Bio


Dr. Arjun Rustagi is an Instructor in Medicine. He is a graduate of the University of Washington Medical Scientist Training Program and Stanford's Internal Medicine Residency and Infectious Diseases Fellowship.
He is dedicated to understanding the early immune responses to infections, and the methods pathogens use to persist despite these responses, in order to inform new prevention and treatment strategies. He studies viral pathogenesis and immunity with Dr. Catherine Blish, under whose mentorship he set up the respiratory viral biosafety level 3 (BSL3) program at Stanford in early 2020 in order to study SARS-CoV-2, contributing to several projects in SARS-CoV-2 virology including a study of intact in vitro lung tissue infection. He also protocoled the first peripheral immune profiling study of severe COVID-19. His ongoing interest is in the full spectrum of in vitro respiratory viral models, particularly those that recapitulate lung epithelium and mucosal immunity.

Clinical Focus


  • Infectious Diseases
  • Critical Illness
  • Viral Diseases
  • Respiratory Infections
  • Infectious Disease

Academic Appointments


Honors & Awards


  • Grant Awardee, Modeling early SARS-CoV-2 pathogenesis, 1K08AI163369, NIH, Stanford University (2022-2026)
  • Grant awardee, Applied Genomics in Infectious Diseases training grant 5T32 AI007502, NIH, Stanford University (2019-2022)
  • Elected, Alpha Omega Alpha (2015)
  • Grant awardee, STD/HIV Predoctoral Fellowship, 5T32 AI07140, NIH, University of Washington (2010-2013)

Boards, Advisory Committees, Professional Organizations


  • Member, Society for Natural Immunity (2019 - Present)
  • Member, Fellows College Council, Department of Medicine, Stanford University (2019 - 2021)
  • Member, Infectious Disease Society of America (2018 - Present)

Professional Education


  • Fellowship, Stanford University, Infectious Disease (2021)
  • Board Certification: American Board of Internal Medicine, Infectious Disease (2020)
  • Board Certification: American Board of Internal Medicine, Internal Medicine (2018)
  • Residency, Stanford University, Internal Medicine (2018)
  • Internship, Stanford University, Internal Medicine (2016)
  • Doctor of Medicine with Honors, University of Washington School of Medicine (2015)
  • Doctor of Philosophy, University of Washington, Department of Global Health, Pathobiology program, Innate antiviral immunity to HIV-1 (2013)
  • Bachelor of Science, Stanford University, Biological Sciences (2005)

Graduate and Fellowship Programs


All Publications


  • An orally bioavailable SARS-CoV-2 main protease inhibitor exhibits improved affinity and reduced sensitivity to mutations. Science translational medicine Westberg, M., Su, Y., Zou, X., Huang, P., Rustagi, A., Garhyan, J., Patel, P. B., Fernandez, D., Wu, Y., Hao, C., Lo, C. W., Karim, M., Ning, L., Beck, A., Saenkham-Huntsinger, P., Tat, V., Drelich, A., Peng, B. H., Einav, S., Tseng, C. K., Blish, C., Lin, M. Z. 2024; 16 (738): eadi0979

    Abstract

    Inhibitors of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) such as nirmatrelvir (NTV) and ensitrelvir (ETV) have proven effective in reducing the severity of COVID-19, but the presence of resistance-conferring mutations in sequenced viral genomes raises concerns about future drug resistance. Second-generation oral drugs that retain function against these mutants are thus urgently needed. We hypothesized that the covalent hepatitis C virus protease inhibitor boceprevir (BPV) could serve as the basis for orally bioavailable drugs that inhibit SARS-CoV-2 Mpro more efficiently than existing drugs. Performing structure-guided modifications of BPV, we developed a picomolar-affinity inhibitor, ML2006a4, with antiviral activity, oral pharmacokinetics, and therapeutic efficacy similar or superior to those of NTV. A crucial feature of ML2006a4 is a derivatization of the ketoamide reactive group that improves cell permeability and oral bioavailability. Last, ML2006a4 was found to be less sensitive to several mutations that cause resistance to NTV or ETV and occur in the natural SARS-CoV-2 population. Thus, anticipatory design can preemptively address potential resistance mechanisms to expand future treatment options against coronavirus variants.

    View details for DOI 10.1126/scitranslmed.adi0979

    View details for PubMedID 38478629

  • Vaccine-Boosted CCP Decreases Virus Replication and Hastens Resolution of Infection Despite Transiently Enhancing Disease in SARS-CoV-2-Infected Hamsters. The Journal of infectious diseases Carroll, T. D., Wong, T., Morris, M. K., Di Germanio, C., Ma, Z. M., Stone, M., Ball, E., Fritts, L., Rustagi, A., Simmons, G., Busch, M., Miller, C. J. 2024

    Abstract

    Definitive data demonstrating the utility of coronavirus disease 2019 (COVID-19) convalescent plasma (CCP) for treating immunocompromised patients remains elusive. To better understand the mechanism of action of CCP, we studied viral replication and disease progression in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected hamsters treated with CCP obtained from recovered COVID-19 patients that were also vaccinated with an mRNA vaccine, hereafter referred to as Vaxplas. Vaxplas transiently enhanced disease severity and lung pathology in hamsters treated near peak viral replication due to immune complex and activated complement deposition in pulmonary endothelium, and recruitment of M1 proinflammatory macrophages into the lung parenchyma. However, aside from one report, transient enhanced disease has not been reported in CCP recipient patients, and the transient enhanced disease in Vaxplas hamsters may have been due to mismatched species IgG-FcR interactions, infusion timing, or other experimental factors. Despite transient disease enhancement, Vaxplas dramatically reduced virus replication in lungs and improved infection outcome in SARS-CoV-2-infected hamsters.

    View details for DOI 10.1093/infdis/jiad568

    View details for PubMedID 38213276

  • Administration of vaccine-boosted COVID-19 convalescent plasma to SARS-CoV-2 infected hamsters decreases virus replication in lungs and hastens resolution of the infection despite transiently enhancing disease and lung pathology. bioRxiv : the preprint server for biology Carroll, T. D., Wong, T., Morris, M. K., Germanio, C. D., Ma, Z., Stone, M., Ball, E., Fritts, L., Rustagi, A., Simmons, G., Busch, M., Miller, C. J. 2023

    Abstract

    The utility of COVID-19 convalescent plasma (CCP) for treatment of immunocompromised patients who are not able to mount a protective antibody response against SARS-CoV-2 and who have contraindications or adverse effects from currently available antivirals remains unclear. To better understand the mechanism of protection in CCP, we studied viral replication and disease progression in SARS-CoV-2 infected hamsters treated with CCP plasma obtained from recovered COVID patients that had also been vaccinated with an mRNA vaccine, hereafter referred to as Vaxplas. We found that Vaxplas dramatically reduced virus replication in the lungs and improved infection outcome in SARS-CoV-2 infected hamsters. However, we also found that Vaxplas transiently enhanced disease severity and lung pathology in treated animals likely due to the deposition of immune complexes, activation of complement and recruitment of increased numbers of macrophages with an M1 proinflammatory phenotype into the lung parenchyma.

