding regions and other features of primary DNA sequence have greatly contributed to experimental biology. Significant challenges remain in genome annotation methods, including the identification of small or overlapping genes and the assessment of mRNA splicing or unconventional translation signals in expression. We have employed a combined analysis of compositional biases and conservation together with frame-specific G+C representation to reevaluate and annotate the genome sequences of mouse and rat cytomegaloviruses. Our analysis predicts that there are at least 34 protein-coding regions in these genomes that were not apparent in earlier annotation efforts. These include 17 single-exon genes, three new exons of previously identified genes, a newly identified four-exon gene for a lectin-like protein (in rat cytomegalovirus), and 10 probable frameshift extensions of previously annotated genes. This expanded set of candidate genes provides an additional basis for investigation in cytomegalovirus biology and pathogenesis.

  View details for DOI 10.1128/JVI.79.12.7570-7596.2005

  View details for Web of Science ID 000229416100030

  View details for PubMedID 15919911

  View details for PubMedCentralID PMC1143683

• Cytomegalovirus infection is associated with acute left ventricular dysfunction in heart transplant recipients after antiviral prohylaxis. 6th American Transplant Congress Potena, L., Holweg, C. T., Luikart, H. I., Levvis, D. B., Cooke, J. P., Mocarski, E. S., Valantine, H. A. WILEY-BLACKWELL. 2005: 167–168

  View details for Web of Science ID 000229231600045

• Aggressive anti-cytomegalovirus prophylaxis and acute rejection in high risk patients: Beat the virus and prevent the rejection Potena, L., Holweg, C., Luikart, H., Mocarski, E., Lewis, D., Cooke, J., Chin, C., Hunt, S., Valantine, H. ELSEVIER SCIENCE INC. 2005: S117–S118

  View details for Web of Science ID 000203407500231

• The carboxyl-terminal region of human cytomegalovirus IE1(491aa) contains an acidic domain that plays a regulatory role and a chromatin-tethering domain that is dispensable during viral replication JOURNAL OF VIROLOGY Reinhardt, J., Smith, G. B., Himmelheber, C. T., Azizkhan-Clifford, J., Mocarski, E. S. 2005; 79 (1): 225-233

  Abstract

  The human cytomegalovirus major immediate-early (alpha) protein IE1(491aa) plays an important role in controlling viral gene expression at low multiplicities of infection. With a transient complementation assay, full-length IE1(491aa) enhanced the growth of ie1 mutant virus CR208 20-fold better than a deletion mutant lacking 71 carboxyl-terminal amino acids (IE1(1-420aa)). A 16-amino-acid domain between amino acids 476 and 491 was both necessary and sufficient for chromatin-tethering activity; however, this domain was completely dispensable for complementation of CR208 replication. The proximal 55-amino-acid acidic domain (amino acids 421 to 475) was found to be most important for function. A deletion mutant lacking only this domain retained chromatin-tethering activity but failed to complement mutant virus. Interestingly, serine phosphorylation (at amino acids 399, 402, 406, 423, 428, 431, 448, 451, and 455) was not required for complementation. These results show that IE1(491aa) is composed of at least two domains that support replication, a region located between amino acids 1 and 399 that complements ie1 mutant virus replication to low levels and an acidic domain between amino acids 421 and 479 that dramatically enhances complementation.

  View details for DOI 10.1128/JVI.79.1.225-233.2005

  View details for Web of Science ID 000225904700022

  View details for PubMedID 15596818

  View details for PubMedCentralID PMC538725

• Global analysis of host cell gene expression late during cytomegalovirus infection reveals extensive dysregulation of cell cycle gene expression and induction of pseudomitosis independent of US28 function JOURNAL OF VIROLOGY Hertel, L., Mocarski, E. S. 2004; 78 (21): 11988-12011

  Abstract

  Replication of human cytomegalovirus (CMV) depends on host cell gene products working in conjunction with viral functions and leads to a dramatic dysregulation of cell cycle gene expression. Comprehensive transcriptional profiling was used to identify pathways most dramatically modulated by CMV at late times during infection and to determine the extent to which expression of the viral chemokine receptor US28 contributed to modulating cellular gene expression. Cells infected with the AD169 strain of virus or a fully replication competent US28-deficient derivative (RV101) were profiled throughout the late phase of infection (50, 72, and 98 h postinfection). Although sensitive statistical analysis showed striking global changes in transcript levels in infected cells compared to uninfected cells, the expression of US28 did not contribute to these alterations. CMV infection resulted in lower levels of transcripts encoding cytoskeletal, extracellular matrix, and adhesion proteins, together with small GTPases and apoptosis regulators, and in higher levels of transcripts encoding cell cycle, DNA replication, energy production, and inflammation-related gene products. Surprisingly, a large number of cellular transcripts encoding mitosis-related proteins were upmodulated at late times in infection, and these were associated with the formation of abnormal mitotic spindles and the appearance of pseudomitotic cells. These data extend our understanding of how broadly CMV alters the regulation of host cell cycle gene products and highlight the establishment of a mitosis-like environment in the absence of cellular DNA replication as important for viral replication and maturation.

  View details for DOI 10.1128/JVI.78.21.11988-12011.2004

  View details for Web of Science ID 000224540900052

  View details for PubMedID 15479839

  View details for PubMedCentralID PMC523267

• Systemic inflammation links impaired glucose metabolism to cardiac allograft vasculopathy development 77th Scientific Meeting of the American-Heart-Association Potena, L., Biadi, O., Fearon, W. B., Holweg, C. T., Luikart, H. I., Mocarski, E. S., Lewis, D. B., Valantine, H. A. LIPPINCOTT WILLIAMS & WILKINS. 2004: 753–53

  View details for Web of Science ID 000224783504063

• Major human cytomegalovirus structural protein pp65 (ppUL83) prevents interferon response factor 3 activation in the interferon response JOURNAL OF VIROLOGY Abate, D. A., Watanabe, S., Mocarski, E. S. 2004; 78 (20): 10995-11006

  Abstract

  We have identified a cytomegalovirus virion protein capable of modulating the rapid induction of an interferon-like response in cells that follows virus binding and penetration. Functional genomics revealed a role for the major cytomegalovirus structural protein, pp65 (ppUL83), in counteracting this response. The underlying mechanism involves a differential impact of this structural protein on the regulation of interferon response factor 3 (IRF-3). In contrast, NF-kappaB is activated independent of pp65, and neither STAT1 nor STAT3 becomes activated by either virus. pp65 is sufficient to prevent the activation of IRF-3 when introduced alone into cells. pp65 acts by inhibiting nuclear accumulation of IRF-3 and is associated with a reduced IRF-3 phosphorylation state. Thus, this investigation shows that the major structural protein of cytomegalovirus is committed to the modulation of the IRF-3 response, a primary mediator of the type I interferon response. By subverting IRF-3, the virus escapes throwing a central alarm devoted to both immediate antiviral control and regulation of the immune response.

  View details for DOI 10.1128/JVI.78.20.10995-11006.2004

  View details for Web of Science ID 000224229000017

  View details for PubMedID 15452220

  View details for PubMedCentralID PMC521853

• Expression of human CXCR2 in murine Neutrophils as a model for assessing cytomegalovirus chemokine vCXCL-1 function in vivo JOURNAL OF INTERFERON AND CYTOKINE RESEARCH Sparer, T. E., Gosling, J., Schall, T. J., Mocarski, E. S. 2004; 24 (10): 611-620

  Abstract

  Human cytomegalovirus (CMV) (Toledo strain) produces a potent chemokine (vCXCL-1) that specifically recognizes human (Hu)CXCR2, one of two human CXCL8 (IL8) receptors found on peripheral blood neutrophils. Thioglycollate-elicited neutrophils from BALB/c mice failed to respond to vCXCL-1 while retaining the capacity to respond to known murine (Mu) CXCR2 ligands, such as hCXCL8 (IL8) and mCXCL1 (KC). A transgenic mouse expressing hCXCR2 under the control of a neutrophil-specific promoter (human myeloid-related protein-8) was generated. Resting or activated neutrophils from transgenic mice were found to express hCXCR2 and to respond to vCXCL-1. vCXCL-1 induced a specific calcium flux and chemotaxis of these cells. Expression of the functional vCXCL-1 receptor in mice will facilitate investigations of the role vCXCL-1 plays during viral infection of an intact host animal. In addition, this work demonstrates the remarkable species specificity of a potent viral chemokine.

  View details for Web of Science ID 000224386500004

  View details for PubMedID 15626158

• Cytomegalovirus and heart transplant atherosclerosis: A likely guilt hidden by weak proofs TRANSPLANTATION Potena, L., Holweg, C., Valantine, H. A., Mocarski, E. S. 2004; 78 (4): 631-631

  View details for DOI 10.1097/01.TP.0000131954.83868.9F

  View details for Web of Science ID 000223486500024

  View details for PubMedID 15446327

• Immune escape and exploitation strategies of cytomegaloviruses: impact on and imitation of the major histocompatibility system CELLULAR MICROBIOLOGY Mocarski, E. S. 2004; 6 (8): 707-717

  Abstract

  Cytomegalovirus (CMV) has yielded many insights into immune escape mechanisms. Both human and mouse CMV encode a diverse array of gene products, many of which appear to modulate the immune response in the host. Some deflect the host response to infection and contribute to lifelong viral persistence while others exploit immune cells that respond to infection. Here, the viral functions that modulate and mimic host major histocompatibility complex (MHC) function will be reviewed. Viral gene products related to both classical and non-classical components of the MHC system assure the virus will persist in immunocompetent individuals. Examples of host countermeasures that neutralize viral immunomodulatory functions have emerged in the characterization of viral functions that contribute to this stand-off in CMVs that infect humans, other primates and rodents. CMV-induced disease occurs when the immune system is not yet developed, such as in the developing fetus, or when it is compromised, such as in allograft transplant recipients, suggesting that the balance between virus escape and host control is central to pathogenesis. Although evidence supports the dominant role of immune escape in CMV pathogenesis and persistence, MHC-related immunomodulatory functions have been ascribed only subtle impact on pathogenesis and the immune response during natural infection. Viral gene products that interface with the MHC system may impact natural killer cell function, antigen presentation, and T lymphocyte immune surveillance. Many also interact with other cells, particularly those in the myeloid lineage, with consequences that have not been explored. Overall, the virus-encoded modulatory functions that have been acquired by CMV likely ensure survival and adaptation to the wide range of mammalian host species in which they are found.

  View details for DOI 10.1111/j.1462-5822.2004.00425.x

  View details for Web of Science ID 000222462200002

  View details for PubMedID 15236638

• Impact of human cytomegalovirus latent infection on myeloid progenitor cell gene expression JOURNAL OF VIROLOGY Slobedman, B., Stern, J. L., Cunningham, A. L., Abendroth, A., Abate, D. A., Mocarski, E. S. 2004; 78 (8): 4054-4062

  Abstract

  Herpesviruses establish lifelong latent infections in their hosts. Human cytomegalovirus (CMV) targets a population of bone marrow-derived myeloid lineage progenitor cells that serve as a reservoir for reactivation; however, the mechanisms by which latent CMV infection is maintained are unknown. To gain insights into mechanisms of maintenance and reactivation, we employed microarrays of approximately 26,900 sequence-verified human cDNAs to assess global changes in cellular gene expression during experimental CMV latent infection of granulocyte-macrophage progenitors (GM-Ps). This analysis revealed at least 29 host cell genes whose expression was increased and six whose expression was decreased during CMV latency. These changes in transcript levels appeared to be authentic, judging on the basis of further analysis of a subset by semiquantitative reverse transcription-PCR. This study provides a comprehensive snapshot of changes in host cell gene expression that result from latent infection and suggest that CMV regulates genes that encode proteins involved in immunity and host defense, cell growth, signaling, and transcriptional regulation. The host genes whose expression we found altered are likely to contribute to an environment that sustains latent infection.

  View details for DOI 10.1128/JVI.78.8.4054-4062.2004

  View details for Web of Science ID 000220641600028

  View details for PubMedID 15047822

  View details for PubMedCentralID PMC374258

• Cytomegalovirus impairs endothelial nitric oxide synthase pathway: Role of oxidative stress and viral entry Iijima, K., Noda, S., Sydow, K., Mocarski, E. S., Cooke, J. P. ELSEVIER SCIENCE INC. 2004: 507A

  View details for DOI 10.1016/S0735-1097(04)92146-8

  View details for Web of Science ID 000189388502149

• Cytomegalovirus infectious burden is proportional to cardiac allograft vasculopathy in heart transplant recipients 53rd Annual Scientific Session of the American-College-of-Cardiology Potena, L., Magelli, C., Ortolani, P., Fearon, W. F., Grigioni, F., Magnani, G., Coccolo, F., Yeung, A. C., Luikart, H. I., Hunt, S. A., Mocarski, E. S., Cooke, J. P., Lewis, D. B., Branzi, A., Valantine, H. A. ELSEVIER SCIENCE INC. 2004: 185A–185A

  View details for Web of Science ID 000189388500790

• Cytomegalovirus infection impairs the nitric oxide synthase pathway - Role of asymmetric dimethylarginine in transplant arteriosclerosis CIRCULATION Weis, M., Kledal, T. N., LIN, K. Y., Panchal, S. N., Gao, S. Z., Valantine, H. A., Mocarski, E. S., Cooke, J. P. 2004; 109 (4): 500-505

  Abstract

  We hypothesized that cytomegalovirus (CMV) may contribute to the vasculopathy observed in cardiac allograft recipients by impairing the endothelial nitric oxide synthase pathway. We focused on asymmetric dimethylarginine (ADMA, the endogenous inhibitor of nitric oxide synthase) as a potential mediator of the adverse vascular effect of CMV.Heart transplant recipients manifested elevated plasma ADMA levels compared with healthy control subjects. Transplant patients with CMV DNA-positive leukocytes had higher plasma ADMA concentrations and more extensive transplant arteriopathy (TA). Human microvascular endothelial cells infected with the CMV isolates elaborated more ADMA. The increase in ADMA was temporally associated with a reduction in the activity of dimethylarginine dimethylaminohydrolase (DDAH, the enzyme that metabolizes ADMA). Infected cultures showed high levels of oxidative stress with enhanced endothelial production of superoxide anion.CMV infection in human heart transplant recipients is associated with higher ADMA elevation and more severe TA. CMV infection in endothelial cells increases oxidative stress, impairs DDAH activity, and increases ADMA elaboration. CMV infection may contribute to endothelial dysfunction and TA by dysregulation of the endothelial nitric oxide synthase pathway.

  View details for DOI 10.1161/01.CIR.0000109692.16004.AF

  View details for Web of Science ID 000188669400011

  View details for PubMedID 14732750

• Asymptomatic cytomegalovirus activation leads to acute rejection in heart transplant recipients despite anti-viral prophylaxis. 5th American Transplant Congress Potena, L., Holweg, C., Luikart, H. I., Mocarski, E. S., Cooke, J. P., Lewis, D. B., Valantine, H. A. WILEY-BLACKWELL. 2004: 453–453

  View details for Web of Science ID 000221322501076

• Differential function and expression of the viral inhibitor of caspase 8-induced apoptosis (vICA) and the viral mitochondria-localized inhibitor of apoptosis (vMIA) cell death suppressors conserved in primate and rodent cytomegaloviruses VIROLOGY McCormick, A. L., Skaletskaya, A., Barry, P. A., Mocarski, E. S., Goldmacher, V. S. 2003; 316 (2): 221-233

  Abstract

  Human cytomegalovirus (CMV) genes UL36 and UL37 encode viral inhibitor of caspase-8-induced apoptosis (vICA) and viral mitochondria inhibitor of apoptosis (vMIA), respectively. Rhesus macaque CMV homologues, denoted Rh-vICA and Rh-vMIA, were identified and found to suppress apoptosis. One of these functions was conserved in MCMV, encoded by the M36 gene and denoted M-vICA. Conserved regions were compared to domains important to vICA- and vMIA-mediated cell death suppression. The conserved sequences of primate CMV vMIA homologues overlapped with the two known functional domains, providing further evidence supporting a crucial role of vMIA in cell death suppression. RNA blot analyses revealed that expression of murine and rhesus macaque CMV UL36 and UL37 homologues started early and continued through late times of infection. Murine CMV homologues were expressed with alpha (immediate early) kinetics, like human CMV UL36 and UL37, whereas rhesus macaque CMV homologues exhibited beta (delayed early) kinetics. Despite differences in organization and transcriptional regulation, this region appears to carry out a conserved role in cell death suppression. When viewed in light of sequence conservation, a functional vMIA homologue appears to be encoded by every primate CMV, whereas a functional vICA homologue appears to be encoded by all cytomegaloviruses for which sequence data are available.

  View details for DOI 10.1016/j.virol.2003.07.003

  View details for PubMedID 14644605

• Common lymphoid progenitors rapidly engraft and protect against lethal murine cytomegalovirus infection after hematopoietic stem cell transplantation BLOOD Arber, C., Bitmansour, A., Sparer, T. E., Higgins, J. P., Mocarski, E. S., Weissman, I. L., Shizuru, J. A., Brown, J. M. 2003; 102 (2): 421-428

  Abstract

  Lymphoid deficiency after allogeneic hematopoietic cell transplantation (HCT) results in increased susceptibility to infection; however, transplantation of mature lymphocytes frequently results in a serious complication known as graft-versus-host disease (GVHD). Here we demonstrate in mice that both congenic as well as allogeneic transplantation of low numbers of highly purified common lymphoid progenitors (CLPs)-a rare population of lymphoid-lineage-committed bone marrow cells-accelerates immune reconstitution after lethal irradiation and rescue with hematopoietic stem cells (HSCs). After congenic transplantation, 3 x 10(3) CLPs protected against murine cytomegalovirus (MCMV) infection at a level roughly equivalent to 107 unfractionated lymph node cells. In the allogeneic model of matched unrelated donor HSC transplantation, cotransplantation of 3 x 10(3) CLPs protected thymus-bearing as well as thymectomized hosts from MCMV infection and attenuated disease severity. Immunohistochemistry in combination with antibody depletion of T and natural killer (NK) cells confirmed that CLP-derived as well as residual host lymphocytes contribute to antiviral protection. Importantly, transplantation of allogeneic CLPs provided a durable antiviral immunity without inducing GVHD. These data support the potential for composing grafts with committed progenitors to reduce susceptibility to viral infection following HCT.

  View details for DOI 10.1182/blood-2002-12-3834

  View details for Web of Science ID 000184083500010

  View details for PubMedID 12663447

• Susceptibility of immature and mature Langerhans cell-type dendritic cells to infection and immunomodulation by human cytomegalovirus JOURNAL OF VIROLOGY Hertel, L., Lacaille, V. G., Strobl, H., Mellins, E. D., Mocarski, E. S. 2003; 77 (13): 7563-7574

  Abstract

  Human cytomegalovirus (CMV) infection initiates in mucosal epithelia and disseminates via leukocytes throughout the body. Langerhans cells (LCs), the immature dendritic cells (DCs) that reside in epithelial tissues, are among the first cells to encounter virus and may play important roles in the immune response, as well as in pathogenesis as hosts for viral replication and as vehicles for dissemination. Here, we demonstrate that CD34(+) progenitor cell-derived LC-type DCs exhibit a differentiation state-dependent susceptibility to CMV infection. In contrast to the small percentage (3 to 4%) of the immature LCs that supported infection, a high percentage (48 to 74%) of mature, LC-derived DCs were susceptible to infection with endotheliotropic strains (TB40/E or VHL/E) of CMV. These cells were much less susceptible to viral strains AD169varATCC, TownevarRIT(3), and Toledo. When exposed to endotheliotropic strains, viral gene expression (IE1/IE2 and other viral gene products) and viral replication proceeded efficiently in LC-derived mature DCs (mDCs). Productive infection was associated with downmodulation of cell surface CD83, CD1a, CD80, CD86, ICAM-1, major histocompatibility complex (MHC) class I, and MHC class II on these cells. In addition, the T-cell proliferative response to allogeneic LC-derived mDCs was attenuated when CMV-infected cultures were used as stimulators. This investigation revealed important characteristics of the interaction between CMV and the LC lineage of DCs, suggesting that LC-derived mDCs are important to viral pathogenesis and immunity through their increased susceptibility to virus replication and virus-mediated immune escape.

