Honors & Awards
Ann Palmenberg Junior Investigator Award, American Society of Virology (2013)
NIH Directors New Innovator Award, NIH (2012)
Fellow, David & Lucile Packard Foundation (2012)
Baxter Faculty Scholar Award, Baxter Foundation (2011)
Current Research and Scholarly Interests
Our research focuses on the identification of host genes that play critical roles in the pathogenesis of infectious agents including viruses. We use haploid genetic screens in human cells as an efficient approach to perform loss-of-function studies. Besides obtaining fundamental insights on how viruses hijack cellular processes and on host defense mechanisms, it might also facilitate the development of new therapeutic strategies.
Independent Studies (6)
- Directed Reading in Microbiology and Immunology
MI 198 (Aut, Win, Spr, Sum)
- Directed Reading in Microbiology and Immunology
MI 299 (Aut, Win, Spr, Sum)
- Graduate Research
IMMUNOL 399 (Win, Spr)
- Graduate Research
MI 399 (Aut, Win, Spr, Sum)
- Medical Scholars Research
MI 370 (Aut, Win, Spr, Sum)
- Undergraduate Research
MI 199 (Aut, Win, Spr, Sum)
- Directed Reading in Microbiology and Immunology
Graduate and Fellowship Programs
Kinetic pathway of 40S ribosomal subunit recruitment to hepatitis C virus internal ribosome entry site.
Proceedings of the National Academy of Sciences of the United States of America
2015; 112 (2): 319-325
Translation initiation can occur by multiple pathways. To delineate these pathways by single-molecule methods, fluorescently labeled ribosomal subunits are required. Here, we labeled human 40S ribosomal subunits with a fluorescent SNAP-tag at ribosomal protein eS25 (RPS25). The resulting ribosomal subunits could be specifically labeled in living cells and in vitro. Using single-molecule Förster resonance energy transfer (FRET) between RPS25 and domain II of the hepatitis C virus (HCV) internal ribosome entry site (IRES), we measured the rates of 40S subunit arrival to the HCV IRES. Our data support a single-step model of HCV IRES recruitment to 40S subunits, irreversible on the initiation time scale. We furthermore demonstrated that after binding, the 40S:HCV IRES complex is conformationally dynamic, undergoing slow large-scale rearrangements. Addition of translation extracts suppresses these fluctuations, funneling the complex into a single conformation on the 80S assembly pathway. These findings show that 40S:HCV IRES complex formation is accompanied by dynamic conformational rearrangements that may be modulated by initiation factors.
View details for DOI 10.1073/pnas.1421328111
View details for PubMedID 25516984
RIP3 Induces Apoptosis Independent of Pronecrotic Kinase Activity.
2014; 56 (4): 481-95
Receptor-interacting protein kinase 3 (RIP3 or RIPK3) has emerged as a central player in necroptosis and a potential target to control inflammatory disease. Here, three selective small-molecule compounds are shown to inhibit RIP3 kinase-dependent necroptosis, although their therapeutic value is undermined by a surprising, concentration-dependent induction of apoptosis. These compounds interact with RIP3 to activate caspase 8 (Casp8) via RHIM-driven recruitment of RIP1 (RIPK1) to assemble a Casp8-FADD-cFLIP complex completely independent of pronecrotic kinase activities and MLKL. RIP3 kinase-dead D161N mutant induces spontaneous apoptosis independent of compound, whereas D161G, D143N, and K51A mutants, like wild-type, only trigger apoptosis when compound is present. Accordingly, RIP3-K51A mutant mice (Rip3(K51A/K51A)) are viable and fertile, in stark contrast to the perinatal lethality of Rip3(D161N/D161N) mice. RIP3 therefore holds both necroptosis and apoptosis in balance through a Ripoptosome-like platform. This work highlights a common mechanism unveiling RHIM-driven apoptosis by therapeutic or genetic perturbation of RIP3.
View details for DOI 10.1016/j.molcel.2014.10.021
View details for PubMedID 25459880
Deciphering the Glycosylome of Dystroglycanopathies Using Haploid Screens for Lassa Virus Entry
2013; 340 (6131): 479-483
Glycosylated ?-dystroglycan (?-DG) serves as cellular entry receptor for multiple pathogens, and defects in its glycosylation cause hereditary Walker-Warburg syndrome (WWS). At least eight proteins are critical to glycosylate ?-DG, but many genes mutated in WWS remain unknown. To identify modifiers of ?-DG, we performed a haploid screen for Lassa virus entry, a hemorrhagic fever virus causing thousands of deaths annually that hijacks glycosylated ?-DG to enter cells. In complementary screens, we profiled cells for absence of ?-DG carbohydrate chains or biochemically related glycans. This revealed virus host factors and a suite of glycosylation units, including all known Walker-Warburg genes and five additional factors critical for the modification of ?-DG. Our findings accentuate the complexity of this posttranslational feature and point out genes defective in dystroglycanopathies.