    View details for DOI 10.1101/2023.08.22.553458

    View details for PubMedID 37662344

  • Organoid modeling of lung-resident immune responses to SARS-CoV-2 infection. Research square Choi, S. S., van Unen, V., Zhang, H., Rustagi, A., Alwahabi, S. A., Santos, A. J., Chan, J. E., Lam, B., Solis, D., Mah, J., Röltgen, K., Trope, W., Guh-Siesel, A., Lin, Z., Beck, A., Edwards, C., Mallajosyula, V., Martin, B. A., Dunn, J. C., Shrager, J., Baric, R. A., Pinsky, B., Boyd, S. D., Blish, C. A., Davis, M. M., Kuo, C. J. 2023

    Abstract

    Tissue-resident immunity underlies essential host defenses against pathogens, but analysis in humans has lacked in vitro model systems where epithelial infection and accompanying resident immune cell responses can be observed en bloc. Indeed, human primary epithelial organoid cultures typically omit immune cells, and human tissue resident-memory lymphocytes are conventionally assayed without an epithelial infection component, for instance from peripheral blood, or after extraction from organs. Further, the study of resident immunity in animals can be complicated by interchange between tissue and peripheral immune compartments. To study human tissue-resident infectious immune responses in isolation from secondary lymphoid organs, we generated adult human lung three-dimensional air-liquid interface (ALI) lung organoids from intact tissue fragments that co-preserve epithelial and stromal architecture alongside endogenous lung-resident immune subsets. These included T, B, NK and myeloid cells, with CD69+CD103+ tissue-resident and CCR7- and/or CD45RA- TRM and conservation of T cell receptor repertoires, all corresponding to matched fresh tissue. SARS-CoV-2 vigorously infected organoid lung epithelium, alongside secondary induction of innate cytokine production that was inhibited by antiviral agents. Notably, SARS-CoV-2-infected organoids manifested adaptive virus-specific T cell activation that was specific for seropositive and/or previously infected donor individuals. This holistic non-reconstitutive organoid system demonstrates the sufficiency of lung to autonomously mount adaptive T cell memory responses without a peripheral lymphoid component, and represents an enabling method for the study of human tissue-resident immunity.

    View details for DOI 10.21203/rs.3.rs-2870695/v1

    View details for PubMedID 37205380

    View details for PubMedCentralID PMC10187413

  • Viral immunity: Basic mechanisms and therapeutic applications-a Keystone Symposia report. Annals of the New York Academy of Sciences Cable, J., Balachandran, S., Daley-Bauer, L. P., Rustagi, A., Antony, F., Frere, J. J., Strampe, J., Kedzierska, K., Cannon, J. L., McGargill, M. A., Weiskopf, D., Mettelman, R. C., Niessl, J., Thomas, P. G., Briney, B., Valkenburg, S. A., Bloom, J. D., Bjorkman, P. J., Iketani, S., Rappazzo, C. G., Crooks, C. M., Crofts, K. F., Pöhlmann, S., Krammer, F., Sant, A. J., Nabel, G. J., Schultz-Cherry, S. 2023

    Abstract

    Viruses infect millions of people each year. Both endemic viruses circulating throughout the population as well as novel epidemic and pandemic viruses pose ongoing threats to global public health. Developing more effective tools to address viruses requires not only in-depth knowledge of the virus itself but also of our immune system's response to infection. On June 29 to July 2, 2022, researchers met for the Keystone symposium "Viral Immunity: Basic Mechanisms and Therapeutic Applications." This report presents concise summaries from several of the symposium presenters.

    View details for DOI 10.1111/nyas.14960

    View details for PubMedID 36718537

  • SARS-CoV-2 escapes direct NK cell killing through Nsp1-mediated downregulation of ligands for NKG2D. Cell reports Lee, M. J., Leong, M. W., Rustagi, A., Beck, A., Zeng, L., Holmes, S., Qi, L. S., Blish, C. A. 2022: 111892

    Abstract

    Natural killer (NK) cells are cytotoxic effector cells that target and lyse virally infected cells; many viruses therefore encode mechanisms to escape such NK cell killing. Here, we interrogate the ability of SARS-CoV-2 to modulate NK cell recognition and lysis of infected cells. We find that NK cells exhibit poor cytotoxic responses against SARS-CoV-2-infected targets, preferentially killing uninfected bystander cells. We demonstrate that this escape is driven by downregulation of ligands for the activating receptor NKG2D (NKG2D-L). Indeed, early in viral infection, prior to NKG2D-L downregulation, NK cells are able to target and kill infected cells; however, this ability is lost as viral proteins are expressed. Finally, we find that SARS-CoV-2 non-structural protein 1 (Nsp1) mediates downregulation of NKG2D-L and that Nsp1 alone is sufficient to confer resistance to NK cell killing. Collectively, our work demonstrates that SARS-CoV-2 evades direct NK cell cytotoxicity and describes a mechanism by which this occurs.

    View details for DOI 10.1016/j.celrep.2022.111892

    View details for PubMedID 36543165

  • SARS-CoV-2 infection drives an inflammatory response in human adipose tissue through infection of adipocytes and macrophages. Science translational medicine Martínez-Colón, G. J., Ratnasiri, K., Chen, H., Jiang, S., Zanley, E., Rustagi, A., Verma, R., Chen, H., Andrews, J. R., Mertz, K. D., Tzankov, A., Azagury, D., Boyd, J., Nolan, G. P., Schürch, C. M., Matter, M. S., Blish, C. A., McLaughlin, T. L. 2022: eabm9151

    Abstract

    Obesity, characterized by chronic low-grade inflammation of the adipose tissue, is associated with adverse coronavirus disease 2019 (COVID-19) outcomes, yet the underlying mechanism is unknown. To explore whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of adipose tissue contributes to pathogenesis, we evaluated COVID-19 autopsy cases and deeply profiled the response of adipose tissue to SARS-CoV-2 infection in vitro. In COVID-19 autopsy cases, we identified SARS-CoV-2 RNA in adipocytes with an associated inflammatory infiltrate. We identified two distinct cellular targets of infection: adipocytes and a subset of inflammatory adipose tissue-resident macrophages. Mature adipocytes were permissive to SARS-CoV-2 infection; although macrophages were abortively infected, SARS-CoV-2 initiated inflammatory responses within both the infected macrophages and bystander preadipocytes. These data suggest that SARS-CoV-2 infection of adipose tissue could contribute to COVID-19 severity through replication of virus within adipocytes and through induction of local and systemic inflammation driven by infection of adipose tissue-resident macrophages.

    View details for DOI 10.1126/scitranslmed.abm9151

    View details for PubMedID 36137009

  • The human disease gene LYSET is essential for lysosomal enzyme transport and viral infection. Science (New York, N.Y.) Richards, C. M., Jabs, S., Qiao, W., Varanese, L. D., Schweizer, M., Mosen, P. R., Riley, N. M., Klüssendorf, M., Zengel, J. R., Flynn, R. A., Rustagi, A., Widen, J. C., Peters, C. E., Ooi, Y. S., Xie, X., Shi, P. Y., Bartenschlager, R., Puschnik, A. S., Bogyo, M., Bertozzi, C. R., Blish, C. A., Winter, D., Nagamine, C. M., Braulke, T., Carette, J. E. 2022: eabn5648

    Abstract

    Lysosomes are key degradative compartments of the cell. Transport to lysosomes relies on GlcNAc-1-phosphotransferase-mediated tagging of soluble enzymes with mannose 6-phosphate (M6P). GlcNAc-1-phosphotransferase deficiency leads to the severe lysosomal storage disorder mucolipidosis II (MLII). Several viruses require lysosomal cathepsins to cleave structural proteins and thus depend on functional GlcNAc-1-phosphotransferase. Here, we used genome-scale CRISPR screens to identify Lysosomal Enzyme Trafficking factor (LYSET) as essential for infection by cathepsin-dependent viruses including SARS-CoV-2. LYSET deficiency resulted in global loss of M6P tagging and mislocalization of GlcNAc-1-phosphotransferase from the Golgi complex to lysosomes. Lyset knockout mice exhibited MLII-like phenotypes and human pathogenic LYSET alleles failed to restore lysosomal sorting defects. Thus, LYSET is required for correct functioning of the M6P trafficking machinery, and mutations in LYSET can explain the phenotype of the associated disorder.