  View details for DOI 10.1128/JVI.77.13.7563-7574.2003

  View details for Web of Science ID 000183598600043

  View details for PubMedID 12805456

  View details for PubMedCentralID PMC164783

• NKG2D-mediated natural killer cell protection against cytomegalovirus is impaired by viral gp40 modulation of retinoic acid early inducible 1 gene molecules JOURNAL OF EXPERIMENTAL MEDICINE Lodoen, M., Ogasawara, K., Hamerman, J. A., Arase, H., Houchins, J. P., Mocarski, E. S., Lanier, L. L. 2003; 197 (10): 1245-1253

  Abstract

  Natural killer (NK) cells play a critical role in the innate immune response against cytomegalovirus (CMV) infections. Although CMV encodes several gene products committed to evasion of adaptive immunity, viral modulation of NK cell activity is only beginning to be appreciated. A previous study demonstrated that the mouse CMV m152-encoded gp40 glycoprotein diminished expression of ligands for the activating NK cell receptor NKG2D on the surface of virus-infected cells. Here we have defined the precise ligands that are affected and have directly implicated NKG2D in immune responses to CMV infection in vitro and in vivo. Murine CMV (MCMV) infection potently induced transcription of all five known retinoic acid early inducible 1 (RAE-1) genes (RAE-1alpha, RAE-1beta, RAE-1delta, RAE-1 epsilon, and RAE-1gamma), but not H-60. gp40 specifically down-regulated the cell surface expression of all RAE-1 proteins, but not H-60, and diminished NK cell interferon gamma production against CMV-infected cells. Consistent with previous findings, a m152 deletion mutant virus (Deltam152) was less virulent in vivo than the wild-type Smith strain of MCMV. Treatment of BALB/c mice with a neutralizing anti-NKG2D antibody before infection increased titers of Deltam152 virus in the spleen and liver to levels seen with wild-type virus. These experiments demonstrate that gp40 impairs NK cell recognition of virus-infected cells through disrupting the RAE-1-NKG2D interaction.

  View details for DOI 10.1084/jem.20021973

  View details for Web of Science ID 000183090200003

  View details for PubMedID 12756263

  View details for PubMedCentralID PMC2193789

• Disruption of mitochondrial networks by the human cytomegalovirus UL37 gene product viral mitochondrion-localized inhibitor of apoptosis JOURNAL OF VIROLOGY McCormick, A. L., Smith, V. L., Chow, D., Mocarski, E. S. 2003; 77 (1): 631-641

  Abstract

  By 24 h after infection with human cytomegalovirus, the reticular mitochondrial network characteristic of uninfected fibroblasts was disrupted as mitochondria became punctate and dispersed. These alterations were associated with expression of the immediate-early (alpha) antiapoptotic UL37x1 gene product viral mitochondrion-localized inhibitor of apoptosis (vMIA). Similar alterations in mitochondrial morphology were induced directly by vMIA in transfected cells. A 68-amino-acid antiapoptotic derivative of vMIA containing the mitochondrial localization and antiapoptotic domains also induced disruption, whereas a mutant lacking the antiapoptotic domain failed to cause disruption. These data suggest that the fission and/or fusion process that normally controls mitochondrial networks is altered by vMIA. Mitochondrial fission has been implicated in the induction of apoptosis and vMIA-mediated inhibition of apoptosis may occur subsequent to this event.

  View details for DOI 10.1128/JVI.77.1.631-641.2003

  View details for Web of Science ID 000179855400062

  View details for PubMedID 12477866

  View details for PubMedCentralID PMC140587

• NK cell-mediated lysis of autologous HCMV-infected skin fibroblasts is highly variable among NK cell clones and polyclonal NK cell lines CLINICAL IMMUNOLOGY Carr, W. H., Little, A. M., Mocarski, E., Parham, P. 2002; 105 (2): 126-140

  Abstract

  Lysis of human cytomegalovirus (HCMV)-infected fibroblasts by autologous natural killer (NK) cells was examined in vitro. For NK cell clones, receptor expression was determined at the level of mRNA and cell-surface protein and compared to the lysis of HCMV AD169 strain-infected fibroblasts in which HLA class I was >70% downregulated. The clones ranged broadly in their ability to lyse AD169-infected fibroblasts, correlating neither with the expression of inhibitory KIR, leukocyte inhibitory receptor-1, or CD94:NKG2A receptors nor with the number of different inhibitory KIR expressed per clone. Some lines of polyclonal NK cells preferentially lysed AD169-infected cells and similarly lysed fibroblasts infected with mutant virus RV798, which lacks the genes for downregulating HLA class I. These results demonstrate that NK cell lysis of HCMV-infected autologous fibroblasts is more complex than a simple missing-self mechanism involving downregulation of HLA class I and failure to engage inhibitory self-specific KIR.

  View details for DOI 10.1006/clim.2002.5273

  View details for Web of Science ID 000179807400003

  View details for PubMedID 12482387

• Latent cytomegalovirus down-regulates major histocompatibility complex class II expression on myeloid progenitors BLOOD Slobedman, B., Mocarski, E. S., Arvin, A. M., Mellins, E. D., Abendroth, A. 2002; 100 (8): 2867-2873

  Abstract

  Following primary infection, human cytomegalovirus (CMV) establishes a lifelong latent infection in bone marrow-derived myeloid lineage cells. Although downmodulation of major histocompatibility complex (MHC) class I and class II protein levels occurs during active viral replication, little is known about the modulation of these proteins during latent infection. When analyzed by flow cytometry, latently infected adherent cells collected from granulocyte macrophage progenitor (GM-P) cultures exhibited a striking reduction in MHC class II antigen present on the cell surface starting very early after exposure to virus that continued for more than 2 weeks. In comparison, cell surface levels of the monocyte cell surface marker CD14 remained unaltered in these cells. A recombinant virus (RV798) lacking the virus genes US2-US11 retained the ability to downmodulate MHC class II levels during latent infection. Immunoblot and immunofluorescent antibody staining analyses showed that the reduction in MHC class II surface levels during latency was associated with a block in protein trafficking. HLA-DR was retained within cytoplasmic vesicles that also contained HLA-DM. Thus, downmodulation remained independent of all previously characterized MHC class I and class II immunomodulatory viral gene products and involved a mechanism not previously ascribed to any viral function. These data show that latent infection is accompanied by reduced cell surface expression of MHC class II proteins, a strategy that would afford the virus escape from immunosurveillance and increase the chances for lifelong latent infection.

  View details for Web of Science ID 000178519100030

  View details for PubMedID 12351397

• Immunomodulation by cytomegaloviruses: manipulative strategies beyond evasion TRENDS IN MICROBIOLOGY Mocarski, E. S. 2002; 10 (7): 332-339

  Abstract

  Human cytomegalovirus (CMV) remains the major infectious cause of birth defects as well as an important opportunistic pathogen. Individuals infected with CMV mount a strong immune response that suppresses persistent viral replication and maintains life-long latency. Loss of immune control opens the way to virus reactivation and disease. The large number of immunomodulatory functions encoded by CMV increases the efficiency of infection, dissemination, reactivation and persistent infection in hosts with intact immune systems and could contribute to virulence in immunocompromised hosts. These functions modulate both the innate and adaptive arms of the immune response and appear to target cellular rather than humoral responses preferentially. CMV encodes a diverse arsenal of proteins focused on altering and/or mimicking: (1) classical and non-classical major histocompatibility complex (MHC) protein function; (2) leukocyte migration, activation and cytokine responses; and (3) host cell susceptibility to apoptosis. Evidence that the host evolves mechanisms to counteract virus immune modulation is also accumulating. Although immune evasion is certainly one clear goal of the virus, the pro-inflammatory impact of certain viral functions suggests that increased inflammation benefits viral dissemination. The ability of such viral functions to successfully 'face off' against the host immune system ensures the success of this pathogen in the human population and could provide key insights into disease mechanisms.

  View details for Web of Science ID 000176847300011

  View details for PubMedID 12110212

• Direct recognition of cytomegalovirus by activating and inhibitory NK cell receptors SCIENCE Arase, H., Mocarski, E. S., Campbell, A. E., Hill, A. B., Lanier, L. L. 2002; 296 (5571): 1323-1326

  Abstract

  Natural killer (NK) cells express inhibitory receptors for major histocompatibility complex (MHC) class I antigens, preventing attack against healthy cells. Mouse cytomegalovirus (MCMV) encodes an MHC-like protein (m157) that binds to an inhibitory NK cell receptor in certain MCMV-susceptible mice. In MCMV-resistant mice, this viral protein engages a related activating receptor (Ly49H) and confers host protection. These activating and inhibitory receptors are highly homologous, suggesting the possibility that one evolved from the other in response to selective pressure imposed by the pathogen.

  View details for DOI 10.1126/science.1070884

  View details for Web of Science ID 000175713000057

  View details for PubMedID 11950999

• Modulation of MHC class II by human cytomegalovirus in dendritic cells Lacaille, V. G., Hertel, L., Strobl, H., Mocarski, E. S., Mellins, E. D. FEDERATION AMER SOC EXP BIOL. 2002: A1037

  View details for Web of Science ID 000174593901719

• Virus self-improvement through inflammation: No pain, no gain PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Mocarski, E. S. 2002; 99 (6): 3362-3364

  View details for DOI 10.1073/pnas.072075899

  View details for Web of Science ID 000174511000003

  View details for PubMedID 11904398

  View details for PubMedCentralID PMC122527

• Fatal attraction: Cytomegalovirus-encoded chemokine homologs VIRAL PROTEINS COUNTERACTING HOST DEFENSES Saederup, N., Mocarski, E. S. 2002; 269: 235-256

  Abstract

  Members of the cytomegalovirus (CMV) subfamily of betaherpesviruses infecting primates and rodents encode divergent proteins with sequence characteristics and activities of chemokines, a class of small, secreted proteins that control leukocyte migration and trafficking behavior. Human CMV genes UL146 and UL147 encode proteins with sequence characteristics of CXC chemokines, whereas, murine CMV encodes a CC chemokine homolog (MCK-2). Human CMV UL146 encodes a neutrophil-attracting chemokine denoted viral CXC chemokine-1 (vCXCL1) that is as potent as host IL-8 and functions via the CXCR2 receptor, one of two human IL-8 receptors. Murine CMV MCK-2 is composed of a chemokine domain derived from open reading frame (ORF) m131 (and denoted MCK-1) as well as a domain derived from m129 that does not have sequence similarity to any known class of proteins. A synthetic version of murine CMV m131 (MCK-1) protein carries out many of the activities of a positive-acting chemokine, including transient release of intracellular calcium stores and cell adhesion of peritoneal macrophage populations. In the context of the viral genome and infection of the mouse host, the m131-m129 (MCK-2) gene product confers increased inflammation, higher levels of viremia, and higher titers of virus in salivary glands, consistent with a role in promoting dissemination by attracting an important mononuclear leukocyte population. Other characterized primate CMVs, but not other primate betaherpesviruses, encode gene products similar to human UL146 and UL147. Other characterized rodent CMVs encode a gene product similar to the murine CMV chemokine homolog, although not as a spliced gene product. Thus chemokines, like viral proteins that downmodulate MHC class I expression or have sequence homology to host MHC class I proteins, have evolved in primate and rodent CMVs to carry out an analogous set of immunomodulatory functions during infection of the host even though they arise from distinct origins.

  View details for Web of Science ID 000179605900014

  View details for PubMedID 12224512

• The common lymphoid progenitor protects against lethal MCMV infection in a murine model of matched unrelated hematopoietic stem cell transplantation. Arber, C., Bitmansour, A., Mocarski, E. S., Shizuru, J. A., Brown, J. M. AMER SOC HEMATOLOGY. 2001: 476A–476A

  View details for Web of Science ID 000172134101992

• Multicenter evaluation of PCR methods for detecting CMV DNA in blood donors TRANSFUSION Roback, J. D., Hillyer, C. D., Drew, W. L., Laycock, M. E., Luka, J., Mocarski, E. S., Slobedman, B., Smith, J. W., Soderberg-Naucler, C., Todd, D. S., Woxenius, S., Busch, M. P. 2001; 41 (10): 1249-1257

  Abstract

  CMV DNA screening may be a useful adjunct to serologic tests in distinguishing potentially infectious blood donations from those that are "CMV-safe." However, there is currently no consensus on the optimal assay method for accurate detection of CMV DNA in donors.A blinded multicenter evaluation of seven CMV PCR assays was performed by five laboratories by using coded sets of analytical controls and donor blood samples.Five assays displayed sufficient sensitivity for donor screening, as judged by consistent detection of a minimum of 25 CMV genome equivalents (geq) in analytical controls constructed to contain from 1 to 100 CMV geq in background DNA from 250,000 cells, while the other two assays displayed inadequate sensitivity. Three sensitive assays, two based on nested PCR directed at the UL93 and UL32 regions of the CMV genome and another test (Monitor Assay, Roche), did not detect CMV DNA in samples from any of 20 pedigreed CMV-seronegative, Western blot-negative (S-/WB-) donors. Two other assays based on nested PCR occasionally detected CMV DNA in S-WB- samples, and one sensitive nested PCR assay directed at UL123 detected CMV DNA in a large proportion (85%) of S-WB- samples.Seven CMV PCR assays currently used for research and/or diagnostic applications displayed marked variations in sensitivity, specificity, and reproducibility when applied to coded analytical and clinical control samples containing cellular DNA from the equivalent of 250,000 WBCs. These results will be useful in the selection of assays with performance characteristics appropriate to donor screening objectives. They may also help explain discrepant findings from previous studies that used PCR to determine CMV DNA prevalence in seronegative and seropositive blood donors.

  View details for Web of Science ID 000171789600015

  View details for PubMedID 11606824

• Murine cytomegalovirus CC chemokine homolog MCK-2 (m131-129) is a determinant of dissemination that increases inflammation at initial sites of infection JOURNAL OF VIROLOGY Saederup, N., Aguirre, S. A., Sparer, T. E., Bouley, D. M., Mocarski, E. S. 2001; 75 (20): 9966-9976

  Abstract

  The murine cytomegalovirus CC chemokine homolog MCK-2 (m131-129) is an important determinant of dissemination during primary infection. Reduced peak levels of viremia at day 5 were followed by reduced levels of virus in salivary glands starting at day 7 when mck insertion (RM461) and point (RM4511) mutants were compared to mck-expressing viruses. A dramatic MCK-2-enhanced inflammation occurred at the inoculation site over the first few days of infection, preceding viremia. The data further reinforce the role of MCK-2 as a proinflammatory signal that recruits leukocytes to increase the efficiency of viral dissemination in the host.

  View details for Web of Science ID 000171102900043

  View details for PubMedID 11559829

• Requirement for uracil-DNA glycosylase during the transition to late-phase cytomegalovirus DNA replication JOURNAL OF VIROLOGY Courcelle, C. T., Courcelle, J., Prichard, M. N., Mocarski, E. S. 2001; 75 (16): 7592-7601

  Abstract

  Cytomegalovirus gene UL114, a homolog of mammalian uracil-DNA glycosylase (UNG), is required for efficient viral DNA replication. In quiescent fibroblasts, UNG mutant virus replication is delayed for 48 h and follows the virus-induced expression of cellular UNG. In contrast, mutant virus replication proceeds without delay in actively growing fibroblasts that express host cell UNG. In the absence of viral or host cell UNG expression, mutant virus fails to proceed to late-phase DNA replication, characterized by rapid DNA amplification. The data suggest that uracil incorporated early during wild-type viral DNA replication must be removed by virus or host UNG prior to late-phase amplification and encapsidation into progeny virions. The process of uracil incorporation and excision may introduce strand breaks to facilitate the transition from early-phase replication to late-phase amplification.

  View details for Web of Science ID 000170093000037

  View details for PubMedID 11462031

  View details for PubMedCentralID PMC114994

• A cytomegalovirus-encoded inhibitor of apoptosis that suppresses caspase-8 activation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Skaletskaya, A., Bartle, L. M., Chittenden, T., McCormick, A. L., Mocarski, E. S., Goldmacher, V. S. 2001; 98 (14): 7829-7834

  Abstract

  We have identified a human cytomegalovirus cell-death suppressor, denoted vICA, encoded by the viral UL36 gene. vICA inhibits Fas-mediated apoptosis by binding to the pro-domain of caspase-8 and preventing its activation. vICA does not share significant sequence homology with FLIPs or other known suppressors of apoptosis, suggesting that this protein represents a new class of cell-death suppressors. Notably, resistance to Fas-mediated apoptosis is delayed in fibroblasts infected with viruses that encode mutant vICA, suggesting that vICA suppresses death-receptor-induced cell death in the context of viral infection. Although vICA is dispensable for viral replication in vitro, the common targeting of caspase-8 activation by diverse herpesviruses argues for an important role for this antiapoptotic mechanism in the pathogenesis of viral infection in the host, most likely in avoiding immune clearance by cytotoxic lymphocytes and natural killer cells.

  View details for Web of Science ID 000169744200035

  View details for PubMedID 11427719

  View details for PubMedCentralID PMC35427

• Distinct and separate roles for herpesvirus-conserved UL97 kinase in cytomegalovirus DNA synthesis and encapsidation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wolf, D. G., Courcelle, C. T., Prichard, M. N., Mocarski, E. S. 2001; 98 (4): 1895-1900

  Abstract

  The human cytomegalovirus UL97 kinase, an important target of antiviral therapy, has an impact on at least two distinct phases of viral replication. Compared with wild-type virus, the UL97 deletion mutant exhibits an early replication defect that reduces DNA accumulation by 4- to 6-fold, as well as a late capsid maturation defect responsible for most of the observed 100- to 1000-fold reduction in replication. Block-release experiments with the antiviral 2-bromo-5,6-dichloro-1-(beta-D-ribofuranosyl)-benzimidazole revealed an important role for UL97 kinase in capsid assembly. Although cleavage of concatemeric DNA intermediates to unit-length genomes remained unaffected, progeny mutant virus maturation was delayed, with accumulation of progeny at significantly reduced levels compared with wild type after release of this block. Transmission electron microscopy confirmed the aberrant accumulation of empty A-like capsids containing neither viral DNA nor an internal scaffold structure, consistent with a failure to stably package DNA in mutant virus-infected cells. The function of UL97 in DNA synthesis as well as capsid assembly suggests that protein phosphorylation mediated by this herpesvirus-conserved kinase increases the efficiency of these two distinct phases of virus replication.

  View details for Web of Science ID 000166949200104

  View details for PubMedID 11172047

  View details for PubMedCentralID PMC29353

• Cytomegalovirus requires uracil-DNA glycosylase activity for transition to late phase viral DNA replication J. Virol E.S. Mocarski, Courcelle, C.T., J. Courcelle, M.N. Prichard 2001
• Human cytomegalovirus latency-associated protein pORF94 is dispensable for productive and latent infection JOURNAL OF VIROLOGY White, K. L., Slobedman, B., Mocarski, E. S. 2000; 74 (19): 9333-9337

  Abstract

  Human cytomegalovirus latency in bone marrow-derived myeloid progenitors is characterized by the presence of latency-associated transcripts encoded in the ie1/ie2 region of the viral genome. To assess the role of ORF94 (UL126a), a conserved open reading frame on these transcripts, a recombinant virus (RC2710) unable to express this gene was constructed. This virus replicated at wild-type levels and expressed productive as well as latency-associated ie1/ie2 region transcripts. During latency in granulocyte-macrophage progenitors, RC2710 DNA was detected at levels indistinguishable from wild-type virus, latent-phase transcription was present, and RC2710 reactivated when latently infected cells were cocultured with permissive fibroblasts. These data suggest pORF94 is not required for either productive or latent infection as assayed in cultured cells despite being the only known nuclear latency-associated protein.