View details for DOI 10.1126/science.1233675
View details for Web of Science ID 000318016700042
View details for PubMedID 23519211
Ebola virus entry requires the cholesterol transporter Niemann-Pick C1
2011; 477 (7364): 340-U115
Infections by the Ebola and Marburg filoviruses cause a rapidly fatal haemorrhagic fever in humans for which no approved antivirals are available. Filovirus entry is mediated by the viral spike glycoprotein (GP), which attaches viral particles to the cell surface, delivers them to endosomes and catalyses fusion between viral and endosomal membranes. Additional host factors in the endosomal compartment are probably required for viral membrane fusion; however, despite considerable efforts, these critical host factors have defied molecular identification. Here we describe a genome-wide haploid genetic screen in human cells to identify host factors required for Ebola virus entry. Our screen uncovered 67 mutations disrupting all six members of the homotypic fusion and vacuole protein-sorting (HOPS) multisubunit tethering complex, which is involved in the fusion of endosomes to lysosomes, and 39 independent mutations that disrupt the endo/lysosomal cholesterol transporter protein Niemann-Pick C1 (NPC1). Cells defective for the HOPS complex or NPC1 function, including primary fibroblasts derived from human Niemann-Pick type C1 disease patients, are resistant to infection by Ebola virus and Marburg virus, but remain fully susceptible to a suite of unrelated viruses. We show that membrane fusion mediated by filovirus glycoproteins and viral escape from the vesicular compartment require the NPC1 protein, independent of its known function in cholesterol transport. Our findings uncover unique features of the entry pathway used by filoviruses and indicate potential antiviral strategies to combat these deadly agents.
View details for DOI 10.1038/nature10348
View details for Web of Science ID 000294852400033
View details for PubMedID 21866103
Haploid Genetic Screens in Human Cells Identify Host Factors Used by Pathogens
2009; 326 (5957): 1231-1235
Loss-of-function genetic screens in model organisms have elucidated numerous biological processes, but the diploid genome of mammalian cells has precluded large-scale gene disruption. We used insertional mutagenesis to develop a screening method to generate null alleles in a human cell line haploid for all chromosomes except chromosome 8. Using this approach, we identified host factors essential for infection with influenza and genes encoding important elements of the biosynthetic pathway of diphthamide, which are required for the cytotoxic effects of diphtheria toxin and exotoxin A. We also identified genes needed for the action of cytolethal distending toxin, including a cell-surface protein that interacts with the toxin. This approach has both conceptual and practical parallels with genetic approaches in haploid yeast.
View details for DOI 10.1126/science.1178955
View details for Web of Science ID 000272117900039
View details for PubMedID 19965467
Human induced pluripotent stem cell-derived cardiomyocytes as an in vitro model for coxsackievirus b3-induced myocarditis and antiviral drug screening platform.
2014; 115 (6): 556-566
Rationale: Viral myocarditis is a life-threatening illness that may lead to heart failure or cardiac arrhythmias. A major causative agent for viral myocarditis is the B3 strain of coxsackievirus, a positive-sense RNA enterovirus. However, human cardiac tissues are difficult to procure in sufficient enough quantities for studying the mechanisms of cardiac-specific viral infection. Objective: This study examined whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) could be used to model the pathogenic processes of coxsackievirus-induced viral myocarditis and to screen antiviral therapeutics for efficacy. Methods and Results: Human iPSC-CMs were infected with a luciferase-expressing coxsackievirus B3 strain (CVB3-Luc). Brightfield microscopy, immunofluorescence, and calcium imaging were utilized to characterize virally-infected hiPSC-CMs for alterations in cellular morphology and calcium handling. Viral proliferation in hiPSC-CMs was quantified using bioluminescence imaging. Antiviral compounds including interferon beta 1 (IFNβ1), ribavirin, pyrrolidine dithiocarbamate, and fluoxetine were tested for their capacity to abrogate CVB3-Luc proliferation in hiPSC-CMs in vitro. The ability of these compounds to reduce CVB3-Luc proliferation in hiPSC-CMs was consistent with reported drug effects in previous studies. Mechanistic analyses via gene expression profiling of hiPSC-CMs infected with CVB3-Luc revealed an activation of viral RNA and protein clearance pathways after IFNβ1 treatment. Conclusions: This study demonstrates that hiPSC-CMs express the coxsackievirus and adenovirus receptor, are susceptible to coxsackievirus infection, and can be used to predict antiviral drug efficacy. Our results suggest that the hiPSC-CM/CVB3-Luc assay is a sensitive platform that can screen novel antiviral therapeutics for their effectiveness in a high-throughput fashion.