    View details for DOI 10.1126/science.abn5648

    View details for PubMedID 36074821

  • Programmable antivirals targeting critical conserved viral RNA secondary structures from influenza A virus and SARS-CoV-2. Nature medicine Hagey, R. J., Elazar, M., Pham, E. A., Tian, S., Ben-Avi, L., Bernardin-Souibgui, C., Yee, M. F., Moreira, F. R., Rabinovitch, M. V., Meganck, R. M., Fram, B., Beck, A., Gibson, S. A., Lam, G., Devera, J., Kladwang, W., Nguyen, K., Xiong, A., Schaffert, S., Avisar, T., Liu, P., Rustagi, A., Fichtenbaum, C. J., Pang, P. S., Khatri, P., Tseng, C., Taubenberger, J. K., Blish, C. A., Hurst, B. L., Sheahan, T. P., Das, R., Glenn, J. S. 2022

    Abstract

    Influenza A virus's (IAV's) frequent genetic changes challenge vaccine strategies and engender resistance to current drugs. We sought to identify conserved and essential RNA secondary structures within IAV's genome that are predicted to have greater constraints on mutation in response to therapeutic targeting. We identified and genetically validated an RNA structure (packaging stem-loop 2 (PSL2)) that mediates in vitro packaging and in vivo disease and is conserved across all known IAV isolates. A PSL2-targeting locked nucleic acid (LNA), administered 3d after, or 14d before, a lethal IAV inoculum provided 100% survival in mice, led to the development of strong immunity to rechallenge with a tenfold lethal inoculum, evaded attempts to select for resistance and retained full potency against neuraminidase inhibitor-resistant virus. Use of an analogous approach to target SARS-CoV-2, prophylactic administration of LNAs specific for highly conserved RNA structures in the viral genome, protected hamsters from efficient transmission of the SARS-CoV-2 USA_WA1/2020 variant. These findings highlight the potential applicability of this approach to any virus of interest via a process we term 'programmable antivirals', with implications for antiviral prophylaxis and post-exposure therapy.

    View details for DOI 10.1038/s41591-022-01908-x

    View details for PubMedID 35982307

  • An intranasal ASO therapeutic targeting SARS-CoV-2. Nature communications Zhu, C., Lee, J. Y., Woo, J. Z., Xu, L., Nguyenla, X., Yamashiro, L. H., Ji, F., Biering, S. B., Van Dis, E., Gonzalez, F., Fox, D., Wehri, E., Rustagi, A., Pinsky, B. A., Schaletzky, J., Blish, C. A., Chiu, C., Harris, E., Sadreyev, R. I., Stanley, S., Kauppinen, S., Rouskin, S., Näär, A. M. 2022; 13 (1): 4503

    Abstract

    The COVID-19 pandemic is exacting an increasing toll worldwide, with new SARS-CoV-2 variants emerging that exhibit higher infectivity rates and that may partially evade vaccine and antibody immunity. Rapid deployment of non-invasive therapeutic avenues capable of preventing infection by all SARS-CoV-2 variants could complement current vaccination efforts and help turn the tide on the COVID-19 pandemic. Here, we describe a novel therapeutic strategy targeting the SARS-CoV-2 RNA using locked nucleic acid antisense oligonucleotides (LNA ASOs). We identify an LNA ASO binding to the 5' leader sequence of SARS-CoV-2 that disrupts a highly conserved stem-loop structure with nanomolar efficacy in preventing viral replication in human cells. Daily intranasal administration of this LNA ASO in the COVID-19 mouse model potently suppresses viral replication (>80-fold) in the lungs of infected mice. We find that the LNA ASO is efficacious in countering all SARS-CoV-2 "variants of concern" tested both in vitro and in vivo. Hence, inhaled LNA ASOs targeting SARS-CoV-2 represents a promising therapeutic approach to reduce or prevent transmission and decrease severity of COVID-19 in infected individuals. LNA ASOs are chemically stable and can be flexibly modified to target different viral RNA sequences and could be stockpiled for future coronavirus pandemics.

    View details for DOI 10.1038/s41467-022-32216-0

    View details for PubMedID 35922434

  • Genome-wide bidirectional CRISPR screens identify mucins as host factors modulating SARS-CoV-2 infection. Nature genetics Biering, S. B., Sarnik, S. A., Wang, E., Zengel, J. R., Leist, S. R., Schafer, A., Sathyan, V., Hawkins, P., Okuda, K., Tau, C., Jangid, A. R., Duffy, C. V., Wei, J., Gilmore, R. C., Alfajaro, M. M., Strine, M. S., Nguyenla, X., Van Dis, E., Catamura, C., Yamashiro, L. H., Belk, J. A., Begeman, A., Stark, J. C., Shon, D. J., Fox, D. M., Ezzatpour, S., Huang, E., Olegario, N., Rustagi, A., Volmer, A. S., Livraghi-Butrico, A., Wehri, E., Behringer, R. R., Cheon, D., Schaletzky, J., Aguilar, H. C., Puschnik, A. S., Button, B., Pinsky, B. A., Blish, C. A., Baric, R. S., O'Neal, W. K., Bertozzi, C. R., Wilen, C. B., Boucher, R. C., Carette, J. E., Stanley, S. A., Harris, E., Konermann, S., Hsu, P. D. 2022

    Abstract

    Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a range of symptoms in infected individuals, from mild respiratory illness to acute respiratory distress syndrome. A systematic understanding of host factors influencing viral infection is critical to elucidate SARS-CoV-2-host interactions and the progression of Coronavirus disease 2019 (COVID-19). Here, we conducted genome-wide CRISPR knockout and activation screens in human lung epithelial cells with endogenous expression of the SARS-CoV-2 entry factors ACE2 and TMPRSS2. We uncovered proviral and antiviral factors across highly interconnected host pathways, including clathrin transport, inflammatory signaling, cell-cycle regulation, and transcriptional and epigenetic regulation. We further identified mucins, a family of high molecular weight glycoproteins, as a prominent viral restriction network that inhibits SARS-CoV-2 infection in vitro and in murine models. These mucins also inhibit infection of diverse respiratory viruses. This functional landscape of SARS-CoV-2 host factors provides a physiologically relevant starting point for new host-directed therapeutics and highlights airway mucins as a host defense mechanism.

    View details for DOI 10.1038/s41588-022-01131-x

    View details for PubMedID 35879412

  • Broad-spectrum CRISPR-mediated inhibition of SARS-CoV-2 variants and endemic coronaviruses in vitro. Nature communications Zeng, L., Liu, Y., Nguyenla, X. H., Abbott, T. R., Han, M., Zhu, Y., Chemparathy, A., Lin, X., Chen, X., Wang, H., Rane, D. A., Spatz, J. M., Jain, S., Rustagi, A., Pinsky, B., Zepeda, A. E., Kadina, A. P., Walker, J. A., Holden, K., Temperton, N., Cochran, J. R., Barron, A. E., Connolly, M. D., Blish, C. A., Lewis, D. B., Stanley, S. A., La Russa, M. F., Qi, L. S. 2022; 13 (1): 2766

    Abstract

    A major challenge in coronavirus vaccination and treatment is to counteract rapid viral evolution and mutations. Here we demonstrate that CRISPR-Cas13d offers a broad-spectrum antiviral (BSA) to inhibit many SARS-CoV-2 variants and diverse human coronavirus strains with >99% reduction of the viral titer. We show that Cas13d-mediated coronavirus inhibition is dependent on the crRNA cellular spatial colocalization with Cas13d and target viral RNA. Cas13d can significantly enhance the therapeutic effects of diverse small molecule drugs against coronaviruses for prophylaxis or treatment purposes, and the best combination reduced viral titer by over four orders of magnitude. Using lipid nanoparticle-mediated RNA delivery, we demonstrate that the Cas13d system can effectively treat infection from multiple variants of coronavirus, including Omicron SARS-CoV-2, in human primary airway epithelium air-liquid interface (ALI) cultures. Our study establishes CRISPR-Cas13 as a BSA which is highly complementary to existing vaccination and antiviral treatment strategies.