  View details for Web of Science ID 000089244300064

  View details for PubMedID 10982383

  View details for PubMedCentralID PMC102135

• Humoral immune response to proteins of human cytomegalovirus latency-associated transcripts BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Landini, M. P., Lazzarotto, T., Xu, J., Geballe, A. P., Mocarski, E. S. 2000; 6 (2): 100-108

  Abstract

  Latent human cytomegalovirus (CMV) infection of hematopoietic progenitor cells is associated with the presence of latency-associated transcripts that may express 6 proteins larger than 44 amino acids in size (open reading frame [ORF] 55, ORF45, ORF94, ORF59, ORF154, ORF152/UL124). The serologic response to these proteins was evaluated in healthy seropositive individuals as well as in individuals undergoing active CMV infection. Individual recombinant GST-fusion proteins, prepared from bacteria, were found by enzyme-linked immunosorbent assay to be recognized by between 8% and 44% long-term healthy seropositive individuals, with ORF94 and ORF55 being the most broadly and significantly recognized. Although nearly all of serum samples (85%) recognized at least 1 of these proteins, none reacted with all 6. Patterns of antibody prevalence to these proteins in long-term seropositive individuals were similar to many antigens expressed during productive replication (IE1, ppUL57, ppUL83/pp65), but none were broadly detected by a majority of individuals, a characteristic of only a few productive-phase antigens, including ppUL44/ICP36 and ppUL32/pp150. Consistent with prevalence in long-term seropositive individuals, commercial preparations of pooled human gamma globulin were also found to recognize latency-associated proteins. Serologic reactivity to latency-associated proteins was slow to develop following primary infection, in a pattern distinct from any of the characterized replication-phase proteins tested here, and was boosted late after secondary infection or reactivation in solid-organ transplant recipients without showing a correlation with viremia or disease. These results provide evidence that proteins expressed from the latent region during natural infection exhibit immunogenicity comparable with most other characterized viral antigens, although the narrow response to individual latency-associated proteins likely precludes their use in serologic assays to investigate clinical correlates or outcome in transplant recipients.

  View details for Web of Science ID 000090022100006

  View details for PubMedID 10741618

• Cytomegalovirus cell tropism, replication, and gene transfer in brain JOURNAL OF NEUROSCIENCE van den Pol, A. N., Mocarski, E., Saederup, N., VIEIRA, J., Meier, T. J. 1999; 19 (24): 10948-10965

  Abstract

  Cytomegalovirus (CMV) infects a majority of adult humans. During early development and in the immunocompromised adult, CMV causes neurological deficits. We used recombinant murine cytomegalovirus (mCMV) expressing either green fluorescent protein (GFP) or beta-galactosidase under control of human elongation factor 1 promoter or CMV immediate early-1 promoter as reporter genes for infected brain cells. In vivo and in vitro studies revealed that neurons and glial cells supported strong reporter gene expression after CMV exposure. Brain cultures selectively enriched in either glia or neurons supported viral replication, leading to process degeneration and cell death within 2 d of viral exposure. In addition, endothelial cells, tanycytes, radial glia, ependymal cells, microglia, and cells from the meninges and choroid were infected. Although mCMV showed no absolute brain cell preference, relative cell preferences were detected. Radial glia cells play an important role in guiding migrating neurons; these were viral targets in the developing brain, suggesting that cortical problems including microgyria that are a consequence of CMV may be caused by compromised radial glia. Although CMV is a species-specific virus, recombinant mCMV entered and expressed reporter genes in both rat and human brain cells, suggesting that mCMV might serve as a vector for gene transfer into brain cells of non-murine species. GFP expression was sufficiently strong that long axons, dendrites, and their associated spines were readily detected in both living and fixed tissue, indicating that mCMV reporter gene constructs may be useful for labeling neurons and their pathways.

  View details for Web of Science ID 000084333100040

  View details for PubMedID 10594076

• A cytomegalavirus-encoded mitochondria-localized inhibitor of apoptosis structurally unrelated to Bcl-2 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Goldmacher, V. S., Bartle, L. M., Skaletskaya, A., Dionne, C. A., Kedersha, N. L., Vater, C. A., Han, J. W., Lutz, R. J., Watanabe, S., McFarland, E. D., KIEFF, E. D., Mocarski, E. S., Chittenden, T. 1999; 96 (22): 12536-12541

  Abstract

  Human cytomegalovirus (CMV), a herpesvirus that causes congenital disease and opportunistic infections in immunocompromised individuals, encodes functions that facilitate efficient viral propagation by altering host cell behavior. Here we show that CMV blocks apoptosis mediated by death receptors and encodes a mitochondria-localized inhibitor of apoptosis, denoted vMIA, capable of suppressing apoptosis induced by diverse stimuli. vMIA, a product of the viral UL37 gene, inhibits Fas-mediated apoptosis at a point downstream of caspase-8 activation and Bid cleavage but upstream of cytochrome c release, while residing in mitochondria and associating with adenine nucleotide translocator. These functional properties resemble those ascribed to Bcl-2; however, the absence of sequence similarity to Bcl-2 or any other known cell death suppressors suggests that vMIA defines a previously undescribed class of anti-apoptotic proteins.

  View details for Web of Science ID 000083373000062

  View details for PubMedID 10535957

  View details for PubMedCentralID PMC22976

• Cytomegalovirus-encoded beta chemokine promotes monocyte-associated viremia in the host PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Saederup, N., Lin, Y. C., Dairaghi, D. J., Schall, T. J., Mocarski, E. S. 1999; 96 (19): 10881-10886

  Abstract

  Chemokine homologs are encoded by many large DNA viruses, suggesting that they contribute to control of host leukocyte transmigration and trafficking during viral infection. Murine cytomegalovirus carries a CC (beta) chemokine homolog gene giving rise to two related proteins, murine cytomegalovirus chemokine 1 and 2 (MCK-1 and MCK-2). MCK-1 peptide was found to induce calcium signaling and adherence in murine peritoneal macrophages. Cells bearing human chemokine receptor CCR3 and the human macrophage THP1 cell line were responsive to MCK-1. This pattern suggested that MCK-1 might act as an agonist, promoting leukocyte trafficking during viral infection. Consistent with this prediction, MCK-1/MCK-2 mutant viruses exhibit dramatically reduced peak levels of monocyte-associated viremia in experimentally infected mice. Thus, MCK-1/MCK-2 appears to promote host leukocyte migration to initial sites of infection and may be responsible for attracting monocytes or macrophages that efficiently disseminate virus in the host.

  View details for Web of Science ID 000082574100066

  View details for PubMedID 10485920

  View details for PubMedCentralID PMC17977

• Cytomegalovirus encodes a potent alpha chemokine PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Penfold, M. E., Dairaghi, D. J., Duke, G. M., Saederup, N., Mocarski, E. S., Kemble, G. W., Schall, T. J. 1999; 96 (17): 9839-9844

  Abstract

  Cytomegalovirus is a widespread opportunistic pathogen affecting immunocompromised individuals in whom neutrophils may mediate virus dissemination and contribute to progression of disease. Recent sequence analysis suggests that genes absent or altered in attenuated strains may influence pathogenesis. We have found two genes, UL146 and UL147, whose products have sequence similarity to alpha (CXC) chemokines. UL146 encodes a protein, designated vCXC-1, that is a 117-aa glycoprotein secreted into the culture medium as a late gene product, where its presence correlates with the ability to attract human neutrophils. Recombinant vCXC-1 is a fully functional chemokine, inducing calcium mobilization, chemotaxis, and degranulation of neutrophils. High-affinity vCXC-1 binding is shown to be mediated via CXCR2, but not CXCR1. vCXC-1 exhibits a potency approaching that of human IL-8. As the first example of a virus-encoded alpha chemokine, vCXC-1 may ensure the active recruitment of neutrophils during cytomegalovirus infection, thereby providing for efficient dissemination during acute infection and accounting for the prominence of this leukocyte subset in cytomegalovirus disease.

  View details for Web of Science ID 000082098500072

  View details for PubMedID 10449781

  View details for PubMedCentralID PMC22297

• Tetracycline-mediated regulation of gene expression within the human cytomegalovirus genome VIROLOGY McVoy, M. A., Mocarski, E. S. 1999; 258 (2): 295-303

  Abstract

  To evaluate the utility of tetracycline gene regulation in the study of human cytomegalovirus gene functions, expression of luciferase under the control of tetracycline-regulatable promoters was studied following transient plasmid transfections and from within recombinant human cytomegalovirus genomes. The tetracycline-regulatable promoter PhCMV*-1 contains sequences from the human cytomegalovirus ie1/ie2 promoter and seven upstream tet operator sites which bind the activator protein tTA only in the absence of tetracycline (Gossen and Bujard (1992). Proc. Natl. Acad. Sci. USA 89, 5547-5551). Two modifications of PhCMV*-1 were also studied: P1129, in which the tet operator sites were reduced from seven to one; and P1125, in which human cytomegalovirus sequences were replaced by adenovirus major late promoter and terminal deoxynucleotidyltransferase initiator sequences. In transient assays, PhCMV*-1 and P1125 exhibited modest differential regulation but were strongly activated by viral infection. P1129 exhibited less viral activation and narrower regulation. In the viral genome, PhCMV*-1 exhibited regulation up to 7-fold during late times of infection, whereas P1125 displayed nearly 100-fold regulation. Regulation of P1125 was fully reversed within 12 to 24 h of adding or removing tetracycline. These results suggest that P1125 may provide sufficient conditional expression to effectively regulate human cytomegalovirus late genes.

  View details for Web of Science ID 000080908300010

  View details for PubMedID 10366566

• Quantitative analysis of latent human cytomegalovirus JOURNAL OF VIROLOGY Slobedman, B., Mocarski, E. S. 1999; 73 (6): 4806-4812

  Abstract

  Cytomegalovirus latency depends on an interaction with hematopoietic cells in bone marrow and peripheral blood. The distribution of viral DNA was investigated by PCR-driven in situ hybridization (PCR-ISH), and the number of viral genomes per cell was estimated by quantitative competitive PCR during both experimental and natural latent infection. During experimental latent infection of cultured granulocyte-macrophage progenitors, the viral genome was detected in >90% of cells at a copy number of 1 to 8 viral genomes per cell. During natural infection, viral genomes were detected in 0.004 to 0.01% of mononuclear cells from granulocyte colony-stimulating factor-mobilized peripheral blood or bone marrow from seropositive donors, at a copy number of 2 to 13 genomes per infected cell. When evaluated by reverse transcription-PCR-ISH, only a small proportion of experimentally infected cells (approximately 2%) had detectable latent transcripts. This investigation identifies the small percentage of bone marrow-derived mononuclear cells that become latently infected during natural infection and suggests that latency may proceed in some cells that fail to encode currently identified latent transcripts.

  View details for Web of Science ID 000080271400032

  View details for PubMedID 10233941

  View details for PubMedCentralID PMC112523

• Analysis of host viral interactions in CMV infection using DNA microarrays. Barry, C. T., Watanabe, S., Mocarski, E. S., Brown, P. O. LIPPINCOTT WILLIAMS & WILKINS. 1999: S611–S611

  View details for Web of Science ID 000080624100295

• Peripheral blood CD14(+) cells from healthy subjects carry a circular conformation of latent cytomegalovirus genome BLOOD Bolovan-Fritts, C. A., Mocarski, E. S., Wiedeman, J. A. 1999; 93 (1): 394-398

  Abstract

  The majority of the human population harbors latent cytomegalovirus. Although CD14(+) peripheral blood mononuclear cells have been implicated as sites of latency, the conformation of the latent viral genome in these cells is unknown. In this study, the conformation of viral genomic DNA was assessed in CD14(+) cells from healthy virus seropositive carriers using an electrophoretic separation on native agarose gels in combination with polymerase chain reaction detection. Here we show that the viral genome migrates as a circular plasmid with a mobility equivalent to a circular 230-kb Shigella flexneri megaplasmid marker. Neither linear nor complex or integrated forms of the viral genome were detected. This report provides further evidence that the CD14(+) cell population is an important site of viral latency in the naturally infected human host. Detection of the viral genome as a circular plasmid during latency suggests that this virus maintains its genome in a manner analogous to other herpesviruses where latent viral genome conformation has been studied.

  View details for Web of Science ID 000077809900047

  View details for PubMedID 9864186

• Modulation of natural killer cell cytotoxicity in human cytomegalovirus infection: The role of endogenous class I major histocompatibility complex and a viral class I homolog JOURNAL OF EXPERIMENTAL MEDICINE Leong, C. C., Chapman, T. L., BJORKMAN, P. J., Formankova, D., Mocarski, E. S., Phillips, J. H., Lanier, L. L. 1998; 187 (10): 1681-1687

  Abstract

  Natural killer (NK) cells have been implicated in early immune responses against certain viruses, including cytomegalovirus (CMV). CMV causes downregulation of class I major histocompatibility complex (MHC) expression in infected cells; however, it has been proposed that a class I MHC homolog encoded by CMV, UL18, may act as a surrogate ligand to prevent NK cell lysis of CMV-infected cells. In this study, we examined the role of UL18 in NK cell recognition and lysis using fibroblasts infected with either wild-type or UL18 knockout CMV virus, and by using cell lines transfected with the UL18 gene. In both systems, the expression of UL18 resulted in the enhanced killing of target cells. We also show that the enhanced killing is due to both UL18-dependent and -independent mechanisms, and that the killer cell inhibitory receptors (KIRs) and CD94/NKG2A inhibitory receptors for MHC class I do not play a role in affecting susceptibility of CMV-infected fibroblasts to NK cell-mediated cytotoxicity.

  View details for Web of Science ID 000073765600014

  View details for PubMedID 9584146

  View details for PubMedCentralID PMC2212284

• Enhancement of obliterative airway disease in rat tracheal allografts infected with recombinant rat cytomegalovirus JOURNAL OF HEART AND LUNG TRANSPLANTATION Reichenspurner, H., Soni, V., Nitschke, M., Berry, G. J., Brazelton, T., Shorthouse, R., Huang, X. F., Boname, J., Girgis, R., Raitz, B. A., Mocarski, E., SANDFORD, G., Morris, R. E. 1998; 17 (5): 439-451

  Abstract

  Cytomegalovirus infection has been identified as a significant risk factor for the development of obliterative bronchiolitis in human lung transplant recipients. This study was designed to assess the influence of rat cytomegalovirus (RCMV) on the pathogenesis and development of obliterative bronchiolitis in an experimental model of obliterative airway disease, which occurs after allogenic heterotopic tracheal transplantation in rodents.Sixty Lewis rats were infected intraperitoneally with 10(7) plaque-forming units of recombinant lac-Z-tagged RCMV expressing the gene for beta-galactosidase. Rats were either infected at the time of surgery (acute infection, n = 30) or 56 days before surgery (chronic infection, n = 30). Tracheae from Brown Norway (allograft) or Lewis (isograft) rats were implanted and wrapped in the greater omentum of infected Lewis rats. RCMV infection was verified in different recipient tissues by in vitro plaque-assays and by direct in situ staining for beta-galactosidase activity. The tracheal grafts were harvested on days 7, 14, and 21 after transplantation and stained with hematoxylin-eosin and Masson's trichrome. The peritracheal cellular inflammation was scored visually. The cellular density of the infiltrating cells and the extent of airway obliteration were analyzed by use of computer-digitized morphometry and compared with uninfected allografts as control.Both acute and chronic cytomegalovirus infection produced significantly higher mononuclear cell density values on days 7 and 14 compared with noninfected controls, indicating a more intense immune response in the infected allografts. Tracheal allograft obliteration was also more extensive after acute and, in particular, after chronic cytomegalovirus infection (64% narrowing after 21 days compared with 36% in grafts from noninfected control animals).Our experimental results provide direct evidence that the tracheal grafts were infected with RCMV and that the development of obliterative airway disease was enhanced in the acutely and chronically infected allografts compared with grafts from noninfected control animals.

  View details for Web of Science ID 000073855800001

  View details for PubMedID 9628562

• Cytomegalovirus remains latent in a common precursor of dendritic and myeloid cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Hahn, G., Jores, R., Mocarski, E. S. 1998; 95 (7): 3937-3942

  Abstract

  Hematopoietic cells and their progenitors play important roles in human cytomegalovirus latency and reactivation. Latent infection has been evaluated in defined populations of myeloid-lineage-committed progenitor cells coexpressing CD33 and CD15 or CD33 and CD14 along with the dendritic cell markers CD1a and CD10. These CD33+ cell populations were found to support latency and expression of viral latency-associated transcripts and to undergo reactivation of productive viral replication when differentiated in the presence of human fibroblasts. Reactivation was also observed when myeloid cells were carried in the presence of fibroblast-conditioned medium or medium supplemented with certain cytokines (interferon gamma, tumor necrosis factor alpha, interleukin 4, or granulocyte-macrophage colony-simulating factor), suggesting that cell differentiation pathways act as determinants of reactivation. More primitive CD34+ hematopoietic cells were also found to be susceptible to viral infection and latency was maintained as these cells differentiated into CD33+-lineage-committed populations. Between 0.01% and 0.001% of CD33+ CD14+ or CD33+ CD15+ bone marrow mononuclear cells isolated from naturally infected individuals were found to express latent transcripts. Thus, cytomegalovirus is carried within a small percentage of myeloid and dendritic cell progenitors in the healthy seropositive host. Virus reactivation may be triggered by factors associated with the inflammatory response.

  View details for Web of Science ID 000072848500111

  View details for PubMedID 9520471

  View details for PubMedCentralID PMC19941

• Defective growth correlates with reduced accumulation of a viral DNA replication protein after low-multiplicity infection by a human cytomegalovirus ie1 mutant JOURNAL OF VIROLOGY Greaves, R. F., Mocarski, E. S. 1998; 72 (1): 366-379

  Abstract

  To investigate the importance of the IE1 p72 regulatory protein during human cytomegalovirus replication, a recombinant virus unable to synthesize IE1 p72 was constructed. The Towne strain mutant CR208 lacked exon 4 of the major immediate-early gene and was isolated and complemented in an IE1-expressing immortalized human fibroblast line (ihfie1.3). Replication of CR208 in primary human fibroblasts was completed after an input multiplicity of 10 PFU/cell but was severely-impaired at 0.1 PFU/cell. CR208 formed plaques with lower efficiency on primary fibroblasts than on ihfiel.3 cells, and the relationship between the CR208 inoculum size and the resulting number of undersized plaques was nonlinear, indicating that multiple particles of CR208 were required to initiate lytic replication in a single primary fibroblast. After infection of primary fibroblasts with CR208 at 5 PFU/cell, a normal pattern of viral antigens was detected, although IE1 p72 was absent. During lower-multiplicity infections, IE2 protein was consistently detected at similar levels in a similar proportion of CR208-infected cells relative to the case for a Towne infection, but many fewer CR208-infected cells contained the ppUL44 polymerase accessory protein when evaluated at 24 or 48 h after infection. Furthermore, fibroblasts infected with CR208 at a low multiplicity failed to form viral DNA replication compartments, despite having expressed IE2 p86. These low-multiplicity growth and expression defects were corrected in two rescued derivatives of CR208 able to synthesize IE1 p72. One rescued virus (CR249) carried a deletion removing the large intron between exons 1 and 2 of the ie1-ie2 locus, revealing that this intron was dispensable for growth in cell culture.