View details for DOI 10.1161/CIRCRESAHA.115.303810
View details for PubMedID 25015077
GPR107, a G-protein-coupled Receptor Essential for Intoxication by Pseudomonas aeruginosa Exotoxin A, Localizes to the Golgi and Is Cleaved by Furin.
journal of biological chemistry
2014; 289 (35): 24005-24018
A number of toxins, including exotoxin A (PE) of Pseudomonas aeruginosa, kill cells by inhibiting protein synthesis. PE kills by ADP-ribosylation of the translation elongation factor 2, but many of the host factors required for entry, membrane translocation, and intracellular transport remain to be elucidated. A genome-wide genetic screen in human KBM7 cells was performed to uncover host factors used by PE, several of which were confirmed by CRISPR/Cas9-gene editing in a different cell type. Several proteins not previously implicated in the PE intoxication pathway were identified, including GPR107, an orphan G-protein-coupled receptor. GPR107 localizes to the trans-Golgi network and is essential for retrograde transport. It is cleaved by the endoprotease furin, and a disulfide bond connects the two cleaved fragments. Compromising this association affects the function of GPR107. The N-terminal region of GPR107 is critical for its biological function. GPR107 might be one of the long-sought receptors that associates with G-proteins to regulate intracellular vesicular transport.
View details for DOI 10.1074/jbc.M114.589275
View details for PubMedID 25031321
Inhibition of ATPIF1 Ameliorates Severe Mitochondrial Respiratory Chain Dysfunction in Mammalian Cells
2014; 7 (1): 27-34
Mitochondrial respiratory chain disorders are characterized by loss of electron transport chain (ETC) activity. Although the causes of many such diseases are known, there is a lack of effective therapies. To identify genes that confer resistance to severe ETC dysfunction when inactivated, we performed a genome-wide genetic screen in haploid human cells with the mitochondrial complex III inhibitor antimycin. This screen revealed that loss of ATPIF1 strongly protects against antimycin-induced ETC dysfunction and cell death by allowing for the maintenance of mitochondrial membrane potential. ATPIF1 loss protects against other forms of ETC dysfunction and is even essential for the viability of human ρ° cells lacking mitochondrial DNA, a system commonly used for studying ETC dysfunction. Importantly, inhibition of ATPIF1 ameliorates complex III blockade in primary hepatocytes, a cell type afflicted in severe mitochondrial disease. Altogether, these results suggest that inhibition of ATPIF1 can ameliorate severe ETC dysfunction in mitochondrial pathology.
View details for DOI 10.1016/j.celrep.2014.02.046
View details for Web of Science ID 000334298200004
View details for PubMedID 24685140
Late endosomal transport and tethering are coupled processes controlled by RILP and the cholesterol sensor ORP1L
JOURNAL OF CELL SCIENCE
2013; 126 (15): 3462-3474
Late endosomes and lysosomes are dynamic organelles that constantly move and fuse to acquire cargo from early endosomes, phagosomes and autophagosome. Defects in lysosomal dynamics cause severe neurodegenerative and developmental diseases, such as Niemann-Pick type C disease and ARC syndrome, yet little is known about the regulation of late endosomal fusion in a mammalian system. Mammalian endosomes destined for fusion need to be transported over very long distances before they tether to initiate contact. Here, we describe that lysosomal tethering and transport are combined processes co-regulated by one multi-protein complex: RAB7-RILP-ORP1L. We show that RILP directly and concomitantly binds the tethering HOPS complex and the p150(Glued) subunit of the dynein motor. ORP1L then functions as a cholesterol-sensing switch controlling RILP-HOPS-p150(Glued) interactions. We show that RILP and ORP1L control Ebola virus infection, a process dependent on late endosomal fusion. By combining recruitment and regulation of both the dynein motor and HOPS complex into a single multiprotein complex, the RAB7-RILP-ORP1L complex efficiently couples and regulates the timing of microtubule minus-end transport and fusion, two major events in endosomal biology.
View details for DOI 10.1242/jcs.129270
View details for Web of Science ID 000322570200024
View details for PubMedID 23729732
MCT1-mediated transport of a toxic molecule is an effective strategy for targeting glycolytic tumors.