    View details for DOI 10.1038/s41467-022-30546-7

    View details for PubMedID 35589813

  • Facile discovery of surrogate cytokine agonists. Cell Yen, M., Ren, J., Liu, Q., Glassman, C. R., Sheahan, T. P., Picton, L. K., Moreira, F. R., Rustagi, A., Jude, K. M., Zhao, X., Blish, C. A., Baric, R. S., Su, L. L., Garcia, K. C. 2022

    Abstract

    Cytokines are powerful immune modulators that initiate signaling through receptor dimerization, but natural cytokines have structural limitations as therapeutics. We present a strategy to discover cytokine surrogate agonists by using modular ligands that exploit induced proximity and receptor dimer geometry as pharmacological metrics amenable to high-throughput screening. Using VHH and scFv to human interleukin-2/15, type-I interferon, and interleukin-10 receptors, we generated combinatorial matrices of single-chain bispecific ligands that exhibited diverse spectrums of functional activities, including potent inhibition of SARS-CoV-2 by surrogate interferons. Crystal structures of IL-2R:VHH complexes revealed that variation in receptor dimer geometries resulted in functionally diverse signaling outputs. This modular platform enabled engineering of surrogate ligands that compelled assembly of an IL-2R/IL-10R heterodimer, which does not naturally exist, that signaled through pSTAT5 on T and natural killer (NK) cells. This "cytokine med-chem" approach, rooted in principles of induced proximity, is generalizable for discovery of diversified agonists for many ligand-receptor systems.

    View details for DOI 10.1016/j.cell.2022.02.025

    View details for PubMedID 35325595

  • The B.1.427/1.429 (epsilon) SARS-CoV-2 variants are more virulent than ancestral B.1 (614G) in Syrian hamsters. PLoS pathogens Carroll, T., Fox, D., van Doremalen, N., Ball, E., Morris, M. K., Sotomayor-Gonzalez, A., Servellita, V., Rustagi, A., Yinda, C. K., Fritts, L., Port, J. R., Ma, Z. M., Holbrook, M. G., Schulz, J., Blish, C. A., Hanson, C., Chiu, C. Y., Munster, V., Stanley, S., Miller, C. J. 2022; 18 (2): e1009914

    Abstract

    As novel SARS-CoV-2 variants continue to emerge, it is critical that their potential to cause severe disease and evade vaccine-induced immunity is rapidly assessed in humans and studied in animal models. In early January 2021, a novel SARS-CoV-2 variant designated B.1.429 comprising 2 lineages, B.1.427 and B.1.429, was originally detected in California (CA) and it was shown to have enhanced infectivity in vitro and decreased antibody neutralization by plasma from convalescent patients and vaccine recipients. Here we examine the virulence, transmissibility, and susceptibility to pre-existing immunity for B 1.427 and B 1.429 in the Syrian hamster model. We find that both variants exhibit enhanced virulence as measured by increased body weight loss compared to hamsters infected with ancestral B.1 (614G), with B.1.429 causing the most marked body weight loss among the 3 variants. Faster dissemination from airways to parenchyma and more severe lung pathology at both early and late stages were also observed with B.1.429 infections relative to B.1. (614G) and B.1.427 infections. In addition, subgenomic viral RNA (sgRNA) levels were highest in oral swabs of hamsters infected with B.1.429, however sgRNA levels in lungs were similar in all three variants. This demonstrates that B.1.429 replicates to higher levels than ancestral B.1 (614G) or B.1.427 in the oropharynx but not in the lungs. In multi-virus in-vivo competition experiments, we found that B.1. (614G), epsilon (B.1.427/B.1.429) and gamma (P.1) dramatically outcompete alpha (B.1.1.7), beta (B.1.351) and zeta (P.2) in the lungs. In the nasal cavity, B.1. (614G), gamma, and epsilon dominate, but the highly infectious alpha variant also maintains a moderate size niche. We did not observe significant differences in airborne transmission efficiency among the B.1.427, B.1.429 and ancestral B.1 (614G) and WA-1 variants in hamsters. These results demonstrate enhanced virulence and high relative oropharyngeal replication of the epsilon (B.1.427/B.1.429) variant in Syrian hamsters compared to an ancestral B.1 (614G) variant.

    View details for DOI 10.1371/journal.ppat.1009914

    View details for PubMedID 35143587

  • Multi-omic profiling reveals widespread dysregulation of innate immunity and hematopoiesis in COVID-19. The Journal of experimental medicine Wilk, A. J., Lee, M. J., Wei, B., Parks, B., Pi, R., Martinez-Colon, G. J., Ranganath, T., Zhao, N. Q., Taylor, S., Becker, W., Stanford COVID-19 Biobank, Jimenez-Morales, D., Blomkalns, A. L., O'Hara, R., Ashley, E. A., Nadeau, K. C., Yang, S., Holmes, S., Rabinovitch, M., Rogers, A. J., Greenleaf, W. J., Blish, C. A. 2021; 218 (8)

    Abstract

    Our understanding of protective versus pathological immune responses to SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19), is limited by inadequate profiling of patients at the extremes of the disease severity spectrum. Here, we performed multi-omic single-cell immune profiling of 64 COVID-19 patients across the full range of disease severity, from outpatients with mild disease to fatal cases. Our transcriptomic, epigenomic, and proteomic analyses revealed widespread dysfunction of peripheral innate immunity in severe and fatal COVID-19, including prominent hyperactivation signatures in neutrophils and NK cells. We also identified chromatin accessibility changes at NF-kappaB binding sites within cytokine gene loci as a potential mechanism for the striking lack of pro-inflammatory cytokine production observed in monocytes in severe and fatal COVID-19. We further demonstrated that emergency myelopoiesis is a prominent feature of fatal COVID-19. Collectively, our results reveal disease severity-associated immune phenotypes in COVID-19 and identify pathogenesis-associated pathways that are potential targets for therapeutic intervention.

    View details for DOI 10.1084/jem.20210582

    View details for PubMedID 34128959

  • Occurrence and Timing of Subsequent Severe Acute Respiratory Syndrome Coronavirus 2 Reverse-transcription Polymerase Chain Reaction Positivity Among Initially Negative Patients. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America Long, D. R., Gombar, S., Hogan, C. A., Greninger, A. L., O'Reilly-Shah, V., Bryson-Cahn, C., Stevens, B., Rustagi, A., Jerome, K. R., Kong, C. S., Zehnder, J., Shah, N. H., Weiss, N. S., Pinsky, B. A., Sunshine, J. E. 2021; 72 (2): 323-326

    Abstract

    Using data for 20 912 patients from 2 large academic health systems, we analyzed the frequency of severe acute respiratory syndrome coronavirus 2 reverse-transcription polymerase chain reaction test discordance among individuals initially testing negative by nasopharyngeal swab who were retested on clinical grounds within 7 days. The frequency of subsequent positivity within this window was 3.5% and was similar across institutions.