  View details for Web of Science ID A1998YL01000042

  View details for PubMedID 9420235

  View details for PubMedCentralID PMC109384

• Sequences within the herpesvirus-conserved pac1 and pac2 motifs are required for cleavage and packaging of the murine cytomegalovirus genome JOURNAL OF VIROLOGY McVoy, M. A., Nixon, D. E., Adler, S. P., Mocarski, E. S. 1998; 72 (1): 48-56

  Abstract

  The DNA sequence motifs pac1 [an A-rich region flanked by poly(C) runs] and pac2 (CGCGGCG near an A-rich region) are conserved near herpesvirus genomic termini and are believed to mediate cleavage of genomes from replicative concatemers. To determine their importance in the cleavage process, we constructed a number of recombinant murine cytomegaloviruses with a second cleavage site inserted at an ectopic location within the viral genome. Cleavage at a wild-type ectopic site occurred as frequently as at the natural cleavage site, whereas mutation of this ectopic site revealed that some of the conserved motifs of pac1 and pac2 were essential for cleavage whereas others were not. Within pac1, the left poly(C) region was very important for cleavage and packaging but the A-rich region was not. Within pac2, the A-rich region and adjacent sequences were essential for cleavage and packaging and the CGCGGCG region contributed to, but was not strictly essential for, efficient cleavage and packaging. A second A-rich region was not important at all. Furthermore, mutations that prevented cleavage also blocked duplication and deletion of the murine cytomegalovirus 30-bp terminal repeat at the ectopic site, suggesting that repeat duplication and deletion are consequences of cleavage. Given that the processes of genome cleavage and packaging appear to be highly conserved among herpesviruses, these findings should be relevant to other members of this family.

  View details for Web of Science ID A1998YL01000006

  View details for PubMedID 9420199

  View details for PubMedCentralID PMC109348

• Formation of cytomegalovirus DNA replication compartments defined by localization of viral proteins and DNA synthesis VIROLOGY Penfold, M. E., Mocarski, E. S. 1997; 239 (1): 46-61

  Abstract

  To characterize the formation of replication compartments in human cytomegalovirus-infected cells, and to determine the fate of newly synthesized DNA, we localized viral replication proteins and DNA synthesis at early and late times during infection. As expected, ppUL57 (single-stranded DNA binding protein) and ppUL44 (DNA polymerase processivity factor) both localized to replication compartments beginning at 48 hpi. BrdU was incorporated into viral DNA in these compartments that was found to mature into progeny virus based on our ability to chase the label into the cytoplasm and out of the cell over the ensuing 72-h period. Although the pattern of BrdU incorporation at early times (20 or 24 hpi) was punctate, and distinct from the replication compartment that formed later during infection, viral DNA synthesized at this time also matured into progeny virus during a chase. Interestingly, sites of ppUL57 localization did not overlap completely with sites of BrdU incorporation at early times. Products from the UL112-113 gene localized to subnuclear regions by 6 hpi, earlier than ppUL57. Between 12 and 24 hpi, both ppUL57 and ppUL44 joined UL112-113 gene products at sites that subsequently developed into replication compartments. When infection was carried out in the presence of phosphonoformate or ganciclovir, replication compartment formation was blocked. A viral mutant deficient in uracil DNA glycosidase, previously shown to exhibit a delay in the initial phase of DNA replication, also exhibited delayed formation of replication compartments. These results raise the possibility that subnuclear sites defined by UL112-113 localization orchestrate the assembly of the CMV replication compartment and implicate punctate sites of BrdU incorporation as sites of early viral DNA replication that precedes the formation of the replication compartment.

  View details for Web of Science ID 000071139000005

  View details for PubMedID 9426445

• Reassessing the organization of the UL42-UL43 region of the human cytomegalovirus strain AD169 genome VIROLOGY Mocarski, E. S., Prichard, M. N., TAN, C. S., Brown, J. M. 1997; 239 (1): 169-175

  Abstract

  A polymorphism in the UL42-UL43 region of the human cytomegalovirus genome has been characterized by nucleotide sequence analysis, revealing a 929-bp insertion following nt 54,612 relative to the published strain AD169-UK genome sequence (M.S. Chee et al., 1990, Curr. Top. Microbiol Immunol. 154, 125-170). Although AD169-UK exhibited polymorphism in this genomic region, other CMV strains (Towne, Toledo, and AD169-ATCC) carried only the newly characterized longer form. The additional sequence altered the assignment of UL42 and UL43 open reading frames. UL42 decreased in size from 157 to 125 codons, retaining 76 of the previously reported carboxyl terminal codons, and UL43 increased in size from 187 to 423 codons, retaining 185 of the previously reported amino terminal codons. This additional sequence makes UL43 a more conserved betaherpesvirus US22 family member. Only AD169-UK exhibited restriction fragment length polymorphism in this region, suggesting that a deletion occurred during the propagation of this strain in cell culture. The additional sequence should be considered a bona fide part of the cytomegalovirus genome and the AD169 genome size should be corrected to 230,283 bp.

  View details for Web of Science ID 000071139000016

  View details for PubMedID 9426456

• The location and sequence composition of the murine cytomegalovirus replicator (oriLyt) VIROLOGY MASSE, M. J., Messerle, M., Mocarski, E. S. 1997; 230 (2): 350-360

  Abstract

  DNA replication during human or simian cytomegalovirus (CMV) infection has been shown to be under control of a replicator region referred to as oriLyt. The murine CMV oriLyt has been mapped to a region of the genome located upstream of the gene encoding the herpesvirus-conserved single-stranded DNA binding protein, analogous to human and simian CMV oriLyts. A minimal oriLyt of approximately 1.7 kbp has been identified using a transient replication system. Like occurs with human and simian CMV counterparts, addition of flanking sequences to this minimal origin-stimulated replication efficiency. Analysis of the DNA sequence in this region shows that murine CMV oriLyt is complex and exhibits an asymmetric distribution of nucleotides as well as many repeat sequence elements, including distinct AT- and GC-rich regions and region with arrays of closely spaced direct repeats. Despite similarities in organization of all three CMV oriLyts, no sequence identity and only limited DNA sequence similarity was detectable. Consistent with this sequence divergence, the human and murine CMV oriLyts were unable to substitute for one another in transient replication assays.

  View details for Web of Science ID A1997WV20400022

  View details for PubMedID 9143291

• Human cytomegalovirus infection inhibits G(1)/S transition JOURNAL OF VIROLOGY Dittmer, D., Mocarski, E. S. 1997; 71 (2): 1629-1634

  Abstract

  Cell cycle progression during cytomegalovirus infection was investigated by fluorescence-activated cell sorter (FACS) analysis of the DNA content in growth-arrested as well as serum-stimulated human fibroblasts. Virus-infected cells maintained in either low (0.2%) or high (10%) serum failed to progress into S phase and failed to divide. DNA content analysis in the presence of G1/S (hydroxyurea and mimosine) and G2/M (nocodazole and colcemid) inhibitors demonstrated that upon virus infection of quiescent (G0) cells, the cell cycle did not progress beyond the G1/S border even after serum stimulation. Proteins which normally indicate G1/S transition (proliferating cell nuclear antigen [PCNA]) or G2/M transition (cyclin B1) were elevated by virus infection. PCNA levels were induced in infected cells and exhibited a punctate pattern of nuclear staining instead of the diffuse pattern observed in mock-infected cells. Cyclin B1 was induced in infected cells which exhibited a G1/S DNA content by FACS analysis, suggesting that expression of this key cell cycle function was dramatically altered by viral functions. These data demonstrate that contrary to expectations, cytomegalovirus inhibits normal cell cycle progression. The host cell is blocked prior to S phase to provide a favorable environment for viral replication.

  View details for Web of Science ID A1997WC30500090

  View details for PubMedID 8995690

  View details for PubMedCentralID PMC191221

• The synthesis and immunogenicity of varicella-zoster virus glycoprotein E and immediate-early protein (IE62) expressed in recombinant herpes simplex virus-1 ANTIVIRAL RESEARCH Lowry, P. W., Koropchak, C. M., Choi, C. Y., Mocarski, E. S., Kern, E. R., Kinchington, P. R., Arvin, A. M. 1997; 33 (3): 187-200

  Abstract

  In order to evaluate the conditions for optimal expression and immunogenicity of varicella-zoster virus (VZV) proteins in a herpes simplex virus-1 (HSV-1) vector, we selected the VZV glycoprotein E (gE), encoded by ORF 68 and the VZV product of ORF 62, an immediate-early major tegument protein (IE62). Three HSV/VZV recombinants were generated: (1) VZV gE protein coding sequences along with the promoter region were inserted into the thymidine kinase (TK) gene of HSV-1 strain KOS; (2) VZV gE expressed from the HSV-1 ICP4 promoter was inserted into the glycoprotein C (gC) gene of HSV-1 strain F; and (3) VZV IE62 protein coding sequences under the control of the HSV-1 ICP4 promoter were inserted into the gC gene of HSV-1 strain F. Immunoblot analysis and immunoperoxidase staining of infected cell monolayers demonstrated vector expression of VZV proteins. Following intracranial inoculation in mice, both VZV gE-HSV (TK) and VZV IE62-HSV (gC) induced an IgG response against VZV gE or VZV IE62. When tested in cytotoxicity assays using T-lymphocytes from VZV immune human donors, the range of precursor frequencies for T-lymphocytes that recognized VZV gE or VZV IE62 was similar whether these proteins were expressed by HSV-1 or a vaccinia vector. These experiments demonstrate that HSV-1 is a competent vector for expression of these VZV proteins and support the feasibility of engineering a combined vaccine for these closely related alpha-herpesviruses.

  View details for Web of Science ID A1997WH07400005

  View details for PubMedID 9037375

• Propagating Kaposi's sarcoma-associated herpesvirus NEW ENGLAND JOURNAL OF MEDICINE Mocarski, E. S. 1997; 336 (3): 214-215

  View details for Web of Science ID A1997WC26600010

  View details for PubMedID 8988903

• A deletion mutant in the human cytomegalovirus gene encoding IE1(491aa) is replication defective due to a failure in autoregulation Colloquium on Genetic Engineering of Viruses and of Virus Vectors Mocarski, E. S., Kemble, G. W., Lyle, J. M., Greaves, R. F. NATL ACAD SCIENCES. 1996: 11321–26

  Abstract

  Human cytomegalovirus (CMV) replication begins with the expression of two regulatory proteins, IE1(491aa) and IE2(579aa), produced from differentially spliced transcripts under control of the ie1/ie2 promoter-enhancer. A deletion mutation removing all 406 IE1(491aa)-specific amino acids was engineered into the viral genome and this mutant (RC303 delta Acc) was propagated on an IE1(491aa)-expressing human fibroblast cell line (ihfie1.3). RC303 delta Acc failed to replicate on normal human fibroblasts at low multiplicities of infection (mois). At mois > 3 plaque-forming units per cell, virus replication and production of progeny were comparable to wild type. However, at mois between 0.01 and 1, mutant virus replicated slowly on normal fibroblasts, a pattern that suggested initiation of productive infection required multiple hits. Replication of RC303 delta Acc correlated with the ability to express IE2(579aa), consistent with a role for IE1(491aa) in positive autoregulation of the ie1/ie2 promoter-enhancer and with data suggesting that virion transactivators compensate for the lack of IE1(491aa) under high moi conditions. ie1-deficient CMV should be completely avirulent, suggesting its utility as a gene therapy vector for hematopoietic progenitors that are normal sites of CMV latency.

  View details for Web of Science ID A1996VM68100008

  View details for PubMedID 8876134

  View details for PubMedCentralID PMC38056

• Human cytomegalovirus latent gene expression in granulocyte-macrophage progenitors in culture and in seropositive individuals PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kondo, K., Xu, J. K., Mocarski, E. S. 1996; 93 (20): 11137-11142

  Abstract

  Following infection with cytomegalovirus, human granulocyte-macrophage progenitors carry the viral genome but fail to support productive replication. Viral transcripts arise from a region encompassing the major regulatory gene locus; however, their structure differs significantly from productive phase transcripts. One class, sense transcripts, is encoded in the same direction as productive phase transcripts but uses two novel start sites in the ie1/ie2 promoter/enhancer region. These transcripts have the potential to encode a novel 94 aa protein. The other class, antisense transcript, is unspliced and complimentary to ie1 exons 2-4, and has the potential to encode novel 154 and 152 aa proteins. Consistent with a role in latency, these transcripts are present in bone marrow aspirates from naturally infected, healthy seropositive donors but are not present in seronegative controls. Sense latent transcripts are present in a majority of seropositive individuals. Consistent with the expression of latent transcripts, antibody to the 94 aa and 152 aa proteins is detectable in the serum of seropositive individuals. Thus, latent infection by cytomegalovirus is accompanied by the presence of latency-associated transcripts and expression of immunogenic proteins. Overall, these results suggest that bone marrow-derived myeloid progenitors are an important natural site of viral latency.

  View details for Web of Science ID A1996VL33300112

  View details for PubMedID 8855322

  View details for PubMedCentralID PMC38297

• Recombinant cytomegaloviruses for study of replication and pathogenesis INTERVIROLOGY Mocarski, E. S., Kemble, G. W. 1996; 39 (5-6): 320-330

  Abstract

  Systematic mutagenesis of large viral genomes such as those of the cytomegaloviruses requires strategies for identifying relevant functions as well as for detailed analysis of particular genes. A number of genetic markers that have been developed in other biological systems have been useful for insertion mutagenesis in these viruses. Thus far, 57 of the over 227 genes carried by wild-type human cytomegalovirus have been found to be dispensable for growth in cultured cells. Because of the limitations on studying human cytomegalovirus in an animal host, the closely related murine cytomegalovirus has been used as a surrogate for pathogenesis, tissue tropism and latency studies in the laboratory mouse. Genetic analysis of this virus has paralleled work on human cytomegalovirus, and an understanding of genes that specifically impact viral growth in particular organs has emerged from these studies. Strategies for generation of permissive cell lines able to complement essential human cytomegalovirus replication functions have been described, and sets of cosmid clones have been used to generate recombinant viruses. These methods will enable a systematic functional analysis of the genomes of human and animal cytomegaloviruses.

  View details for Web of Science ID A1996WW70900003

  View details for PubMedID 9130042

• Human cytomegalovirus uracil DNA glycosylase is required for the normal temporal regulation of both DNA synthesis and viral replication JOURNAL OF VIROLOGY Prichard, M. N., Duke, G. M., Mocarski, E. S. 1996; 70 (5): 3018-3025

  Abstract

  Human cytomegalovirus (CMV) encodes a gene, UL114, whose product is homologous to the uracil DNA glycosylase and is highly conserved in all herpesviruses. This DNA repair enzyme excises uracil residues in DNA that result from the misincorporation of dUTP or spontaneous deamination of cytosine. We constructed a recombinant virus, RC2620, that contains a large deletion in the UL114 open reading frame and carries a 1.2-kb insert containing the Escherichia coli gpt gene. RC2620 retains the capacity to replicate in primary human fibroblasts and reaches titers that are similar to those produced by the parent virus but exhibits a significantly longer replication cycle. Although the rate of expression of alpha and beta gene products appears to be unaffected by the mutation, DNA synthesis fails to proceed normally. Once initiated, DNA synthesis in mutant virus-infected cells proceeds at the same rate as with wild-type virus, but initiation is delayed by 48 h. The mutant virus also exhibits two predicted phenotypes: (i) hypersensitivity to the nucleoside analog 5-bromodeoxyuridine and (ii) retention of more uracil residues in genomic DNA than the parental virus. Together, these data suggest UL114 is required for the proper excision of uracil residues from viral DNA but in addition plays some role in establishing the correct temporal progression of DNA synthesis and viral replication. Although such involvement has not been previously observed in herpesviruses, a requirement for uracil DNA glycosylase in DNA replication has been observed in poxviruses.

  View details for Web of Science ID A1996UF24700039

  View details for PubMedID 8627778

  View details for PubMedCentralID PMC190161

• Human cytomegalovirus clinical isolates carry at least 19 genes not found in laboratory strains JOURNAL OF VIROLOGY Cha, T. A., Tom, E., Kemble, G. W., Duke, G. M., Mocarski, E. S., Spaete, R. R. 1996; 70 (1): 78-83

  Abstract

  Nucleotide sequence comparisons were performed on a highly heterogeneous region of three human cytomegalovirus strains, Toledo, Towne, and AD169. The low-passage, virulent Toledo genome contained a DNA segment of approximately 13 kbp that was not found in the Towne genome and a segment of approximately 15 kbp that was not found in the AD169 genome. The Towne strain contained approximately 4.7 kbp of DNA that was absent from the AD169 genome, and only about half of this segment was present, arranged in an inverted orientation, in the Toledo genome. These additional sequences were located at the unique long (UL)/b' (IRL) boundary within the L component of the viral genome. A region representing nucleotides 175082 to 178221 of the AD169 genome was conserved in all three strains; however, substantial reduction in the size of the adjacent b' sequence was found. The additional DNA segment within the Toledo genome contained 19 open reading frames not present in the AD169 genome. The additional DNA segment within the Towne genome contained four new open reading frames, only one of which shared homology with the Toledo genome. This comparison was extended to five additional clinical isolates, and the additional Toledo sequence was conserved in all. These findings reveal a dramatic level of genome sequence complexity that may explain the differences that these strains exhibit in virulence and tissue tropism. Although the additional sequences have not altered the predicted size of the viral genome (230 to 235 kbp), a total of 22 new open reading frames (denoted UL133 to UL154), many of which have sequence characteristics of glycoproteins, are now defined as cytomegalovirus specific. Our work suggests that wild-type virus carries more than 220 genes, some of which are lost by large-scale deletion and rearrangement of the UL/b' region during laboratory passage.

  View details for Web of Science ID A1996TJ65000011

  View details for PubMedID 8523595

  View details for PubMedCentralID PMC189790

• SELECTABLE INSERTION AND DELETION MUTAGENESIS OF THE HUMAN CYTOMEGALOVIRUS GENOME USING THE ESCHERICHIA-COLI GUANOSINE PHOSPHORIBOSYL TRANSFERASE (GPT) GENE JOURNAL OF GENERAL VIROLOGY Greaves, R. F., Brown, J. M., VIEIRA, J., Mocarski, E. S. 1995; 76: 2151-2160

  Abstract

  We describe the mutagenesis of the IRSI-US5 region of the human cytomegalovirus genome, demonstrating the potential of the E. coli guanosine phosphoribosyl transferase (gpt) gene as a selectable marker for insertion and deletion mutagenesis of high passage (AD169, Towne) as well as low passage (Toledo) strains of virus. Despite evidence suggesting that the US3 gene product may play a regulatory role, disruption of this gene with a gpt insert had no effect on growth of any of these strains of virus in resting or dividing human fibroblasts, or in human thymus plus liver implants in SCID-hu mice. Transcripts of the gpt gene, under control of the herpes simplex virus thymidine kinase promoter adjacent to the US3 enhancer in the viral genome, accumulated with delayed early (beta) kinetics. Mutants with deletions in the IRS1 and US3-US5 regions were isolated by back-selection against gpt with the drug 6-thioguanine by growing virus in human Lesch-Nyhan (hypoxanthine-guanine phosphoribosyl transferase deficient) skin fibroblasts immortalized with human papillomavirus oncogenes. Thus, we demonstrate a dependable method for insertion and deletion mutagenesis that can be applied to any region of the viral genome.