    View details for DOI 10.1093/cid/ciaa722

    View details for PubMedID 33501950

  • SARS-CoV-2 subgenomic RNA kinetics in longitudinal clinical samples Open Forum Infectious Diseases Verma, R., Kim, E., Martinez, G., Jagannathan, ., Rustagi, A., Parsonnet, J., Bonilla, H., Khosla, C., Holubar, M., Subramanian, A., Singh, ., Maldonado, Y., Blish, C., Andrews, J. 2021

    View details for DOI 10.1093/ofid/ofab310

  • SARS-CoV-2 RNAemia predicts clinical deterioration and extrapulmonary complications from COVID-19. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America Ram-Mohan, N. n., Kim, D. n., Zudock, E. J., Hashemi, M. M., Tjandra, K. C., Rogers, A. J., Blish, C. A., Nadeau, K. C., Newberry, J. A., Quinn, J. V., O'Hara, R. n., Ashley, E. n., Nguyen, H. n., Jiang, L. n., Hung, P. n., Blomkalns, A. L., Yang, S. n. 2021

    Abstract

    The determinants of COVID-19 disease severity and extrapulmonary complications (EPCs) are poorly understood. We characterized relationships between SARS-CoV-2 RNAemia and disease severity, clinical deterioration, and specific EPCs.We used quantitative (qPCR) and digital (dPCR) PCR to quantify SARS-CoV-2 RNA from plasma in 191 patients presenting to the Emergency Department (ED) with COVID-19. We recorded patient symptoms, laboratory markers, and clinical outcomes, with a focus on oxygen requirements over time. We collected longitudinal plasma samples from a subset of patients. We characterized the role of RNAemia in predicting clinical severity and EPCs using elastic net regression.23.0% (44/191) of SARS-CoV-2 positive patients had viral RNA detected in plasma by dPCR, compared to 1.4% (2/147) by qPCR. Most patients with serial measurements had undetectable RNAemia within 10 days of symptom onset, reached maximum clinical severity within 16 days, and symptom resolution within 33 days. Initially RNAaemic patients were more likely to manifest severe disease (OR 6.72 [95% CI, 2.45 - 19.79]), worsening of disease severity (OR 2.43 [95% CI, 1.07 - 5.38]), and EPCs (OR 2.81 [95% CI, 1.26 - 6.36]). RNA load correlated with maximum severity (r = 0.47 [95% CI, 0.20 - 0.67]).dPCR is more sensitive than qPCR for the detection of SARS-CoV-2 RNAemia, which is a robust predictor of eventual COVID-19 severity and oxygen requirements, as well as EPCs. Since many COVID-19 therapies are initiated on the basis of oxygen requirements, RNAemia on presentation might serve to direct early initiation of appropriate therapies for the patients most likely to deteriorate.

    View details for DOI 10.1093/cid/ciab394

    View details for PubMedID 33949665

  • Progenitor identification and SARS-CoV-2 infection in human distal lung organoids. Nature Salahudeen, A. A., Choi, S. S., Rustagi, A., Zhu, J., van Unen, V., de la O, S. M., Flynn, R. A., Margalef-Catala, M., Santos, A. J., Ju, J., Batish, A., Usui, T., Zheng, G. X., Edwards, C. E., Wagar, L. E., Luca, V., Anchang, B., Nagendran, M., Nguyen, K., Hart, D. J., Terry, J. M., Belgrader, P., Ziraldo, S. B., Mikkelsen, T. S., Harbury, P. B., Glenn, J. S., Garcia, K. C., Davis, M. M., Baric, R. S., Sabatti, C., Amieva, M. R., Blish, C. A., Desai, T. J., Kuo, C. J. 2020

    Abstract

    The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange. Three-dimensional in vitro human distal lung culture systems would strongly facilitate investigation of pathologies including interstitial lung disease, cancer, and SARS-CoV-2-associated COVID-19 pneumonia. We generated long-term feeder-free, chemically defined culture of distal lung progenitors as organoids derived from single adult human alveolar epithelial type II (AT2) or KRT5+ basal cells. AT2 organoids exhibited AT1 transdifferentiation potential while basal cell organoids developed lumens lined by differentiated club and ciliated cells. Single cell analysis of basal organoid KRT5+ cells revealed a distinct ITGA6+ITGB4+ mitotic population whose proliferation further segregated to a TNFRSF12Ahi subfraction comprising ~10% of KRT5+ basal cells, residing in clusters within terminal bronchioles and exhibiting enriched clonogenic organoid growth activity. Distal lung organoids were created with apical-out polarity to display ACE2 on the exposed external surface, facilitating SARS-CoV-2 infection of AT2 and basal cultures and identifying club cells as a novel target population. This long-term, feeder-free organoid culture of human distal lung, coupled with single cell analysis, identifies unsuspected basal cell functional heterogeneity and establishes a facile in vitro organoid model for human distal lung infections including COVID-19-associated pneumonia.

    View details for DOI 10.1038/s41586-020-3014-1

    View details for PubMedID 33238290

  • Profiling of the Human Natural Killer Cell Receptor-Ligand Repertoire. Journal of visualized experiments : JoVE Vendrame, E., McKechnie, J. L., Ranganath, T., Zhao, N. Q., Rustagi, A., Vergara, R., Ivison, G. T., Kronstad, L. M., Simpson, L. J., Blish, C. A. 2020

    Abstract

    Natural killer (NK) cells are among the first responders to viral infections. The ability of NK cells to rapidly recognize and kill virally infected cells is regulated by their expression of germline-encoded inhibitory and activating receptors. The engagement of these receptors by their cognate ligands on target cells determines whether the intercellular interaction will result in NK cell killing. This protocol details the design and optimization of two complementary mass cytometry (CyTOF) panels. One panel was designed to phenotype NK cells based on receptor expression. The other panel was designed to interrogate expression of known ligands for NK cell receptors on several immune cell subsets. Together, these two panels allow for the profiling of the human NK cell receptor-ligand repertoire. Furthermore, this protocol also details the process by which we stain samples for CyTOF. This process has been optimized for improved reproducibility and standardization. An advantage of CyTOF is its ability to measure over 40 markers in each panel, with minimal signal overlap, allowing researchers to capture the breadth of the NK cell receptor-ligand repertoire. Palladium barcoding also reduces inter-sample variation, as well as consumption of reagents, making it easier to stain samples with each panel in parallel. Limitations of this protocol include the relatively low throughput of CyTOF and the inability to recover cells after analysis. These panels were designed for the analysis of clinical samples from patients suffering from acute and chronic viral infections, including dengue virus, human immunodeficiency virus (HIV), and influenza. However, they can be utilized in any setting to investigate the human NK cell receptor-ligand repertoire. Importantly, these methods can be applied broadly to the design and execution of future CyTOF panels.

    View details for DOI 10.3791/61912

    View details for PubMedID 33283785

  • Evaluating the Utility of UV Lamps to Mitigate the Spread of Pathogens in the ICU APPLIED SCIENCES-BASEL Gostine, A., Gostine, D., Short, J., Rustagi, A., Cadnum, J., Donskey, C., Angelotti, T. 2020; 10 (18)
  • SARS-CoV-2 Antibody Responses Correlate with Resolution of RNAemia But Are Short-Lived in Patients with Mild Illness. medRxiv : the preprint server for health sciences Röltgen, K., Wirz, O. F., Stevens, B. A., Powell, A. E., Hogan, C. A., Najeeb, J., Hunter, M., Sahoo, M. K., Huang, C., Yamamoto, F., Manalac, J., Otrelo-Cardoso, A. R., Pham, T. D., Rustagi, A., Rogers, A. J., Shah, N. H., Blish, C. A., Cochran, J. R., Nadeau, K. C., Jardetzky, T. S., Zehnder, J. L., Wang, T. T., Kim, P. S., Gombar, S., Tibshirani, R., Pinsky, B. A., Boyd, S. D. 2020

    Abstract

    SARS-CoV-2-specific antibodies, particularly those preventing viral spike receptor binding domain (RBD) interaction with host angiotensin-converting enzyme 2 (ACE2) receptor, could offer protective immunity, and may affect clinical outcomes of COVID-19 patients. We analyzed 625 serial plasma samples from 40 hospitalized COVID-19 patients and 170 SARS-CoV-2-infected outpatients and asymptomatic individuals. Severely ill patients developed significantly higher SARS-CoV-2-specific antibody responses than outpatients and asymptomatic individuals. The development of plasma antibodies was correlated with decreases in viral RNAemia, consistent with potential humoral immune clearance of virus. Using a novel competition ELISA, we detected antibodies blocking RBD-ACE2 interactions in 68% of inpatients and 40% of outpatients tested. Cross-reactive antibodies recognizing SARS-CoV RBD were found almost exclusively in hospitalized patients. Outpatient and asymptomatic individuals' serological responses to SARS-CoV-2 decreased within 2 months, suggesting that humoral protection may be short-lived.

    View details for DOI 10.1101/2020.08.15.20175794

    View details for PubMedID 32839786

    View details for PubMedCentralID PMC7444305

  • Cytokine profile in plasma of severe COVID-19 does not differ from ARDS and sepsis. JCI insight Wilson, J. G., Simpson, L. J., Ferreira, A., Rustagi, A., Roque, J. A., Asuni, A., Ranganath, T., Grant, P. M., Subramanian, A. K., Rosenberg-Hasson, Y., Maecker, H., Holmes, S., Levitt, J. E., Blish, C., Rogers, A. J. 2020

    Abstract

    BACKGROUND: Elevated levels of inflammatory cytokines have been associated with poor outcomes among COVID-19 patients. It is unknown, however, how these levels compare to those observed in critically ill patients with ARDS or sepsis due to other causes.METHODS: We used a luminex assay to determine expression of 76 cytokines from plasma of hospitalized COVID-19 patients and banked plasma samples from ARDS and sepsis patients. Our analysis focused on detecting statistical differences in levels of 6 cytokines associated with cytokine storm (IL-1b, IL-1RA, IL-6, IL-8, IL-18, and TNFalpha) between patients with moderate COVID-19, severe COVID-19, and ARDS or sepsis.RESULTS: 15 hospitalized COVID-19 patients, 9 of whom were critically ill, were compared to critically ill patients with ARDS (n = 12) or sepsis (n = 16). There were no statistically significant differences in baseline levels of IL-1b, IL-1RA, IL-6, IL-8, IL-18, and TNFalpha between patients with COVID-19 and critically ill controls with ARDS or sepsis.CONCLUSIONS: Levels of inflammatory cytokines were not higher in severe COVID-19 patients than in moderate COVID-19 or critically ill patients with ARDS or sepsis in this small cohort. Broad use of immunosuppressive therapies in ARDS has failed in numerous Phase 3 studies; use of these therapies in unselected patients with COVID-19 may be unwarranted.FUNDING: A.J.R.: Stanford ICU Biobank NHLBI K23 HL125663. C.A.B.: Burroughs Wellcome Fund Investigators in the Pathogenesis of Infectious Diseases #1016687; NIH/NIAID U19AI057229-16 (PI MM Davis); Stanford Maternal Child Health Research Institute; Chan Zuckerberg Biohub.

    View details for DOI 10.1172/jci.insight.140289

    View details for PubMedID 32706339

  • A single-cell atlas of the peripheral immune response in patients with severe COVID-19. Nature medicine Wilk, A. J., Rustagi, A., Zhao, N. Q., Roque, J., Martinez-Colon, G. J., McKechnie, J. L., Ivison, G. T., Ranganath, T., Vergara, R., Hollis, T., Simpson, L. J., Grant, P., Subramanian, A., Rogers, A. J., Blish, C. A. 2020

    Abstract

    There is an urgent need to better understand the pathophysiology of Coronavirus disease 2019 (COVID-19), the global pandemic caused by SARS-CoV-2, which has infected more than three million people worldwide1. Approximately 20% of patients with COVID-19 develop severe disease and 5% of patients require intensive care2. Severe disease has been associated with changes in peripheral immune activity, including increased levels of pro-inflammatory cytokines3,4 that may be produced by a subset of inflammatory monocytes5,6, lymphopenia7,8 and T cell exhaustion9,10. To elucidate pathways in peripheral immune cells that might lead to immunopathology or protective immunity in severe COVID-19, we applied single-cell RNA sequencing (scRNA-seq) to profile peripheral blood mononuclear cells (PBMCs) from seven patients hospitalized for COVID-19, four of whom had acute respiratory distress syndrome, and six healthy controls. We identify reconfiguration of peripheral immune cell phenotype in COVID-19, including a heterogeneous interferon-stimulated gene signature, HLA class II downregulation and a developing neutrophil population that appears closely related to plasmablasts appearing in patients with acute respiratory failure requiring mechanical ventilation. Importantly, we found that peripheral monocytes and lymphocytes do not express substantial amounts of pro-inflammatory cytokines. Collectively, we provide a cell atlas of the peripheral immune response to severe COVID-19.

    View details for DOI 10.1038/s41591-020-0944-y

    View details for PubMedID 32514174

  • Defining the features and duration of antibody responses to SARS-CoV-2 infection associated with disease severity and outcome. Science immunology Röltgen, K. n., Powell, A. E., Wirz, O. F., Stevens, B. A., Hogan, C. A., Najeeb, J. n., Hunter, M. n., Wang, H. n., Sahoo, M. K., Huang, C. n., Yamamoto, F. n., Manohar, M. n., Manalac, J. n., Otrelo-Cardoso, A. R., Pham, T. D., Rustagi, A. n., Rogers, A. J., Shah, N. H., Blish, C. A., Cochran, J. R., Jardetzky, T. S., Zehnder, J. L., Wang, T. T., Narasimhan, B. n., Gombar, S. n., Tibshirani, R. n., Nadeau, K. C., Kim, P. S., Pinsky, B. A., Boyd, S. D. 2020; 5 (54)

    Abstract

    SARS-CoV-2-specific antibodies, particularly those preventing viral spike receptor binding domain (RBD) interaction with host angiotensin-converting enzyme 2 (ACE2) receptor, can neutralize the virus. It is, however, unknown which features of the serological response may affect clinical outcomes of COVID-19 patients. We analyzed 983 longitudinal plasma samples from 79 hospitalized COVID-19 patients and 175 SARS-CoV-2-infected outpatients and asymptomatic individuals. Within this cohort, 25 patients died of their illness. Higher ratios of IgG antibodies targeting S1 or RBD domains of spike compared to nucleocapsid antigen were seen in outpatients who had mild illness versus severely ill patients. Plasma antibody increases correlated with decreases in viral RNAemia, but antibody responses in acute illness were insufficient to predict inpatient outcomes. Pseudovirus neutralization assays and a scalable ELISA measuring antibodies blocking RBD-ACE2 interaction were well correlated with patient IgG titers to RBD. Outpatient and asymptomatic individuals' SARS-CoV-2 antibodies, including IgG, progressively decreased during observation up to five months post-infection.

    View details for DOI 10.1126/sciimmunol.abe0240

    View details for PubMedID 33288645

  • Human B Cell Clonal Expansion and Convergent Antibody Responses to SARS-CoV-2. Cell host & microbe Nielsen, S. C., Yang, F. n., Jackson, K. J., Hoh, R. A., Röltgen, K. n., Jean, G. H., Stevens, B. A., Lee, J. Y., Rustagi, A. n., Rogers, A. J., Powell, A. E., Hunter, M. n., Najeeb, J. n., Otrelo-Cardoso, A. R., Yost, K. E., Daniel, B. n., Nadeau, K. C., Chang, H. Y., Satpathy, A. T., Jardetzky, T. S., Kim, P. S., Wang, T. T., Pinsky, B. A., Blish, C. A., Boyd, S. D. 2020

    Abstract

    B cells are critical for the production of antibodies and protective immunity to viruses. Here we show that patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) who develop coronavirus disease 2019 (COVID-19) display early recruitment of B cells expressing a limited subset of IGHV genes, progressing to a highly polyclonal response of B cells with broader IGHV gene usage and extensive class switching to IgG and IgA subclasses with limited somatic hypermutation in the initial weeks of infection. We identify convergence of antibody sequences across SARS-CoV-2-infected patients, highlighting stereotyped naive responses to this virus. Notably, sequence-based detection in COVID-19 patients of convergent B cell clonotypes previously reported in SARS-CoV infection predicts the presence of SARS-CoV/SARS-CoV-2 cross-reactive antibody titers specific for the receptor-binding domain. These findings offer molecular insights into shared features of human B cell responses to SARS-CoV-2 and SARS-CoV.