  View details for Web of Science ID A1995RT16600005

  View details for PubMedID 7561752

• DRAMATIC INTERSTRAIN DIFFERENCES IN THE REPLICATION OF HUMAN CYTOMEGALOVIRUS IN SCID-HU MICE JOURNAL OF INFECTIOUS DISEASES Brown, J. M., Kaneshima, H., Mocarski, E. S. 1995; 171 (6): 1599-1603

  Abstract

  The ability of a low-passage strain (Toledo) and laboratory strains (AD169 and Towne) of human cytomegalovirus to replicate in SCID-hu (thymus plus liver) mice were compared. At a time of peak replication, 14 days after inoculation, the Toledo strain grew 2-3 orders of magnitude better than any laboratory strain, a difference reflecting the number of infected thymic stromal cells in the implants. The growth property of the Toledo strain was stable through serial passage and plaque purification. The AD169-ATCC strain failed to grow at all, while an independently maintained stock of this strain obtained from the United Kingdom replicated to low levels, suggesting that divergence had occurred during propagation in different locations. This work predicts the existence of viral genetic determinant(s) for growth in tissues that are lost during propagation in culture.

  View details for Web of Science ID A1995RB29800030

  View details for PubMedID 7769298

• MURINE CYTOMEGALOVIRUS IE2, AN ACTIVATOR OF GENE-EXPRESSION, IS DISPENSABLE FOR GROWTH AND LATENCY IN MICE VIROLOGY Cardin, R. D., ABENES, G. B., Stoddart, C. A., Mocarski, E. S. 1995; 209 (1): 236-241

  Abstract

  The murine cytomegalovirus alpha (immediate-early) gene product, IE2(391aa), a protein that is related to the human cytomegalovirus US22 protein family, had previously been shown to be dispensable for viral growth in cell culture. In transient assays, however, this protein was found to transactivate the murine CMV ie1/ie3 and ie2 promoters, as well as a number of other promoters. Transactivation was mediated via promoter-proximal elements rather than through elements located upstream in the enhancer region. This activation predicted that ie2 would play a role in regulating gene expression; however, ie2 mutants did not exhibit altered growth or latency in the mouse. ie2-deficient viruses reached peak titers in spleen, salivary glands, lungs, liver, kidneys, pancreas, peripheral blood leukocytes, and adrenal glands that were comparable to wild-type virus. When assayed by spleen explant culture, ie2-deficient viruses yielded reactivation levels similar to wild type. Thus, the murine CMV ie2 gene encodes a regulatory protein that is dispensable for viral infection of cells in culture as well as for interaction with tissues in the infected BALB/c mouse.

  View details for Web of Science ID A1995QY81900026

  View details for PubMedID 7747475

• Cytomegalovirus latency and latency-specific transcription in hematopoietic progenitors 5th International Cytomegalovirus Conference Kondo, K., Mocarski, E. S. TAYLOR & FRANCIS AS. 1995: 63–67

  Abstract

  Latency-associated transcripts have been detected in human cytomegalovirus (CMV)-infected granulocyte-macrophage progenitors and have been shown to be encoded from both DNA strands in the ie1/ie2 region of the viral genome. One class includes differentially spliced transcripts that extends exon 1 by initiating at two novel start sites 292 and 356 nucleotides in the ie1/ie2 promoter/enhancer region, upstream of the start site used during productive infection. The other class includes an unspliced transcript antisense to ie1 exons 2,3, and 4. These transcripts are specific to latently infected cells and are not detected during productive infection of human fibroblast cells. Several open reading frames are found within these cytomegalovirus latency-specific transcripts (CLTs), including one 94 codons long that begins in the extended exon 1 region and is predicted to remain open through the exon1/2 and exon 2/3 splice junctions. Expression of latent transcripts was detected in bone marrow-derived hematopoietic samples from CMV-seropositive healthy donors but not from seronegative donors.

  View details for Web of Science ID A1995TZ68700013

  View details for PubMedID 8668945

• HUMAN CYTOMEGALOVIRUS LATENT INFECTION OF GRANULOCYTE-MACROPHAGE PROGENITORS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kondo, K., Kaneshima, H., Mocarski, E. S. 1994; 91 (25): 11879-11883

  Abstract

  We have investigated the interaction of human cytomegalovirus (CMV) with cultured primary granulocyte-macrophage progenitors, a suspected natural site of viral latency, and have established conditions for latent infection and reactivation in this cell population. Progenitor cells from human fetal liver or bone marrow maintained a CD14+, CD15+, CD33+ cell surface phenotype during propagation in suspension culture. Exposure to human CMV did not reduce growth or alter the phenotype of these cells during a 4-week culture period. Viral replication was not detectable in these cells, although viral DNA, as measured by PCR analysis, persisted in a high proportion of cultured cells in the absence of delayed early (beta) gene expression. Viral gene expression was restricted such that only ie1 region transcripts were detected by PCR analysis of cDNA, and these transcripts were estimated to be present in no less than 2-5% of latently infected cells. Most of these transcripts remained unspliced, a result that strikingly contrasts with the splicing pattern normally seen during viral replication in permissive cells. Latent virus reactivated after prolonged, 16- to 21-day cocultivation of infected granulocyte-macrophage progenitors with permissive cells, results that support a role for the myelomonocytic cell population as a biological reservoir of latent human CMV and suggest that these cells may be the source of CMV DNA PCR-positive monocytes found in the peripheral blood of healthy carriers.

  View details for Web of Science ID A1994PW70800024

  View details for PubMedID 7991550

  View details for PubMedCentralID PMC45339

• STRUCTURE AND FUNCTION OF THE MURINE CYTOMEGALOVIRUS SGG1 GENE - A DETERMINANT OF VIRAL GROWTH IN SALIVARY-GLAND ACINAR-CELLS JOURNAL OF VIROLOGY Lagenaur, L. A., Manning, W. C., VIEIRA, J., Martens, C. L., Mocarski, E. S. 1994; 68 (12): 7717-7727

  Abstract

  The salivary gland has long been recognized as an important target organ for cytomegalovirus replication in the infected host. A viral gene, denoted sgg1, plays an important role for replication in the salivary gland even though it is dispensable for growth in other organs or in cultured cells. The nucleotide sequence of this gene and of cDNA clones representing two spliced transcripts (1.5 and 1.8 kb in size) has been determined. The more abundant 1.5-kb transcript contains a 312-amino-acid (aa) open reading frame (ORF) and encodes the corresponding 37-kDa protein (Sgg1) when expressed in transfected COS-7 cells. The 1.8-kb transcript initiates upstream of the 1.5-kb transcript and contains a 108-aa ORF in addition to the 312-aa ORF. This longer cDNA also encodes the 37-kDa protein Sgg1, although at lower abundance than the 1.5-kb cDNA. Sgg1 localizes to the cytoplasm of COS-7 cells, which is consistent with the predicted structural characteristics of the 312-aa ORF: a type 1 integral membrane protein. During viral infection, expression of both sgg1 transcripts is highest at early times (8 to 12 h) after infection; only the 1.5-kb transcript is present, at low levels, late in infection. A recombinant virus, RM868, carrying a lacZ-gpt insertion within sgg1, fails to express Sgg1 protein and exhibits reduced growth in the salivary gland. RM868 retains the capacity to disseminate in the infected mouse and to enter serous acinar cells, although it fails to replicate efficiently in this cell type. These results suggest that sgg1 is critical for high levels of viral replication in the salivary gland.

  View details for Web of Science ID A1994PR43200008

  View details for PubMedID 7966561

  View details for PubMedCentralID PMC237233

• PERIPHERAL-BLOOD MONONUCLEAR PHAGOCYTES MEDIATE DISSEMINATION OF MURINE CYTOMEGALOVIRUS JOURNAL OF VIROLOGY Stoddart, C. A., Cardin, R. D., Boname, J. M., Manning, W. C., ABENES, G. B., Mocarski, E. S. 1994; 68 (10): 6243-6253

  Abstract

  Cytomegalovirus is transmitted with blood and organs from seropositive individuals, although the particular leukocyte population harboring latent or persistent virus remains poorly characterized. Murine cytomegalovirus, tagged with the Escherichia coli lacZ gene, was used to identify cells in which virus replicates during acute infection of immunocompetent mice. Recombinant murine cytomegaloviruses, RM461, RM460, and RM427, were constructed to express beta-galactosidase under control of the human cytomegalovirus ie1/ie2 promoter/enhancer. The lacZ gene was inserted between the ie2 and sgg1 genes in RM461 and RM460, disrupting a 0.85-kb late transcript that was found to be dispensable for replication in cultured cells as well as for infection of mice. In BALB/c mice, lacZ-tagged and wild-type viruses exhibited a similar 50% lethal dose and all had the capacity to latently infect the spleen. Peripheral blood mononuclear phagocytes were the major infected leukocyte cell type, as demonstrated by the ability of infected cells to adhere to glass and to phagocytize latex beads; however, these cells did not exhibit typical monocyte markers. Plaque assay for virus and 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) staining of frozen sections of organs from infected mice revealed that the major target organs included the spleen, adrenal glands, liver, and salivary glands, although tissues as diverse as brown fat and lungs were also involved. Individual blue-staining cells were readily identified in all infected tissues. These studies identified a mononuclear phagocyte, possibly a macrophage or dendritic cell precursor, as the vehicle of virus dissemination during acute infection, and demonstrate the utility of using lacZ-tagged murine cytomegalovirus for tropism, pathogenesis, and latency studies.

  View details for Web of Science ID A1994PG54100012

  View details for PubMedID 8083964

  View details for PubMedCentralID PMC237044

• HUMAN CYTOMEGALOVIRUS LATE PROTEIN ENCODED BY IE2 - A TRANSACTIVATOR AS WELL AS A REPRESSOR OF GENE-EXPRESSION JOURNAL OF GENERAL VIROLOGY Jenkins, D. E., Martens, C. L., Mocarski, E. S. 1994; 75: 2337-2348

  Abstract

  In order to study the function of human cytomegalovirus (HCMV) immediate early gene 2 (ie2) (UL122) gene products made at late times during infection, cDNA clones were isolated from an expression library made with 74 h post-infection mRNA. Based on screening of the library, 1% of transcripts in infected cells at this time were ie2 region-specific, and transcripts encoding gamma IE2(338aa), a 40K late gene product, were more abundant than those encoding IE2(579aa), an alpha gene product made throughout infection. As expected, the cDNA capable of directing the expression of gamma IE2(338aa) was derived from a contiguous genomic region within exon 5 of the ie1/ie2 region. The cDNA clones encoding gamma IE2(338aa) and IE2(579aa) were compared for their ability to trans-activate viral and cellular promoters and to repress expression from the ie1/ie2 promoter via the ie2 cis-repression signal. Unexpectedly, gamma IE2(338aa) trans-activated a variety of test promoters when cotransfected with the major alpha gene product, IE1(491aa). Promoters derived from the cellular beta-actin gene, the simian virus 40 early region and the human immunodeficiency virus were all responsive to gamma IE2(338aa) plus IE1(491aa), although several beta promoters derived from the HCMV genome were unresponsive. Thus, this abundant late product from the ie2 region may play a role in trans-activation in addition to its role as a repressor of alpha gene expression.

  View details for Web of Science ID A1994PF54300024

  View details for PubMedID 8077932

• GENES IN THE HINDIII J-FRAGMENT OF THE MURINE CYTOMEGALOVIRUS GENOME ARE DISPENSABLE FOR GROWTH IN CULTURED-CELLS - INSERTION MUTAGENESIS WITH A LACZ GPT CASSETTE JOURNAL OF VIROLOGY VIEIRA, J., Farrell, H. E., Rawlinson, W. D., Mocarski, E. S. 1994; 68 (8): 4837-4846

  Abstract

  The organization and function of the genes encoded within the HindIII J region of the murine cytomegalovirus genome were analyzed by transcript mapping and cDNA isolation, nucleotide sequence analysis and identification of open reading frames (ORFs), and construction of recombinant viruses carrying insertions disrupting five of the seven ORFs. This region was found to encode five beta transcripts and one gamma transcript in addition to two beta transcripts previously mapped to the sgg1 locus. Seven open reading frames were identified, and one was recognized as a homolog of a human cytomegalovirus US22 gene family. The five largest ORFs contained within the HindIII J fragment (sgg1, HJ4, HJ5, HJ6, and HJ7) were each disrupted by the insertion of a lacZ/gpt genetic marker cassette. The growth kinetics of all recombinant viruses were investigated and found to be the same as wild-type parental virus, indicating that these five ORFs were dispensable for growth in cell culture.

  View details for Web of Science ID A1994NW97800014

  View details for PubMedID 8035482

  View details for PubMedCentralID PMC236423

• MOLECULAR EVOLUTION OF HERPESVIRUSES - GENOMIC AND PROTEIN-SEQUENCE COMPARISONS JOURNAL OF VIROLOGY Karlin, S., Mocarski, E. S., Schachtel, G. A. 1994; 68 (3): 1886-1902

  Abstract

  Phylogenetic reconstruction of herpesvirus evolution is generally founded on amino acid sequence comparisons of specific proteins. These are relevant to the evolution of the specific gene (or set of genes), but the resulting phylogeny may vary depending on the particular sequence chosen for analysis (or comparison). In the first part of this report, we compare 13 herpesvirus genomes by using a new multidimensional methodology based on distance measures and partial orderings of dinucleotide relative abundances. The sequences were analyzed with respect to (i) genomic compositional extremes; (ii) total distances within and between genomes; (iii) partial orderings among genomes relative to a set of sequence standards; (iv) concordance correlations of genome distances; and (v) consistency with the alpha-, beta-, gammaherpesvirus classification. Distance assessments within individual herpesvirus genomes show each to be quite homogeneous relative to the comparisons between genomes. The gammaherpesviruses, Epstein-Barr virus (EBV), herpesvirus saimiri, and bovine herpesvirus 4 are both diverse and separate from other herpesvirus classes, whereas alpha- and betaherpesviruses overlap. The analysis revealed that the most central genome (closest to a consensus herpesvirus genome and most individual herpesvirus sequences of different classes) is that of human herpesvirus 6, suggesting that this genome is closest to a progenitor herpesvirus. The shorter DNA distances among alphaherpesviruses supports the hypothesis that the alpha class is of relatively recent ancestry. In our collection, equine herpesvirus 1 (EHV1) stands out as the most central alphaherpesvirus, suggesting it may approximate an ancestral alphaherpesvirus. Among all herpesviruses, the EBV genome is closest to human sequences. In the DNA partial orderings, the chicken sequence collection is invariably as close as or closer to all herpesvirus sequences than the human sequence collection is, which may imply that the chicken (or other avian species) is a more natural or more ancient host of herpesviruses. In the second part of this report, evolutionary relationships among the 13 herpesvirus genomes are evaluated on the basis of recent methods of amino acid alignment applied to four essential protein sequences. In this analysis, the alignment of the two betaherpesviruses (human cytomegalovirus versus human herpesvirus 6) showed lower scores compared with alignments within alphaherpesviruses (i.e., among EHV1, herpes simplex virus type 1, varicella-zoster virus, pseudorabies virus type 1 and Marek's disease virus) and within gammaherpesviruses (EBV versus herpesvirus saimiri).(ABSTRACT TRUNCATED AT 400 WORDS)

  View details for Web of Science ID A1994MY59600073

  View details for PubMedID 8107249

  View details for PubMedCentralID PMC236651

• ALTERING CENTRAL-NERVOUS-SYSTEM PHYSIOLOGY WITH A DEFECTIVE HERPES-SIMPLEX VIRUS VECTOR EXPRESSING THE GLUCOSE TRANSPORTER GENE PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ho, D. Y., Mocarski, E. S., Sapolsky, R. M. 1993; 90 (8): 3655-3659

  Abstract

  Because of their postmitotic nature, neurons are difficult subjects for gene transfer. To circumvent this, we have used a defective herpes simplex virus vector to overexpress the rat brain glucose transporter (GT) gene under the control of the human cytomegalovirus ie1 promoter. This vector, designated vIE1GT, was propagated using a herpes simplex virus type 1 temperature-sensitive mutant, ts756. GT expressed from vIE1GT was readily immunoprecipitated from membrane fractions of vIE1GT-infected Vero cells. By using indirect double immunofluorescence techniques, vIE1GT was shown to be capable of enhancing GT expression in cultured hippocampal neurons and glia. Glucose transport in such vIE1GT-infected cultures was increased approximately 2-fold relative to controls. The efficacy of this system in vivo was then tested by microinjection of vIE1GT into adult rat hippocampus. When examined 2 days later, GT expression from vIE1GT was demonstrated in hippocampal neurons by in situ hybridization; a small but significant increase in glucose transport was detected in tissue immediately surrounding the injection site by 2-deoxy[14C]glucose uptake and autoradiography. Such injections did not cause marked cytopathology. Thus, this approach can be used to alter central nervous system physiology in vitro and in vivo.

  View details for Web of Science ID A1993KX81600112

  View details for PubMedID 8386379

  View details for PubMedCentralID PMC46360

• HUMAN CYTOMEGALOVIRUS IN A SCID-HU MOUSE - THYMIC EPITHELIAL-CELLS ARE PROMINENT TARGETS OF VIRAL REPLICATION PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Mocarski, E. S., Bonyhadi, M., Salimi, S., McCune, J. M., Kaneshima, H. 1993; 90 (1): 104-108

  Abstract

  Animal models of human cytomegalovirus (CMV) infections have not been available to study pathogenesis or to evaluate antiviral drugs. Severe combined immunodeficient mice implanted with human fetal tissues (SCID-hu) were found to support CMV replication and may provide a model for this species-specific virus. When conjoint implants of human fetal thymus and liver were inoculated with a low-passage-number isolate of CMV, strain Toledo, consistent high-level viral replication was detected 5, 12, 15, 28, and 35 days after inoculation and virus replication continued for up to 9 months. Other human tissue implants, including lung and colon, were also found to support viral growth but with greater variability in levels and for a shorter duration. As expected, the species specificity of human CMV was preserved in this model such that virus was detected in the human conjoint thymus/liver implant but not in surrounding mouse tissues. The majority of virus-infected cells were localized in the thymic medulla rather than cortical region of the implant and immunofluorescence analysis identified epithelial cells rather than any hematopoietic cell population as the principal hosts for viral replication. Finally, treatment of infected animals with ganciclovir reduced viral replication, thereby demonstrating the value of this system for evaluating antiviral therapies. This animal model opens the way for a range of investigations not previously possible with human CMV.

  View details for Web of Science ID A1993KF40700022

  View details for PubMedID 7678330

  View details for PubMedCentralID PMC45608

• RECOMBINATION INVITRO BETWEEN HERPES-SIMPLEX VIRUS TYPE-1 A-SEQUENCES PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bruckner, R. C., Dutch, R. E., Zemelman, B. V., Mocarski, E. S., Lehman, I. R. 1992; 89 (22): 10950-10954

  Abstract

  We have partially purified an activity from extracts of cells infected with herpes simplex virus type 1 that mediates recombination between repeated copies of the 317-base-pair a sequence of herpes simplex virus type 1. Recombination leads to deletion of a lacZ indicator gene situated between two directly repeated copies of the a sequence and is scored by transformation of lacZ- Escherichia coli. The two products of the reaction can be observed directly by restriction enzyme digestion and Southern blot analysis. The recombinase activity is also detectable, but at a lower level, in uninfected cell extracts. The DNA substrate must contain the two a sequences arranged in direct orientation to generate the lacZ deletion. However, when the a sequences are arranged in inverted orientation, an inversion results. A substrate with two homologous sequences of size and G + C content similar to the a sequence undergoes recombination at a much lower frequency. The reaction requires a divalent cation (Mg2+ or Mn2+) but not ATP or any other nucleoside triphosphate. The simple requirements and specificity for the a sequence suggest that the recombination may proceed by a site-specific mechanism.