    View details for DOI 10.1016/j.chom.2020.09.002

    View details for PubMedID 32941787

  • Eremothecium coryli bloodstream infection in a patient with acute myeloid leukemia: first case report of human infection DIAGNOSTIC MICROBIOLOGY AND INFECTIOUS DISEASE Multani, A., Rustagi, A., Epstein, D. J., Gomez, C. A., Budvytiene, I., Banaei, N., Brown, J. M., Liu, A. Y. 2019; 95 (1): 77–79
  • Deltex2 represses MyoD expression and inhibits myogenic differentiation by acting as a negative regulator of Jmjd1c PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Luo, D., de Morree, A., Boutet, S., Quach, N., Natu, V., Rustagi, A., Rando, T. A. 2017; 114 (15): E3071-E3080

    Abstract

    The myogenic regulatory factor MyoD has been implicated as a key regulator of myogenesis, and yet there is little information regarding its upstream regulators. We found that Deltex2 inhibits myogenic differentiation in vitro, and that skeletal muscle stem cells from Deltex2 knockout mice exhibit precocious myogenic differentiation and accelerated regeneration in response to injury. Intriguingly, Deltex2 inhibits myogenesis by suppressing MyoD transcription, and the Deltex2 knockout phenotype can be rescued by a loss-of-function allele for MyoD In addition, we obtained evidence that Deltex2 regulates MyoD expression by promoting the enrichment of histone 3 modified by dimethylation at lysine 9 at a key regulatory region of the MyoD locus. The enrichment is attributed to a Deltex2 interacting protein, Jmjd1c, whose activity is directly inhibited by Deltex2 and whose expression is required for MyoD expression in vivo and in vitro. Finally, we find that Deltex2 causes Jmjd1c monoubiquitination and inhibits its demethylase activity. Mutation of the monoubiquitination site in Jmjd1c abolishes the inhibitory effect of Deltex2 on Jmjd1c demethylase activity. These results reveal a mechanism by which a member of the Deltex family of proteins can inhibit cellular differentiation, and demonstrate a role of Deltex in the epigenetic regulation of myogenesis.

    View details for DOI 10.1073/pnas.1613592114

    View details for Web of Science ID 000398789800012

    View details for PubMedID 28351977

  • Innate Antiviral Immune Signaling, Viral Evasion and Modulation by HIV-1 JOURNAL OF MOLECULAR BIOLOGY Rustagi, A., Gale, M. 2014; 426 (6): 1161-1177

    Abstract

    The intracellular innate antiviral response in human cells is an essential component of immunity against virus infection. As obligate intracellular parasites, all viruses must evade the actions of the host cell's innate immune response in order to replicate and persist. Innate immunity is induced when pathogen recognition receptors of the host cell sense viral products including nucleic acid as "non-self". This process induces downstream signaling through adaptor proteins to activate latent transcription factors that drive the expression of genes encoding antiviral and immune modulatory effector proteins that restrict virus replication and regulate adaptive immunity. The interferon regulatory factors (IRFs) are transcription factors that play major roles in innate immunity. In particular, IRF3 is activated in response to infection by a range of viruses including RNA viruses, DNA viruses and retroviruses. Among these viruses, human immunodeficiency virus type 1 (HIV-1) remains a major global health problem mediating chronic infection in millions of people wherein recent studies show that viral persistence is linked with the ability of the virus to dysregulate and evade the innate immune response. In this review, we discuss viral pathogen sensing, innate immune signaling pathways and effectors that respond to viral infection, the role of IRF3 in these processes and how it is regulated by pathogenic viruses. We present a contemporary overview of the interplay between HIV-1 and innate immunity, with a focus on understanding how innate immune control impacts infection outcome and disease.

    View details for DOI 10.1016/j.jmb.2013.12.003

    View details for Web of Science ID 000333487600004

    View details for PubMedID 24326250

  • Two new monoclonal antibodies for biochemical and flow cytometric analyses of human interferon regulatory factor-3 activation, turnover, and depletion METHODS Rustagi, A., Doehle, B. P., McElrath, M. J., Gale, M. 2013; 59 (2): 225-232

    Abstract

    Interferon regulatory factor-3 (IRF-3) is a master transcription factor that drives the host intracellular innate immune response to virus infection. The importance of IRF-3 in innate immune responses is highlighted by the fact that pathogenic viruses have developed strategies for antagonism of IRF-3. Several tools exist for evaluation of viral regulation of IRF-3 activation and function, but high-quality monoclonal antibodies that mark the differential activation states of human IRF-3 are lacking. To study IRF-3 activation, turnover, and depletion in a high-throughput manner in the context of virus infection, we have developed two new monoclonal antibodies to human IRF-3. These antibodies detect IRF-3 in virus-infected cells in a wide variety of assays and provide a new tool to study virus-host interactions and innate immune signaling.

    View details for DOI 10.1016/j.ymeth.2012.05.011

    View details for Web of Science ID 000316651100008

    View details for PubMedID 22705311

    View details for PubMedCentralID PMC3568209

  • Vpu Mediates Depletion of Interferon Regulatory Factor 3 during HIV Infection by a Lysosome-Dependent Mechanism JOURNAL OF VIROLOGY Doehle, B. P., Chang, K., Rustagi, A., McNevin, J., McElrath, M. J., Gale, M. 2012; 86 (16): 8367-8374

    Abstract

    HIV has evolved sophisticated mechanisms to avoid restriction by intracellular innate immune defenses that otherwise serve to control acute viral infection and virus dissemination. Innate defenses are triggered when pattern recognition receptor (PRR) proteins of the host cell engage pathogen-associated molecule patterns (PAMPs) present in viral products. Interferon regulatory factor 3 (IRF3) plays a central role in PRR signaling of innate immunity to drive the expression of type I interferon (IFN) and interferon-stimulated genes (ISGs), including a variety of HIV restriction factors, that serve to limit viral replication directly and/or program adaptive immunity. Productive infection of T cells by HIV is dependent upon the targeted proteolysis of IRF3 that occurs through a virus-directed mechanism that results in suppression of innate immune defenses. However, the mechanisms by which HIV controls innate immune signaling and IRF3 function are not defined. Here, we examined the innate immune response induced by HIV strains identified through their differential control of PRR signaling. We identified viruses that, unlike typical circulating HIV strains, lack the ability to degrade IRF3. Our studies show that IRF3 regulation maps specifically to the HIV accessory protein Vpu. We define a molecular interaction between Vpu and IRF3 that redirects IRF3 to the endolysosome for proteolytic degradation, thus allowing HIV to avoid the innate antiviral immune response. Our studies reveal that Vpu is an important IRF3 regulator that supports acute HIV infection through innate immune suppression. These observations define the Vpu-IRF3 interface as a novel target for therapeutic strategies aimed at enhancing the immune response to HIV.