  View details for Web of Science ID A1992JY87400080

  View details for PubMedID 1332062

• INFECTION OF HEMATOPOIETIC PROGENITOR CELLS BY HUMAN CYTOMEGALOVIRUS BLOOD Maciejewski, J. P., Bruening, E. E., DONAHUE, R. E., Mocarski, E. S., Young, N. S., STJEOR, S. C. 1992; 80 (1): 170-178

  Abstract

  The susceptibility of hematopoietic progenitor cells to infection by human cytomegalovirus (HCMV) was investigated using several strains of HCMV, including the recombinant strain RC256. RC256 is derived from the laboratory strain Towne and contains the Escherichia coli LacZ gene coding for beta-galactosidase (beta-gal) regulated by an early HCMV promoter. Expression of LacZ allowed the detection of HCMV in individual hematopoietic cells. Clonogeneic bone marrow (BM) progenitors, including CD34+ cells, could be infected with HCMV and would then form normal hematopoietic colonies. By polymerase chain reaction (PCR) amplification of DNA, HCMV could be detected in both erythroid and myeloid colonies. LacZ activity was observed predominantly in cells of myelomonocytic lineage. When cells derived from HCMV-infected progenitors were cocultivated with permissive human fibroblasts, infectious virus expressing LacZ was recovered. Although no characteristic HCMV cytopathology was observed in BM colonies, high virus to cell ratios resulted in a moderate inhibition of colony formation. Since infected hematopoietic progenitors can harbor HCMV for weeks and through several differentiation steps in culture, we postulate that in vivo these cells may serve as a reservoir of latent virus and contribute to HCMV dissemination.

  View details for Web of Science ID A1992JB28000024

  View details for PubMedID 1377049

• HUMAN CYTOMEGALOVIRUS ORIGIN OF DNA-REPLICATION (ORILYT) RESIDES WITHIN A HIGHLY COMPLEX REPETITIVE REGION PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA MASSE, M. J., Karlin, S., Schachtel, G. A., Mocarski, E. S. 1992; 89 (12): 5246-5250

  Abstract

  A global analysis of the 230-kilobase-pair (kbp) human cytomegalovirus genome revealed three regions that were very rich in repeated sequences. The region with the highest content of inverted and direct repeats lies between 92,100 and 93,500 bp, upstream of the gene encoding the single-stranded DNA binding protein. Cloned restriction fragments containing this region were able to replicate when trans-acting factors were provided by virus infection in a transient replication assay. With this assay, the region between 92,210 and 93,715 bp on the viral genome was defined as the minimal replication origin, oriLyt. The sequence composition and repeats within oriLyt were used to divide the region into two domains that may be important in origin function. Sequences flanking either the left or right side of the minimal oriLyt contributed to efficient replication; however, these sequences were not essential for origin function. Thus, the region of the viral genome with the most striking concentration of direct and inverted repeats corresponds to the oriLyt of human cytomegalovirus.

  View details for Web of Science ID A1992HY05300010

  View details for PubMedID 1319057

• CYTOMEGALOVIRUS DETERMINANT OF REPLICATION IN SALIVARY-GLANDS JOURNAL OF VIROLOGY Manning, W. C., Stoddart, C. A., Lagenaur, L. A., ABENES, G. B., Mocarski, E. S. 1992; 66 (6): 3794-3802

  Abstract

  Murine cytomegalovirus carrying a deletion mutation disrupting the expression of a gene dispensable for growth in cultured cells was found to disseminate poorly in the mouse. The mutation resulted in a dramatic decrease in the expression of a 1.5-kb major and a 1.8-kb minor beta transcript from a region adjacent to the ie2 gene in the viral genome. Nucleotide sequence determination indicated that 323 bp, including a predicted polyadenylation signal, was deleted from this beta gene. In cultured cells, the plaque morphology and growth characteristics of the mutant were similar to those of parental or rescued wild-type viruses. Following intraperitoneal inoculation of BALB/c mice, growth of the mutant in the salivary gland was dramatically reduced 10,000-fold, while growth in the liver and spleen was not dramatically affected. The beta gene was thus denoted sgg1 (salivary gland growth gene 1). Neither intranasal infection nor direct inoculation into the salivary glands completely overcame the restriction of growth in this organ, suggesting that the sgg1 gene encoded a determinant of tissue tropism. To investigate the impact of the sgg1 mutation on virus dissemination via the blood, the virus titer in peripheral blood leukocytes was determined. No difference was found between the sgg1 mutant and rescued wild-type virus. Thus, murine cytomegalovirus sgg1 gene products appear to be involved in entry or replication of virus in salivary gland cells.

  View details for Web of Science ID A1992HU59900062

  View details for PubMedID 1316482

• NEUTRALIZING EPITOPES ON HERPES-SIMPLEX VIRUS-1-EXPRESSED ROTAVIRUS-VP7 ARE DEPENDENT ON COEXPRESSION OF OTHER ROTAVIRUS PROTEINS VIROLOGY Dormitzer, P. R., Ho, D. Y., MACKOW, E. R., Mocarski, E. S., Greenberg, H. B. 1992; 187 (1): 18-32

  Abstract

  We constructed a recombinant thymidine kinase-negative herpes simplex virus type 1 (HSV-1) that expressed the rotavirus major outer capsid glycoprotein, VP7. In the recombinant HSV-1, a promoter from the 5' noncoding region of the HSV-1 glycoprotein B locus regulated the expression of VP7 as a HSV-1 gamma 1 gene product. HSV-1-expressed VP7 resembled rotavirus-expressed VP7 in its SDS-PAGE mobility, high mannose-type glycosylation, disulfide bonding, perinuclear to cytoplasmic localization, intracellular retention, and reactivity with polyclonal antisera and nonneutralizing antibodies. Unlike rotavirus-expressed VP7, HSV-1-expressed VP7 lacked several neutralizing epitopes by immuno-histochemical staining and by ELISA. One neutralizing epitope identified on HSV-1-expressed VP7 by ELISA was masked by paraformaldehyde fixation of recombinant HSV-1- but not rotavirus-infected cells. Neutralizing epitopes were restored to HSV-1-expressed VP7 by coinfection of cells with the HSV-1 recombinant and a heterologous rotavirus that lack the neutralizing epitopes. The recovered neutralizing epitopes were detected on double-shelled rotavirus particles produced in the coinfected cells. This study indicates that the formation of several neutralizing epitopes on rotavirus VP7 requires interaction of VP7 with other rotavirus proteins. In addition, HSV-1 was a useful vector for studying the localization, processing, and antigenicity of an RNA virus glycoprotein.

  View details for Web of Science ID A1992HD62800003

  View details for PubMedID 1371025

• TRANSACTIVATION OF THE CYTOMEGALOVIRUS ICP36 GENE PROMOTER REQUIRES THE ALPHA-GENE PRODUCT TRS1 IN ADDITION TO IE1 AND IE2 JOURNAL OF VIROLOGY STASIAK, P. C., Mocarski, E. S. 1992; 66 (2): 1050-1058

  Abstract

  Very little is known about the human cytomegalovirus functions that activate gamma (late) gene expression. We have investigated the regulation of the human cytomegalovirus gamma gene encoding the ICP36 major late DNA-binding protein family (UL44). Transactivation of the ICP36 gene promoter was found to be absolutely dependent on the trs1 gene product when expressed in cells in conjunction with ie1 and ie2 gene products. Transactivation occurred poorly or not at all when any one of these three transactivators was omitted. TRS1 is a member of the US22 family of proteins and is encoded by a region near the L-S junction of the viral genome within the c repeat and adjacent Us sequences. TRS1 is highly homologous to IRS1, which is encoded from the other copy of the c repeat, and plasmid constructs carrying the irs1 gene were also able to mediate transactivation of the ICP36 promoter. RNA blot analysis of steady-rate RNA throughout infection showed that the trs1 transcript was expressed with the kinetics of an alpha gene but its accumulation was delayed relative to that of ie1 and ie2 transcripts. On the basis of these experiments, TRS1 and IRS1 are proposed to be important intermediaries in the cascade of cytomegalovirus gene expression.

  View details for Web of Science ID A1992GY96500050

  View details for PubMedID 1370547

• HERPES-SIMPLEX VIRUS TYPE-1 RECOMBINATION - ROLE OF DNA-REPLICATION AND VIRAL-A SEQUENCES JOURNAL OF VIROLOGY Dutch, R. E., Bruckner, R. C., Mocarski, E. S., Lehman, I. R. 1992; 66 (1): 277-285

  Abstract

  During the course of infection, elements of the herpes simplex virus type 1 (HSV-1) genome undergo inversion, a process that is believed to occur through the viral a sequences. To investigate the mechanism of this recombinational event, we have developed an assay that detects the deletion of DNA segments flanked by directly repeated a sequences in plasmids transiently maintained in Vero cells. With this assay, we have observed a high frequency of recombination (approximately 8%) in plasmids that undergo replication in HSV-1-infected cells. We also found a low level of recombination between a sequences in plasmids introduced into uninfected cells and in unreplicated plasmids in HSV-1-infected cells. In replicating plasmids, recombination between a sequences occurs at twice the frequency seen with directly repeated copies of a different sequence of similar size. Recombination between a sequences appears to occur at approximately the same time as replication, suggesting that the processes of replication and recombination are closely linked.

  View details for Web of Science ID A1992GU97100034

  View details for PubMedID 1309247

• EVIDENCE FOR SELECTIVE EVOLUTION IN CODON USAGE IN CONSERVED AMINO-ACID SEGMENTS OF HUMAN ALPHAHERPESVIRUS PROTEINS JOURNAL OF MOLECULAR EVOLUTION Schachtel, G. A., Bucher, P., Mocarski, E. S., Blaisdell, B. E., Karlin, S. 1991; 33 (6): 483-494

  Abstract

  The genomes of human viruses herpes simplex 1 (HSV1) and varicella zoster (VZV), although similar in biology, largely concordant in gene order, and identical in many amino acid segments, differ widely in their genomic G + C (abbreviated S) content, which is high in HSV1 (68%) and low in VZV (46%). This paper analyzes several striking codon usage contrasts. The S difference in coding regions is dramatically large in codon site 3, S3, about 42%. The large difference in S3 is maintained at the same level in a subset of closely similar genes and even in corresponding identical amino acid blocks. A similar difference in S levels in silent site 1 (S1) is found in leucine and arginine. The difference in S3 levels occurs in every gene and in every multicodon amino acid form. The S difference also exists in amino acid usage, with HSV1 using significantly more codon types SSN, while VZV uses more codon types WWN (where W stands for A or T). The nonoverlapping and narrow histograms of S3 gene frequencies in both viruses suggest that the difference has arisen and been maintained by a process of selective rather than nonselective effects. This is in sharp contrast to the relatively large variance seen for highly similar genes in the human versus yeast analysis. Interpretations and hypotheses to explain the HSV1 vs VZV codon usage disparity relate to virus-host interactions, to the role of viral genes in DNA metabolism, to availability of molecular resources (molecular Gause exclusion principle), and to differences in genomic structure.

  View details for Web of Science ID A1991GT50800003

  View details for PubMedID 1663999

• INITIAL EVENTS INVOLVED IN CYTOMEGALOVIRUS CELL-INTERACTIONS SYMP ON PATHOGENESIS OF HUMAN CYTOMEGALOVIRUS-ASSOCIATED DISEASES Mocarski, E. S. ELSEVIER SCIENCE INC. 1991: 43–47

  View details for Web of Science ID A1991FQ90900011

  View details for PubMedID 1648834

• HUMAN CYTOMEGALOVIRUS IE2 NEGATIVELY REGULATES ALPHA-GENE EXPRESSION VIA A SHORT TARGET SEQUENCE NEAR THE TRANSCRIPTION START SITE JOURNAL OF VIROLOGY Cherrington, J. M., KHOURY, E. L., Mocarski, E. S. 1991; 65 (2): 887-896

  Abstract

  Repression of human cytomegalovirus alpha (immediate-early) gene expression is under the control of the viral ie2 gene. Here we show that ie2 negatively regulates gene expression directed by the strong cytomegalovirus enhancer via a specific 15-bp target sequence (which we term cis repression signal [crs]). This crs is located between -14 and +1 relative to the transcription start site and will function in an orientation-independent fashion, consistent with repression occurring at the transcriptional level. Repression is dominant over transactivation by ie1 gene products. The crs (5'-CGTTTAGTGAACCGT-3') does not contain previously recognized binding sites for cellular transcription factors, and a precise copy is not found elsewhere in the human cytomegalovirus genome. The position of the signal near the transcription start site appears to be important in function; addition of the crs near the transcription start site of a heterologous promoter, from the thymidine kinase gene of herpes simplex virus type 1, conferred cytomegalovirus ie2-dependent repression upon this promoter. Thus, we propose that an ie2 gene product or an induced cellular protein mediates repression by binding to crs. Negative regulation of alpha gene expression may be important during viral replication or latency.

  View details for Web of Science ID A1991ET44500038

  View details for PubMedID 1846203

• TRANSACTIVATION OF A CYTOMEGALOVIRUS GAMMA-GENE PROMOTER REQUIRES TRS1, A MEMBER OF THE US22 FAMILY 3RD INTERNATIONAL WORKSHOP ON CYTOMEGALOVIRUS STASIAK, P. C., Mocarski, E. S. ELSEVIER SCIENCE PUBL B V. 1991: 63–79

  View details for Web of Science ID A1991BU78M00010

• THE PRODUCTS OF HUMAN CYTOMEGALOVIRUS GENES UL1-UL7, INCLUDING GP48, ARE DISPENSABLE FOR GROWTH IN CELL-CULTURE 3RD INTERNATIONAL WORKSHOP ON CYTOMEGALOVIRUS Ripalti, A., Mocarski, E. S. ELSEVIER SCIENCE PUBL B V. 1991: 57–60

  View details for Web of Science ID A1991BU78M00009

• MOLECULAR GENETIC-ANALYSIS OF CYTOMEGALOVIRUS GENE-REGULATION IN GROWTH, PERSISTENCE AND LATENCY CURRENT TOPICS IN MICROBIOLOGY AND IMMUNOLOGY Mocarski, E. S., ABENES, G. B., Manning, W. C., Sambucetti, L. C., Cherrington, J. M. 1990; 154: 47-74

  View details for Web of Science ID A1990EC74700003

  View details for PubMedID 2161325

• NF-KAPPA-B ACTIVATION OF THE CYTOMEGALO-VIRUS ENHANCER IS MEDIATED BY A VIRAL TRANSACTIVATOR AND BY T-CELL STIMULATION EMBO JOURNAL Sambucetti, L. C., Cherrington, J. M., Wilkinson, G. W., Mocarski, E. S. 1989; 8 (13): 4251-4258

  Abstract

  The expression of cytomegalovirus alpha (immediate early) genes is under control of an enhancer that carries signals for strong constitutive expression as well as response elements for transactivation by viral proteins. We have used synthetic oligonucleotides representing the 16, 18 and 19 bp repeat elements within the enhancer to investigate the role of virus-induced cellular transcription factors in enhancer activation. We show that the transcription factor NF-kappa B, which binds to the 18 bp repeat, plays a central role in enhancer activation in infected human fibroblasts and that activation is mediated by the product of the viral gene ie1. The simian immunodeficiency virus kappa B site can functionally substitute for the 18 bp element in transient transactivation assays and can also compete efficiently for specific binding to the 18 bp repeat element in vitro. Point mutations in the NF-kappa B site within the 18 bp element disrupt ie1-mediated transactivation and binding. We have found that the characteristics of the 18 bp binding factor from human fibroblasts are indistinguishable from NF-kappa B induced by phorbol ester plus mitogen treatment of T lymphocytes, as determined by gel mobility shift assay as well as protection of the binding site from chemical cleavage. Furthermore, T cell stimulation mediates activation of the viral enhancer via kappa B sites, an observation that may be important in the interaction of cytomegalovirus with the naturally infected human host. Thus, NF-kappa B plays a central role as a target for enhancer activation via viral and cellular factors.

  View details for Web of Science ID A1989CE21700034

  View details for PubMedID 2556267

• A HOST-CELL PROTEIN BINDS TO A HIGHLY CONSERVED SEQUENCE ELEMENT (PAC-2) WITHIN THE CYTOMEGALOVIRUS-A SEQUENCE JOURNAL OF VIROLOGY Kemble, G. W., Mocarski, E. S. 1989; 63 (11): 4715-4728

  Abstract

  The human cytomegalovirus (CMV) a sequence has significant homology to two regions, pac-1 and pac-2, within the a sequence of herpes simplex virus type 1 (HSV-1). Both regions have been shown to be important cis-acting signals in HSV-1 genome maturation. We have demonstrated that a small fragment from within the CMV a sequence, containing the pac-1 and pac-2 motifs, carries all of the signals necessary for generation of genomic termini and for inversion. These observations indicated that the function of these highly conserved sequence motifs was similar in CMV and HSV-1. We have identified and partially purified a host cell protein with affinity for the sequence 5'-GGCGGCGGCGCATAAAA-3' within CMV pac-2. This partially purified protein has an apparent molecular weight of 89,000 under denaturing conditions and could be renatured after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the capacity to bind DNA was the property of a single polypeptide chain. This activity was found in a wide variety of human cell lines, including those that are permissive as well as those that are nonpermissive for CMV growth, but not in cell lines from monkey, mouse, or drosophila origins. Our work implicates a host cell protein in a sequence function.

  View details for Web of Science ID A1989AU74000031

  View details for PubMedID 2552148

• HERPES-SIMPLEX VIRUS LATENT RNA (LAT) IS NOT REQUIRED FOR LATENT INFECTION IN THE MOUSE PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ho, D. Y., Mocarski, E. S. 1989; 86 (19): 7596-7600

  Abstract

  During latent infection by herpes simplex virus (HSV), an abundant latency-associated transcript (LAT) that is antisense to a predominant viral alpha gene (ICP0) is found localized in the nucleus of sensory neurons. We disrupted both copies of the LAT gene in the HSV-1 genome by insertion of the Escherichia coli lacZ gene under LAT promoter control. The resulting recombinant virus, RH142, does not express any detectable LAT in either latently or productively infected cells, although beta-galactosidase expression is readily detectable in sensory neurons of latently infected mice. Expression was first detectable 3 days postinoculation and continued at approximately the same level for the entire experimental period (56 days). beta-Galactosidase expression was not detectable at any time during RH142 replication in Vero cells. Thus, the kinetics of expression and cell-type specificity of the LAT gene are distinct from other HSV-1 genes that are expressed during productive growth. When latently infected trigeminal ganglia were explanted, RH142 reactivated from latency with the kinetics and an efficiency indistinguishable from the parental wild-type virus. These studies argue against any possible antisense regulatory mechanism of LAT in the regulation of viral gene expression or any role of LAT-encoded protein during the establishment or maintenance of latency in the mouse.

  View details for Web of Science ID A1989AT78200066

  View details for PubMedID 2552449

• IDENTIFICATION AND PRELIMINARY USE OF RECOMBINANT LAMBDA GT11 FUSION PROTEINS IN HUMAN CYTOMEGALO-VIRUS DIAGNOSIS JOURNAL OF GENERAL VIROLOGY Ripalti, A., Landini, M. P., Mocarski, E. S., Laplaca, M. 1989; 70: 1247-1251

  Abstract

  We have isolated reactive clones from a lambda gt11 expression library of human cytomegalovirus (HCMV) DNA using HCMV-positive human sera. Among the recombinant clones obtained, one carried a fragment encoding a portion of p52, the major non-structural DNA-binding protein of 52K (p52) and another carried a part of the gene coding for pl50, the major structural phosphoprotein. These two fusion proteins were examined by immunoblot analysis to test their ability to bind specific antibodies in human sera. The results showed that high titres of antibody to the DNA-binding protein are present in sera of patients undergoing acute HCMV infection, whereas high titres of antibodies to the structural phosphoprotein are widespread in the healthy HCMV-seropositive population. The use of these fusion proteins as antigens for differential screening of serum as a way of detecting recent HCMV infection is discussed.

  View details for Web of Science ID A1989U619900021

  View details for PubMedID 2543778

• REGULATION OF CYTOMEGALO-VIRUS LATE-GENE EXPRESSION - DIFFERENTIAL USE OF 3 START SITES IN THE TRANSCRIPTIONAL ACTIVATION OF ICP36 GENE-EXPRESSION JOURNAL OF VIROLOGY Leach, F. S., Mocarski, E. S. 1989; 63 (4): 1783-1791

  Abstract

  We have investigated the transcriptional regulation of the human cytomegalovirus gamma gene encoding the ICP36 family (p52, the major late DNA-binding protein). The ICP36 transcription unit initiates at three distinct sites which are separated by approximately 50 nucleotides and are differentially regulated during infection. At early times (8 h postinfection), only two of these start sites, the most proximal and distal site, were active whereas at late times (36 h postinfection), the middle start site was activated. Expression from this late start site was dependent upon DNA replication. Consensus TATA elements were located upstream of all three start sites, although the element upstream of the late start site was unusual in both sequence and position when compared with conventional TATA elements. Deletion analysis was used in conjunction with transient assays to define independent promoters in this region. The two early start sites and associated TATA elements functioned as separable independently regulated promoters. The region containing the late start site and TATA element but excluding either of the flanking TATA elements was inactive in transient assays. Our work establishes that the ICP36 gene is under complex early and late transcriptional regulation and that the sequences regulating transcriptional activation are temporally and spatially distinct.

  View details for Web of Science ID A1989T678700033

  View details for PubMedID 2538657

• HERPES-SIMPLEX VIRUS-1 HELICASE PRIMASE - A COMPLEX OF 3 HERPES-ENCODED GENE-PRODUCTS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Crute, J. J., Tsurumi, T., Zhu, L., Weller, S. K., Olivo, P. D., Challberg, M. D., Mocarski, E. S., Lehman, I. R. 1989; 86 (7): 2186-2189

  Abstract

  In an earlier report, we described a DNA helicase that is specifically induced upon infection of Vero cells with herpes simplex virus 1. We have purified this enzyme to near homogeneity and found it to consist of three polypeptides with molecular weights of 120,000, 97,000, and 70,000. Immunochemical analysis has shown these polypeptides to be the products of three of the genes UL52, UL5, and UL8 that are required for replication of a plasmid containing a herpes simplex 1 origin (oriS). In addition to helicase activity, the enzyme contains a tightly associated DNA primase. Thus, the three-subunit enzyme is a helicase-primase complex that may prime lagging-strand synthesis as it unwinds DNA at the viral replication fork.

  View details for Web of Science ID A1989U042300014

  View details for PubMedID 2538835

• HUMAN CYTOMEGALOVIRUS-IE1 TRANSACTIVATES THE ALPHA-PROMOTER-ENHANCER VIA AN 18-BASE-PAIR REPEAT ELEMENT JOURNAL OF VIROLOGY Cherrington, J. M., Mocarski, E. S. 1989; 63 (3): 1435-1440

  Abstract

  The expression of alpha (immediate-early) genes of cytomegalovirus is regulated via a complex enhancer that consists of several different repeat elements. We describe here the autoinduction of expression from the alpha promoter-enhancer by the most abundant alpha gene product, a 491-amino-acid nuclear phosphoprotein referred to as ie1. We defined the 18-base-pair repeat element within the alpha enhancer as the signal through which ie1 acts to regulate gene expression. This element contains an NF kappa B site that may play an important role in ie1 autoregulation. Our analysis, which relied on deletions through the enhancer as well as reconstitution of responsiveness to a promoter with synthetic 18-base-pair repeats, strongly implicated ie1 in the transcriptional transactivation of the alpha promoter through its enhancer.

  View details for Web of Science ID A1989T291000054

  View details for PubMedID 2536844

• A METHOD TO IDENTIFY DISTINCTIVE CHARGE CONFIGURATIONS IN PROTEIN SEQUENCES, WITH APPLICATION TO HUMAN HERPESVIRUS POLYPEPTIDES JOURNAL OF MOLECULAR BIOLOGY Karlin, S., Blaisdell, B. E., Mocarski, E. S., Brendel, V. 1989; 205 (1): 165-177

  Abstract

  Charge interactions are of great importance for protein function and structure, and for a variety of cellular and biochemical processes. We present a systematic approach to the detection of distinctive clusters, runs and periodic patterns of charged residues in a protein sequence. Criteria and formulae are set forth to assess statistical significance of these charge configurations. For the 80-odd proteins potentially encoded by the Epstein-Barr virus, only the major nuclear antigens of the latent state and the transactivator of the lytic cycle contain separated charge clusters of opposite sign as well as periodic charge patterns. From our studies of the polypeptides of the human herpesviruses and of a broad collection of human and other viral protein sequences, distinctive charge configurations appear to be associated with viral capsid and core proteins (positive clusters or runs, mostly at the carboxyl terminus), with many viral glycoproteins and membrane-associated proteins (negative charge clusters), and with transactivators and transforming proteins (multiple charge structures). The statistics developed in this paper apply more generally to other than charge properties of a protein and should aid in the evaluation of a large variety of sequence features.

  View details for Web of Science ID A1989R914200013

  View details for PubMedID 2538622

• INSERTIONAL MUTAGENESIS OF THE MURINE CYTOMEGALO-VIRUS GENOME - ONE PROMINENT ALPHA-GENE (IE2) IS DISPENSABLE FOR GROWTH VIROLOGY Manning, W. C., Mocarski, E. S. 1988; 167 (2): 477-484

  Abstract

  We have utilized insertional mutagenesis to investigate the functional importance of murine cytomegalovirus (MCMV) immediate early (IE or alpha) region 2 (ie2) in replication. We constructed a recombinant virus, RM408, that carries the Escherichia coli lacZ gene under transcriptional control of the MCMV alpha promoter/enhancer inserted within the ie2 transcription unit. RM408 carries the first defined genetic mutation in a specific CMV gene. After infection of NIH3T3 cells, RM408 expressed beta-galactosidase in abundance and with kinetics indistinguishable from those of the natural ie1 gene product which is left intact in the recombinant. We detected no expression from the ie2 region in RM408-infected cells. Growth kinetics, yield, and expression of other viral genes in RM408 was similar to wild-type MCMV, indicating that the ie2 gene product was nonessential for growth in cell culture.

  View details for Web of Science ID A1988R459200018

  View details for PubMedID 2849236

• Biology and replication of cytomegalovirus. Transfusion medicine reviews Mocarski, E. S. 1988; 2 (4): 229-234

  View details for PubMedID 2856527

• BETA-GALACTOSIDASE AS A MARKER IN THE PERIPHERAL AND NEURAL TISSUES OF THE HERPES-SIMPLEX VIRUS-INFECTED MOUSE VIROLOGY Ho, D. Y., Mocarski, E. S. 1988; 167 (1): 279-283

  Abstract

  We have inserted a modified Escherichia coli lacZ gene, placed under the control of herpes simplex virus alpha 4 or beta 8 regulatory signals, into the HSV-1 genome disrupting the viral thymidine kinase gene. Using beta-galactosidase as an in situ indicator of viral gene expression, we detected expression from these recombinant HSV in dermal and neural tissues of the BALB/c mouse. Our detection of beta-galactosidase expression in neuronal cells indicates that TK-deficient viruses are capable of invading mouse neuronal cells and expressing up to the beta class of gene product.

  View details for Web of Science ID A1988Q895500033

  View details for PubMedID 2847416

• THE CLEAVAGE RECOGNITION SIGNAL IS CONTAINED WITHIN SEQUENCES SURROUNDING AN A-A JUNCTION IN HERPES-SIMPLEX VIRUS-DNA VIROLOGY NASSERI, M., Mocarski, E. S. 1988; 167 (1): 25-30

  Abstract

  Herpesvirus genome maturation involves site-specific cleavage of viral DNA concatemers and encapsidation of unit-length molecules, processes that are apparently coupled. Here, applying a transfection-infection approach, we have investigated the arrangement of the DNA sequence elements involved in cleavage and shown that specific cleavage occurs independently of DNA replication. We show that the cis-acting signal for cleavage is located within a 179-bp fragment from across an a-a junction formed as part of the genome maturation process of herpes simplex virus 1. Plasmids carrying the 179-bp fragment are cleaved at the appropriate site even though they are unable to replicate in HSV-infected cells. When linked to an origin, the same 179-bp a-a fragment will replicate and package into progeny virus as a defective genome. Two highly conserved homologies, pac1 and pac2, that have been observed in all herpesviruses examined, including cytomegalovirus, Epstein-Barr virus, varicella-zoster virus, and herpes simplex virus 2 as well as the herpes simplex virus 1 genome, are contained within the 179-bp fragment. This suggests that a common mechanism is utilized for genome maturation in the herpesvirus group.

  View details for Web of Science ID A1988Q895500004

  View details for PubMedID 2847414

• HUMAN CYTOMEGALO-VIRUS ICP22, THE PRODUCT OF THE HWLF1 READING FRAME, IS AN EARLY NUCLEAR-PROTEIN THAT IS RELEASED FROM CELLS JOURNAL OF GENERAL VIROLOGY Mocarski, E. S., Pereira, L., McCormick, A. L. 1988; 69: 2613-2621

  Abstract

  We employed a murine monoclonal antibody (CH41) and a lambda gt11 library of human cytomegalovirus (CMV) DNA fragments to map the gene for a viral protein, denoted infected cell protein (ICP) 22, to the HWLF1 open reading frame in the S component of the CMV genome (0.92 to 0.93 map units). By using antibody CH41 in immunofluorescence, immunoprecipitation and immunoblotting analyses, ICP22 was readily detected as a beta (delayed early) gene product during viral growth. The cellular localization of this protein was found to be nuclear by immunofluorescence analysis; however, it partitioned with the cytoplasm when cells were fractionated with non-ionic detergents. Analysis of cell-free medium showed that a proportion of ICP22 was released from cells as a soluble protein at both early (24 h) and late (72 to 120h) times in infection. The function of this protein which has such diverse characteristics remains unknown.

  View details for Web of Science ID A1988Q333200018

  View details for PubMedID 2844970

• TRANSLATIONAL CONTROL OF CYTOMEGALO-VIRUS GENE-EXPRESSION IS MEDIATED BY UPSTREAM AUG CODONS JOURNAL OF VIROLOGY Geballe, A. P., Mocarski, E. S. 1988; 62 (9): 3334-3340

  Abstract

  Human cytomegalovirus regulates gene expression at both the transcriptional and posttranscriptional levels. We have characterized posttranscriptional signals carried on the RNA of one beta (or delayed-early) gene, signals that we show delay the translation of an actively transcribed gene. The cis-acting components of this signal map to two upstream AUG codons in the 5' leader of the beta gene transcript that act to delay the kinetics and reduce the levels of gene expression. Both upstream AUG codons are part of short open reading frames, and the presence of either is sufficient to suppress expression of a downstream open reading frame. These upstream natural AUG codons appear to act in cis at the level of translation, consistent with the scanning hypothesis of eucaryotic translation. The occurrence of natural upstream AUG codons in other cytomegalovirus transcripts suggests their physiological significance in influencing the kinetics and steady-state levels of gene expression during viral growth.

  View details for Web of Science ID A1988P689000033

  View details for PubMedID 2841486

• VIRAL VECTORS - A MEETING REVIEW GENES & DEVELOPMENT Mocarski, E. 1988; 2 (8): 926-928

  View details for Web of Science ID A1988P720200002

• A DNA helicase induced by herpes simplex virus type 1. Nucleic acids research Crute, J. J., Mocarski, E. S., Lehman, I. R. 1988; 16 (14A): 6585-6596

  Abstract

  We have identified and partially purified a DNA-dependent ATPase that is present specifically in herpes simplex virus type 1-infected Vero cells. The enzyme which has a molecular weight of approximately 440,000 differs from the comparable host enzyme in its elution from phosphocellulose columns and in its nucleoside triphosphate specificity. The partially purified DNA-dependent ATPase is also a DNA helicase that couples ATP or GTP hydrolysis to the displacement of an oligonucleotide annealed to M13 single-stranded DNA. The enzyme requires a 3' single-stranded tail on the duplex substrate, suggesting that the polarity of unwinding is 5'----3' relative to the M13 DNA. The herpes specific DNA helicase may therefore translocate on the lagging strand in the semidiscontinuous replication of the herpes virus 1 genome.

  View details for PubMedID 2840645

• A DNA HELICASE INDUCED BY HERPES-SIMPLEX VIRUS TYPE-1 NUCLEIC ACIDS RESEARCH Crute, J. J., Mocarski, E. S., Lehman, I. R. 1988; 16 (14): 6585-6596

  Abstract

  We have identified and partially purified a DNA-dependent ATPase that is present specifically in herpes simplex virus type 1-infected Vero cells. The enzyme which has a molecular weight of approximately 440,000 differs from the comparable host enzyme in its elution from phosphocellulose columns and in its nucleoside triphosphate specificity. The partially purified DNA-dependent ATPase is also a DNA helicase that couples ATP or GTP hydrolysis to the displacement of an oligonucleotide annealed to M13 single-stranded DNA. The enzyme requires a 3' single-stranded tail on the duplex substrate, suggesting that the polarity of unwinding is 5'----3' relative to the M13 DNA. The herpes specific DNA helicase may therefore translocate on the lagging strand in the semidiscontinuous replication of the herpes virus 1 genome.

  View details for Web of Science ID A1988P435100023

  View details for PubMedCentralID PMC338315

• INSERTION AND DELETION MUTAGENESIS OF THE HUMAN CYTOMEGALOVIRUS GENOME PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Spaete, R. R., Mocarski, E. S. 1987; 84 (20): 7213-7217

  Abstract

  Studies on human cytomegalovirus (CMV) have been limited by a paucity of molecular genetic techniques available for manipulating the viral genome. We have developed methods for site-specific insertion and deletion mutagenesis of CMV utilizing a modified Escherichia coli lacZ gene as a genetic marker. The lacZ gene was placed under the control of the major beta gene regulatory signals and inserted into the viral genome by homologous recombination, disrupting one of two copies of this beta gene within the L-component repeats of CMV DNA. We observed high-level expression of beta-galactosidase by the recombinant in a temporally authentic manner, with levels of this enzyme approaching 1% of total protein in infected cells. Thus, CMV is an efficient vector for high-level expression of foreign gene products in human cells. Using back selection of lacZ-deficient virus in the presence of the chromogenic substrate 5-bromo-4-chloro-3-indolyl beta-D-galactoside, we generated random endpoint deletion mutants. Analysis of these mutants revealed that CMV DNA sequences flanking the insert had been removed, thereby establishing this approach as a means of determining whether sequences flanking a lacZ insertion are dispensable for viral growth. In an initial test of the methods, we have shown that 7800 base pairs of one copy of L-component repeat sequences can be deleted without affecting viral growth in human fibroblasts.

  View details for Web of Science ID A1987K504900053

  View details for PubMedID 2823255

• A CYTOMEGALOVIRUS PROTEIN WITH PROPERTIES OF HERPES-SIMPLEX VIRUS ICP8 - PARTIAL-PURIFICATION OF THE POLYPEPTIDE AND MAP POSITION OF THE GENE JOURNAL OF VIROLOGY Kemble, G. W., McCormick, A. L., Pereira, L., Mocarski, E. S. 1987; 61 (10): 3143-3151

  Abstract

  We demonstrated the presence of a single-stranded DNA-binding protein in human cytomegalovirus (CMV)-infected cells with properties analogous to those of herpes simplex virus (HSV) ICP8. Using monoclonal antibody specific for the CMV protein, we analyzed its fluorescence pattern and time of synthesis, mapped the gene encoding it by using a lambda gt11 library of CMV DNA fragments, and monitored its purification by phosphocellulose and DNA-Sepharose chromatography. In all characteristics we examined, the CMV protein behaved analogously to HSV ICP8. Our results are consistent with a functional role of CMV ICP8 in viral replication that is similar to that of HSV ICP8 and with the evolutionary conservation of the gene of interest in two divergent herpesviruses.

  View details for Web of Science ID A1987J994200026

  View details for PubMedID 3041036

• STRUCTURE AND VARIABILITY OF THE A-SEQUENCE IN THE GENOME OF HUMAN CYTOMEGALOVIRUS (TOWNE STRAIN) JOURNAL OF GENERAL VIROLOGY Mocarski, E. S., Liu, A. C., Spaete, R. R. 1987; 68: 2223-2230

  Abstract

  We have defined the boundaries of the a sequence from human cytomegalovirus (CMV) strain Towne, characterized internal variability and determined the position of the cleavage site used to generate genomic termini. The cleavage site is positioned a fixed distance from two stretches of sequence homology that have been observed near the ends of many herpesvirus genomes. Unlike a comparable region in CMV (AD169), the CMV (Towne) a sequence has a relatively low level of variability within the a sequence and its structure is stable through repeated virus passage.

  View details for Web of Science ID A1987J777800019

  View details for PubMedID 3039048

• A CIS-ACTING ELEMENT WITHIN THE 5' LEADER OF A CYTOMEGALOVIRUS BETA-TRANSCRIPT DETERMINES KINETIC CLASS CELL Geballe, A. P., Spaete, R. R., Mocarski, E. S. 1986; 46 (6): 865-872

  Abstract

  During cytomegalovirus (CMV) infection, gene expression is regulated by transcriptional and posttranscriptional events. Although recent studies have established that posttranscriptional controls are important determinants of gene expression in several eukaryotic systems, the precise signals and mechanisms have not been clearly identified. We present evidence for a cis-acting signal, contained within the 5' leader region of a CMV beta (or early) gene, that acts posttranscriptionally in the regulation of gene expression. Addition of this signal to an alpha (or immediate early) gene construct converted expression of the indicator protein to the beta class, even though the gene remained under alpha transcriptional control. Deletion of a portion of the beta gene leader sequence (nucleotides +62 to +142) reverted expression to the alpha class. This cis-dominant signal appears to act by blocking expression posttranscriptionally until a viral function activates full gene expression at the appropriate time in infection.

  View details for Web of Science ID A1986E058200008

  View details for PubMedID 3019554

• A DNA-BINDING PROTEIN-SPECIFIC FOR AN ORIGIN OF REPLICATION OF HERPES-SIMPLEX VIRUS TYPE-1 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Elias, P., ODONNELL, M. E., Mocarski, E. S., Lehman, I. R. 1986; 83 (17): 6322-6326

  Abstract

  We have identified a protein that binds specifically to an origin of replication (oris) of the herpes simplex virus type 1 genome. The oris binding protein, detectable only in nuclear extracts of infected cells, shows the same time course of appearance as the herpesvirus-induced DNA polymerase and the DNA binding protein ICP8. The partially purified oris binding protein generates a DNase I "footprint" that spans 18- of the 90-base-pair minimal oris sequence. The oris binding protein may, therefore, be analogous to other origin-specific binding proteins that are required for the initiation of viral and chromosomal DNA replication.

  View details for Web of Science ID A1986D837900020

  View details for PubMedID 3018724

  View details for PubMedCentralID PMC386495

• CYTOMEGALOVIRUS VIREMIA INCREASES WITH PROGRESSIVE IMMUNE-DEFICIENCY IN PATIENTS INFECTED WITH HTLV-III AIDS RESEARCH Fiala, M., Cone, L. A., CHANG, C. M., Mocarski, E. S. 1986; 2 (3): 175-181

  Abstract

  Cytomegalovirus (CMV) viremia was studied in 15 homosexual patients [two healthy, nine with the acquired immune deficiency syndrome (AIDS), five with the AIDS-related complex (ARC)], in one patient with transfusion-related AIDS, and in four individuals with cancer or connective tissue disorders. While CMV viremia was absent in the latter patients, in the healthy homosexuals, and in three stable ARC patients, it was present in clinically-deteriorating ARC patients and in all nine AIDS patients. Some CMV isolates caused limited proliferation of fibroblasts in the culture medium. The rapidity and ease of CMV isolation from leukocytes of patients at different stages of infection with human T-lymphocytotropic virus type III (HTLV-III) indicate that the CMV titer in leukocytes increases as immune deficiency worsens. In ARC patients CMV viremia may be predictive of clinical complications in the next 3 months. In AIDS patients CMV viremia at high titer was an unfavorable prognostic sign.