    View details for DOI 10.1128/JVI.00423-12

    View details for Web of Science ID 000307198300003

    View details for PubMedID 22593165

    View details for PubMedCentralID PMC3421752

  • HIV infection of dendritic cells subverts the IFN induction pathway via IRF-1 and inhibits type 1 IFN production BLOOD Harman, A. N., Lai, J., Turville, S., Samarajiwa, S., Gray, L., Marsden, V., Mercier, S., Jones, K., Nasr, N., Rustagi, A., Cumming, H., Donaghy, H., Mak, J., Gale, M., Churchill, M., Hertzog, P., Cunningham, A. L. 2011; 118 (2): 298-308

    Abstract

    Many viruses have developed mechanisms to evade the IFN response. Here, HIV-1 was shown to induce a distinct subset of IFN-stimulated genes (ISGs) in monocyte-derived dendritic cells (DCs), without detectable type I or II IFN. These ISGs all contained an IFN regulatory factor 1 (IRF-1) binding site in their promoters, and their expression was shown to be driven by IRF-1, indicating this subset was induced directly by viral infection by IRF-1. IRF-1 and -7 protein expression was enriched in HIV p24 antigen-positive DCs. A HIV deletion mutant with the IRF-1 binding site deleted from the long terminal repeat showed reduced growth kinetics. Early and persistent induction of IRF-1 was coupled with sequential transient up-regulation of its 2 inhibitors, IRF-8, followed by IRF-2, suggesting a mechanism for IFN inhibition. HIV-1 mutants with Vpr deleted induced IFN, showing that Vpr is inhibitory. However, HIV IFN inhibition was mediated by failure of IRF-3 activation rather than by its degradation, as in T cells. In contrast, herpes simplex virus type 2 markedly induced IFNβ and a broader range of ISGs to higher levels, supporting the hypothesis that HIV-1 specifically manipulates the induction of IFN and ISGs to enhance its noncytopathic replication in DCs.

    View details for DOI 10.1182/blood-2010-07-297721

    View details for Web of Science ID 000292735100015

    View details for PubMedID 21411754

    View details for PubMedCentralID PMC4123420

  • Enhanced gene repair mediated by methyl-CpG-modified single-stranded oligonucleotides NUCLEIC ACIDS RESEARCH Bertoni, C., Rustagi, A., Rando, T. A. 2009; 37 (22): 7468-7482

    Abstract

    Gene editing mediated by oligonucleotides has been shown to induce stable single base alterations in genomic DNA in both prokaryotic and eukaryotic organisms. However, the low frequencies of gene repair have limited its applicability for both basic manipulation of genomic sequences and for the development of therapeutic approaches for genetic disorders. Here, we show that single-stranded oligodeoxynucleotides (ssODNs) containing a methyl-CpG modification and capable of binding to the methyl-CpG binding domain protein 4 (MBD4) are able to induce >10-fold higher levels of gene correction than ssODNs lacking the specific modification. Correction was stably inherited through cell division and was confirmed at the protein, transcript and genomic levels. Downregulation of MBD4 expression using RNAi prevented the enhancement of gene correction efficacy obtained using the methyl-CpG-modified ssODN, demonstrating the specificity of the repair mechanism being recruited. Our data demonstrate that efficient manipulation of genomic targets can be achieved and controlled by the type of ssODN used and by modulation of the repair mechanism involved in the correction process. This new generation of ssODNs represents an important technological advance that is likely to have an impact on multiple applications, especially for gene therapy where permanent correction of the genetic defect has clear advantages over viral and other nonviral approaches currently being tested.

    View details for DOI 10.1093/nar/gkp757

    View details for Web of Science ID 000272935000020

    View details for PubMedID 19854937

    View details for PubMedCentralID PMC2794159

  • Enhanced Level of Gene Correction Mediated by Oligonucleotides Containing CpG Modification in the mdx Mouse Model for Duchenne Muscular Dystrophy Bertoni, C., Rustagi, A., Rando, T. A. NATURE PUBLISHING GROUP. 2006: S221
  • An inwardly rectifying potassium channel in apical membrane of Calu-3 cells JOURNAL OF BIOLOGICAL CHEMISTRY Wu, J. V., Krouse, M. E., Rustagi, A., Joo, N. S., Wine, J. J. 2004; 279 (45): 46558-46565

    Abstract

    Patch clamp methods and reverse transcription-polymerase chain reaction (RT-PCR) were used to characterize an apical K+ channel in Calu-3 cells, a widely used model of human airway gland serous cells. In cell-attached and excised apical membrane patches, we found an inwardly rectifying K+ channel (Kir). The permeability ratio was PNa/PK = 0.058. In 30 patches with both cystic fibrosis transmembrane conductance regulator and Kir present, we observed 79 cystic fibrosis transmembrane conductance regulator and 58 Kir channels. The average chord conductance was 24.4 +/- 0.5 pS (n = 11), between 0 and -200 mV, and was 9.6 +/- 0.7 pS (n = 8), between 0 and 50 mV; these magnitudes and their ratio of approximately 2.5 are most similar to values for rectifying K+ channels of the Kir4.x subfamilies. We attempted to amplify transcripts for Kir4.1, Kir4.2, and Kir5.1; of these only Kir4.2 was present in Calu-3 lysates. The channel was only weakly activated by ATP and was relatively insensitive to internal pH. External Cs+ and Ba2+ blocked the channel with Kd values in the millimolar range. Quantitative modeling of Cl- secreting epithelia suggests that secretion rates will be highest and luminal K+ will rise to 16-28 mm if 11-25% of the total cellular K+ conductance is placed in the apical membrane (Cook, D. I., and Young, J. A. (1989) J. Membr. Biol. 110, 139-146). Thus, we hypothesize that the K+ channel described here optimizes the rate of secretion and is involved in K+ recycling for the recently proposed apical H+ -K+ -ATPase in Calu-3 cells.

    View details for DOI 10.1074/jbc.M406058200

    View details for Web of Science ID 000224832400028

    View details for PubMedID 15328350

  • Regulation of antiprotease and antimicrobial protein secretion by airway submucosal gland serous cells JOURNAL OF BIOLOGICAL CHEMISTRY Joo, N. S., Lee, D. J., Winges, K. M., Rustagi, A., Wine, J. J. 2004; 279 (37): 38854-38860

    Abstract

    Airway submucosal gland serous cells express the cystic fibrosis transmembrane conductance regulator (CFTR) and secrete antimicrobial, anti-inflammatory, and antioxidant molecules. In cystic fibrosis, diminished gland secretion may impair innate airway host defenses. We used Calu-3 cells as a serous cell model to study the types of proteins released, the pathways that release them, and the possible involvement of CFTR activity in protein release. Many proteins were secreted constitutively into the apical fluid and showed increased release to agonists. We identified some of them by high pressure liquid chromatography-mass spectrometry and reverse transcriptase PCR, including lysozyme, siderocalin (the protein NGAL), which inhibits bacterial growth by binding iron-containing siderophores, HSC-71, which is thought to have anti-inflammatory properties, and the serine protease inhibitors alpha-1-antitrypsin and alpha-1-antichymotrypsin, which may function as antimicrobials as well as play a potential role in diminishing the activation of epithelial Na(+) channels by serine proteases. We used an enzyme-linked immunosorbent assay to quantify lysozyme secretion by Calu-3 cells in response to various agonists and inhibitors. Forskolin increased the lysozyme secretion rate (J(lyz)) from 32 to 77 ng/hr/cm(2) (n = 36, p < 0.005). Thapsigargin increased J(lyz) from 40 to 63 ng/h/cm(2) (n = 16, p < 0.005), and forskolin plus thapsigargin further increased the forskolin-stimulated J(lyz) by 48% (n = 9, p < 0.05). 1-Ethyl-benzimidazolinone and carbachol were less effective. Glibenclamide inhibited basal and stimulated J(lyz), but clotrimazole was without effect. CFTR(inh)172 caused a small (15%) but significant inhibition of forskolin-stimulated J(lyz) without affecting basal J(lyz). Thus, Calu-3 cells secrete diverse proteins that in aggregate would be expected to suppress microbial growth, protect the airways from damage, and limit the activation of epithelial Na(+) channels via serine proteases.

    View details for DOI 10.1074/jbc.M407077200

    View details for Web of Science ID 000223684100098

    View details for PubMedID 15234967