  View details for Web of Science ID A1986E059600003

  View details for PubMedID 3019362

• REGULATION OF CYTOMEGALOVIRUS LATE GENE-EXPRESSION - GAMMA-GENES ARE CONTROLLED BY POSTTRANSCRIPTIONAL EVENTS JOURNAL OF VIROLOGY Geballe, A. P., Leach, F. S., Mocarski, E. S. 1986; 57 (3): 864-874

  Abstract

  We investigated the control of human cytomegalovirus (CMV) late (gamma)-gene expression in human fibroblast cells. Transcriptional activity of two gamma genes, encoding ICP27, a structural component (matrix or tegument) of virions, and ICP36, a major DNA-binding protein family, was followed by analysis of steady-state RNA levels during viral infection. Synthesis of the protein products of these genes was analyzed with specific monoclonal antibodies in conjunction with sensitive immunoblot or immunoprecipitation analysis. Although accumulation of ICP27 and ICP36 was not abundant until late times, both late genes were as transcriptionally active at early times (4 h postinfection) as at late times (48 h postinfection). Reduced amounts (less than 5% of late levels) of the protein products were detected at early times, demonstrating that a small proportion of the ICP27 and ICP36 RNA made at this time was translated. These observations establish that expression of at least two CMV gamma genes is regulated through posttranscriptional events. The very early transcriptional activation of late genes and the relative importance of posttranscriptional regulation to late-gene expression distinguishes CMV from other well-studied herpesviruses and does not appear analogous to late-gene regulation in any other DNA animal virus.

  View details for Web of Science ID A1986A038400020

  View details for PubMedID 3005633

• REGULATION OF CYTOMEGALO-VIRUS GENE-EXPRESSION - ALPHA-PROMOTERS AND BETA-PROMOTERS ARE TRANS ACTIVATED BY VIRAL FUNCTIONS IN PERMISSIVE HUMAN-FIBROBLASTS JOURNAL OF VIROLOGY Spaete, R. R., Mocarski, E. S. 1985; 56 (1): 135-143

  Abstract

  We have fused immediate (alpha) and delayed (beta) early promoter-regulatory sequences taken from the cytomegalovirus (CMV) genome to Escherichia coli lacZ (beta-galactosidase) as an indicator gene to study regulated expression of these promoters. After transfection of human fibroblast cells with plasmid constructs carrying beta-galactosidase fusions, and subsequent infection with CMV, we have demonstrated that viral trans-acting functions up-regulate the expression of these genes in a temporally authentic manner. The alpha promoter is activated even when de novo protein synthesis is blocked and when UV-inactivated virus is used, suggesting that, as for herpes simplex virus type 1 (HSV-1), a virion structural protein is responsible for its up-regulation. We have found that HSV-1, as well as CMV, is capable of trans activating the CMV alpha promoter. The beta promoter is activated by CMV but is completely unresponsive to HSV-1 infection. The temporal synthesis of the alpha and beta promoters in the transient expression system conforms with their natural regulation during viral replication. The beta-galactosidase fusions we describe provide a most exquisitely sensitive indicator system for the study of cis- and trans-acting viral regulatory functions.

  View details for Web of Science ID A1985AQY6700018

  View details for PubMedID 2993644

• NUCLEOTIDE-SEQUENCE AND STRUCTURAL FEATURES OF A NOVEL US-A JUNCTION PRESENT IN A DEFECTIVE HERPES-SIMPLEX VIRUS GENOME JOURNAL OF VIROLOGY Mocarski, E. S., Deiss, L. P., Frenkel, N. 1985; 55 (1): 140-146

  Abstract

  Defective genomes generated during serial propagation of herpes simplex virus type 1 (Justin) consist of tandem reiterations of sequences that are colinear with a portion of the S component of the standard viral genome. We determined the structure of the novel US-a junction, at which the US sequences of one repeat unit join the a sequences of the adjacent repeat unit. Comparison of the nucleotide sequence at this junction with the nucleotide sequence of the corresponding US region of the standard virus genome indicated that the defective genome repeat unit arose by a single recombinational event between an L-S junction a sequence and the US region. The recombinational process might have been mediated by limited sequence homology. The sequences retained within the US-a junction further define the signal for cleavage and packaging of viral DNA.

  View details for Web of Science ID A1985AKH8700019

  View details for PubMedID 2989551

• PRECISE LOCALIZATION OF GENES ON LARGE ANIMAL VIRUS GENOMES - USE OF LAMBDA-GT11 AND MONOCLONAL-ANTIBODIES TO MAP THE GENE FOR A CYTOMEGALO-VIRUS PROTEIN FAMILY PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Mocarski, E. S., Pereira, L., Michael, N. 1985; 82 (4): 1266-1270

  Abstract

  We describe an efficient procedure, which uses monoclonal antibodies directed against specific viral proteins, for the precise mapping of genes on large DNA virus genomes. We have used the technique to locate the gene encoding a family of antigenically related DNA-binding proteins on the 240-kilobase-pair human cytomegalovirus (CMV) genome. A random library of CMV DNA fragments was generated using the prokaryotic vector lambda gt11, which expresses open reading frames as beta-galactosidase fusion proteins in infected Escherichia coli. The library was screened with a mixture of monoclonal antibodies directed against the gene products of interest. The coding sequence for infected cell protein 36 (ICP36) was localized to a 2800-base-pair EcoRI fragment (map coordinates 0.228-0.240) on the CMV(Towne) and CMV(AD169) genomes by using DNA from immunoreactive lambda gt11 as probe. A 5000-nucleotide transcript from this region was detected during the early and late phases of the CMV growth cycle. This transcript directed the synthesis of the predominant member of the ICP36 family when hybrid-selected and translated in vitro. Immunoprecipitation of the in vitro translation product with the same monoclonal antibodies used in the initial mapping confirmed the location of the ICP36 gene. These studies establish the utility of the lambda gt11 expression system for rapid and precise mapping of CMV genes (or other large animal virus genes) that encode proteins for which serological reagents exist.

  View details for Web of Science ID A1985ADL2500066

  View details for PubMedID 2983334

• THE A-SEQUENCE OF THE CYTOMEGALO-VIRUS GENOME FUNCTIONS AS A CLEAVAGE PACKAGING SIGNAL FOR HERPES-SIMPLEX VIRUS DEFECTIVE GENOMES JOURNAL OF VIROLOGY Spaete, R. R., Mocarski, E. S. 1985; 54 (3): 817-824

  Abstract

  Although herpes simplex virus (HSV) 1 and human cytomegalovirus (CMV) differ remarkably in their biological characteristics and do not share nucleotide sequence homology, they have in common a genome structure that undergoes sequence isomerization of the long (L) and short (S) components. We have demonstrated that the similarity in their genome structures extends to the existence of an alpha sequence in the CMV genome as previously defined for the HSV genome. As such, the alpha sequence is predicted to participate as a cis-replication signal in four viral functions: (i) inversion, (ii) circularization, (iii) amplification, and (iv) cleavage and packaging of progeny viral DNA. We have constructed a chimeric HSV-CMV amplicon (herpesvirus cis replication functions carried on an Escherichia coli plasmid vector) substituting CMV DNA sequences for the HSV cleavage/packaging signal in a test of the ability of this CMV L-S junction sequence to provide the cis signal for cleavage/packaging in HSV 1-infected cells. We demonstrate that the alpha sequence of CMV DNA functions as a cleavage/packaging signal for HSV defective genomes. We show the structure of this sequence and provide a functional demonstration of cross complementation in replication signals which have been preserved over evolutionary time in these two widely divergent human herpesviruses.

  View details for Web of Science ID A1985AHT1900022

  View details for PubMedID 2987533

• MULTIPLE INFECTIONS BY CYTOMEGALOVIRUS IN PATIENTS WITH ACQUIRED IMMUNODEFICIENCY SYNDROME - DOCUMENTATION BY SOUTHERN BLOT HYBRIDIZATION JOURNAL OF INFECTIOUS DISEASES Drew, W. L., SWEET, E. S., Miner, R. C., Mocarski, E. S. 1984; 150 (6): 952-953

  View details for Web of Science ID A1984TT57700023

  View details for PubMedID 6094678

• HERPES-VIRUS-DEPENDENT AMPLIFICATION AND INVERSION OF CELL-ASSOCIATED VIRAL THYMIDINE KINASE GENE FLANKED BY VIRAL-A SEQUENCES AND LINKED TO AN ORIGIN OF VIRAL-DNA REPLICATION PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES Mocarski, E. S., Roizman, B. 1982; 79 (18): 5626-5630

  Abstract

  The genome of herpes simplex virus 1 or 2 consists of two components, L and S, which invert relative to each other during infection. As a result, viral DNA consists of four equimolar populations of molecules differing solely in the relative orientations of the L and S components. Previous studies have shown that the a sequences, located in the same orientation at the genomic termini and in inverted orientation at the L-S junction, play a key role in the inversion of L and S components. In this report we describe a virus-dependent system designed to allow identification of the viral genes capable of acting in trans to invert DNA flanked by inverted copies of a sequences. In this system, cells are converted to the thymidine kinase-positive phenotype with a chimeric plasmid carrying the thymidine kinase gene flanked by inverted copies of the a sequence and linked to an origin of viral DNA replication derived from the S component. The DNA introduced into the cells is retained and propagated in its original sequence arrangement as head-to-tail concatemers. Infection of these cells with herpes simplex virus 1 or 2 results in as much as 100-fold amplification of the plasmid sequences and inversion of the DNA flanked by copies of the a sequence. In infected cells, the amplified resident DNA accumulates in head-to-tail concatemers and no rearrangement other than the inversions could be detected. These results suggest that the a sequence-dependent inversions required trans-acting viral gene products.

  View details for Web of Science ID A1982PH57400038

  View details for PubMedID 6291055

• STRUCTURE AND ROLE OF THE HERPES-SIMPLEX VIRUS-DNA TERMINI IN INVERSION, CIRCULARIZATION AND GENERATION OF VIRION DNA CELL Mocarski, E. S., Roizman, B. 1982; 31 (1): 89-97

  Abstract

  The herpes simplex virus genome consists of two components, L and S, which invert relative to each other during viral replication. The a sequence is present at the genomic termini in direct orientation and at the L-S junction in inverted orientation. Previously, we showed that insertion of a fragment spanning the L-S junction into the viral genome causes additional inversions. In this study, we determine the nucleotide sequence of the genomic termini and show that insertion of either the free S terminus or the L terminus causes inversions in the viral genome. We conclude that the a sequence is the inversion-specific sequence, that linear unit-length molecules packaged in virions are generated by cleavage between adjacent copies of the a sequence, that cleavage produces 3' single-base extensions on the genomic termini and that the signal for cleavage is contained within the a sequence.

  View details for Web of Science ID A1982PQ46100011

  View details for PubMedID 6297756

• SITE-SPECIFIC INVERSION SEQUENCE OF THE HERPES-SIMPLEX VIRUS GENOME - DOMAIN AND STRUCTURAL FEATURES PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES Mocarski, E. S., Roizman, B. 1981; 78 (11): 7047-7051

  Abstract

  The genome of herpes simplex virus-1 consists of two covalently linked components, L and S, that invert relative to each other. The L and S components consist of unique DNA sequences bracketed by inverted repeats. The inverted repeats of the L component are designated ab and b' a' and those of the S component are designated a' c' and ca. The number of a sequences at the termini and at the L-S component junction varies from one to several copies. Insertion into the middle of the L component of a DNA fragment consisting of 156 base pairs (bp) of the b sequence, an entire a sequence of 501 bp, and 618 bp of the c sequence created a new site through which additional inversions in the genome occurred. Comparison of the nucleotide sequences of DNA fragments containing one and two a sequences defined the domain of the a sequence. The single a sequence consists of two 20-bp direct repeats (designated as DR1) bracketing a region that contains 19 tandem direct repeats of a 12-bp sequence (DR2) adjacent to three direct repeats of a 37-bp sequence (DR4), in addition to short stretches of unique sequences. The fragment with two tandem a sequences contained three copies of DR1-i.e., the intervening DR1 was shared by the two a sequences. Furthermore, one a sequence contained 22 copies of DR2 and two copies of DR4 whereas the second a sequence contained 19 copies of DR2 and two copies of DR4. These observations suggest that (i) amplification of the number of terminal and internal a sequences is the consequence of intramolecular or intermolecular recombination through DR1, (ii) the number of copies of DR2 and DR4 within the a sequence is not fixed and may vary as a consequence of unequal crossing over or slippage, and (iii) inversion results from intramolecular recombination between terminal and inverted a sequences.

  View details for Web of Science ID A1981MQ92600095

  View details for PubMedID 6273905

• MOLECULAR ENGINEERING OF THE HERPES-SIMPLEX VIRUS GENOME - INSERTION OF A SECOND L-S JUNCTION INTO THE GENOME CAUSES ADDITIONAL GENOME INVERSIONS CELL Mocarski, E. S., Post, L. E., Roizman, B. 1980; 22 (1): 243-255

  Abstract

  We have developed a technique for the insertion of any DNA fragment into the herpes simplex virus (HSV) genome at specific sites. This technique was used to resolve a specific problem concerning the isomerization of the HSV genome. Briefly, HSV DNA consists of four isomers differing in the orientation of two covalently linked components, L and S, relative to each other. Each component consists of unique sequences flanked by inverted repeats. To determine whether the isomerization of HSV DNA is the result of generalized recombinatin between homologous reiterated sequences in the inverted repeats or the result of site-specific recombination, we constructed plasmids in which DNA fragments derived from various regions of the viral genome were inserted in both orientations into the thymidine kinase gene, rendering it nonfunctional. The HSV DNA sequences in the plasmids were then recombined into the viral genome, and viral recombinants were selected for their thymidine kinase-deficient phenotype. The insertion of these fragments by homologous recombination was highly efficient in that all the viral clones isolated contained the inserted fragment at the expected location. The only fragments that promoted additional inversions of the viral genome were those spanning the junction between the L and S components. Furthermore, analysis of isomers formed by these recombinants indicates that the inversions occur only when sequences in the inserted fragment are in inverted orientation in relation to homologous sequences at the termini or at the authentic junction.

  View details for Web of Science ID A1980KP49400028

  View details for PubMedID 6253078

• CLONING OF REITERATED AND NONREITERATED HERPES-SIMPLEX VIRUS-1 SEQUENCES AS BAMHI FRAGMENTS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES Post, L. E., Conley, A. J., Mocarski, E. S., Roizman, B. 1980; 77 (7): 4201-4205

  Abstract

  Over 95% of the herpes simplex virus type 1 (strain F) DNA sequences have been cloned as BamHI fragments in the pBR322 plasmid. With one exception, all of the cloned fragments have the same electrophoretic mobilities and restriction enzyme cleavage sites as do the authentic fragments derived from the BamHI digests of the viral genome. The exception is the BamHI B fragment mapping at the right end of L component in the prototype arrangement of the DNA. Thus, a small deletion mapping near the left end of the fragment was present in two independently derived plasmids. Included in the collection of plasmids are several clones containing DNA sequences that span the junction between the L and S components of the virus DNA. Several plasmids containing the junction fragment were found to be sufficiently stable to permit the preparation of large amounts of the DNA fragment for fine-structure mapping of the restriction enzyme cleavage sites. Preliminary studies on one cloned fragment (BamHI G) have shown that it is biologically active in marker rescue of a temperature-sensitive mutation and in transfer of a plaque morphology marker.

  View details for Web of Science ID A1980KB88900094

  View details for PubMedID 6254015

• Membrane glycoproteins and antigens induced by human cytomegalovirus. journal of general virology Stinski, M. F., Mocarski, E. S., Thomsen, D. R., Urbanowski, M. L. 1979; 43 (1): 119-129

  Abstract

  Early after infection of cells with cytomegalovirus, the membranes were modified with respect to both glycoprotein composition and immunological specificity. Virus-specified antigens were inserted into the plasma membrane at 24 h after infection, as much as 2 days before virion and dense body maturation. Although at least four virus-induced glycoproteins were synthesized and bound to plasma and microsomal membranes between 20 and 24 h after infection, virus-specified antigen accumulated primarily on the plasma membrane. In contrast, at late times (72 h) after infection when virus nucleocapsids can be detected in the necleus, virus-specified antigen was prominent on the plasma, endoplasmic reticulum and nuclear membranes. It is proposed that the virus-specified glycoproteins and antigens of this herpesvirus accumulate first on the plasma membrane and then on internal membranes. The appearance of virus-specified antigen on internal membranes coincides with the commencement of virion and dense body envelopment.

  View details for PubMedID 225413

• DNA OF HUMAN CYTOMEGALO-VIRUS - SIZE HETEROGENEITY AND DEFECTIVENESS RESULTING FROM SERIAL UNDILUTED PASSAGE JOURNAL OF VIROLOGY Stinski, M. F., Mocarski, E. S., Thomsen, D. R. 1979; 31 (1): 231-239

  Abstract

  The majority of DNA molecules associated with plaque-purified, low-multiplicity-passaged human cytomegalovirus (Towne strain) had a molecular weight of approximately 150 x 10(6) and a molecular complexity of approximately 140 x 10(6). Serial high-multiplicity passage resulted in the production of defective cytomegalovirions. An accumulation of smaller DNA molecules packaged into virions was directly correlated with a decrease in infectivity and an increase in the particle-to-PFU ratio. The majority of defective DNA molecules had a molecular weight of approximately 100 x 10(6). In addition, there were some viral DNA molecules of approximately 60 x 10(6). Restriction enzyme analysis of defective cytomegalovirus DNA detected unique DNA fragments, suggesting a specific alteration in the nucleotide sequence. Some reasons for the generation of defective cytomegalovirus DNA are discussed.

  View details for Web of Science ID A1979HB43800025

  View details for PubMedID 228055

• PERSISTENCE OF THE CYTOMEGALO-VIRUS GENOME IN HUMAN-CELLS JOURNAL OF VIROLOGY Mocarski, E. S., Stinski, M. F. 1979; 31 (3): 761-775

  Abstract

  A small percentage of human fibroblast cells survived high-multiplicity infection by cytomegalovirus and were isolated as persistently infected cultures. Approximately 30% of the cells were in the productive phase of infection, since virus-specific structural antigens and virions were associated with these cells. The remaining cells contained neither viral structural antigens nor particles. Nuclear DNA from these nonproductive cells contained approximately 120 genome equivalents of viral DNA per cell as determined by reassociation kinetics. In situ hybridization confirmed that nuclei from nonproductive cells contained a significant amount of viral DNA that was distributed in most of these cells. Early virus-induced proteins and antigens were also detected. Nonproductive cells continued to grow, and there was a slow, spontaneous transition of some of these cells to productive viral replication. The majority of the viral DNA in nonproductive cells persisted with restricted gene expression. When infectious virus production was eliminated by growing the persistently infected cultures in the presence of anticytomegalovirus serum, approximately 45 genome equivalents of the viral DNA persisted per cell. The reassociation reaction approached completion. After removal of the antiserum and subculturing, infectious virus production resumed. Therefore, it was assumed that all sequences of the viral genome remained associated with these cells. Restriction of cytomegalovirus gene expression in persistently infected cell cultures is discussed.

  View details for Web of Science ID A1979HJ33900020

  View details for PubMedID 229251

• MEMBRANE-GLYCOPROTEINS AND ANTIGENS INDUCED BY HUMAN CYTOMEGALO-VIRUS JOURNAL OF GENERAL VIROLOGY Stinski, M. F., Mocarski, E. S., Thomsen, D. R., Urbanowski, M. L. 1979; 43 (APR): 119-129

  View details for Web of Science ID A1979GZ36600010