2013; 45 (1): 104-108
There is increasing evidence that oncogenic transformation modifies the metabolic program of cells. A common alteration is the upregulation of glycolysis, and efforts to target glycolytic enzymes for anticancer therapy are under way. Here, we performed a genome-wide haploid genetic screen to identify resistance mechanisms to 3-bromopyruvate (3-BrPA), a drug candidate that inhibits glycolysis in a poorly understood fashion. We identified the SLC16A1 gene product, MCT1, as the main determinant of 3-BrPA sensitivity. MCT1 is necessary and sufficient for 3-BrPA uptake by cancer cells. Additionally, SLC16A1 mRNA levels are the best predictor of 3-BrPA sensitivity and are most elevated in glycolytic cancer cells. Furthermore, forced MCT1 expression in 3-BrPA-resistant cancer cells sensitizes tumor xenografts to 3-BrPA treatment in vivo. Our results identify a potential biomarker for 3-BrPA sensitivity and provide proof of concept that the selectivity of cancer-expressed transporters can be exploited for delivering toxic molecules to tumors.
View details for DOI 10.1038/ng.2471
View details for PubMedID 23202129
Attachment of Chlamydia trachomatis L2 to host cells requires sulfation
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2012; 109 (25): 10059-10064
Chlamydia trachomatis is a pathogen responsible for a prevalent sexually transmitted disease. It is also the most common cause of infectious blindness in the developing world. We performed a loss-of-function genetic screen in human haploid cells to identify host factors important in C. trachomatis L2 infection. We identified and confirmed B3GAT3, B4GALT7, and SLC35B2, which encode glucuronosyltransferase I, galactosyltransferase I, and the 3'-phosphoadenosine 5'-phosphosulfate transporter 1, respectively, as important in facilitating Chlamydia infection. Knockout of any of these three genes inhibits Chlamydia attachment. In complementation studies, we found that the introduction of functional copies of these three genes into the null clones restored full susceptibility to Chlamydia infection. The degree of attachment of Chlamydia strongly correlates with the level of sulfation of the host cell, not simply with the amount of heparan sulfate. Thus, other, as-yet unidentified sulfated macromolecules must contribute to infection. These results demonstrate the utility of screens in haploid cells to study interactions of human cells with bacteria. Furthermore, the human null clones generated can be used to investigate the role of heparan sulfate and sulfation in other settings not limited to infectious disease.
View details for DOI 10.1073/pnas.1120244109
View details for Web of Science ID 000306061400079
View details for PubMedID 22675117
Ebola virus entry requires the host-programmed recognition of an intracellular receptor
2012; 31 (8): 1947-1960
Ebola and Marburg filoviruses cause deadly outbreaks of haemorrhagic fever. Despite considerable efforts, no essential cellular receptors for filovirus entry have been identified. We showed previously that Niemann-Pick C1 (NPC1), a lysosomal cholesterol transporter, is required for filovirus entry. Here, we demonstrate that NPC1 is a critical filovirus receptor. Human NPC1 fulfills a cardinal property of viral receptors: it confers susceptibility to filovirus infection when expressed in non-permissive reptilian cells. The second luminal domain of NPC1 binds directly and specifically to the viral glycoprotein, GP, and a synthetic single-pass membrane protein containing this domain has viral receptor activity. Purified NPC1 binds only to a cleaved form of GP that is generated within cells during entry, and only viruses containing cleaved GP can utilize a receptor retargeted to the cell surface. Our findings support a model in which GP cleavage by endosomal cysteine proteases unmasks the binding site for NPC1, and GP-NPC1 engagement within lysosomes promotes a late step in entry proximal to viral escape into the host cytoplasm. NPC1 is the first known viral receptor that recognizes its ligand within an intracellular compartment and not at the plasma membrane.
View details for DOI 10.1038/emboj.2012.53
View details for Web of Science ID 000303108600010
View details for PubMedID 22395071
Identification of host cell factors required for intoxication through use of modified cholera toxin
JOURNAL OF CELL BIOLOGY
2011; 195 (5): 751-764
We describe a novel labeling strategy to site-specifically attach fluorophores, biotin, and proteins to the C terminus of the A1 subunit (CTA1) of cholera toxin (CTx) in an otherwise correctly assembled and active CTx complex. Using a biotinylated N-linked glycosylation reporter peptide attached to CTA1, we provide direct evidence that ~12% of the internalized CTA1 pool reaches the ER. We also explored the sortase labeling method to attach the catalytic subunit of diphtheria toxin as a toxic warhead to CTA1, thus converting CTx into a cytolethal toxin. This new toxin conjugate enabled us to conduct a genetic screen in human cells, which identified ST3GAL5, SLC35A2, B3GALT4, UGCG, and ELF4 as genes essential for CTx intoxication. The first four encode proteins involved in the synthesis of gangliosides, which are known receptors for CTx. Identification and isolation of the ST3GAL5 and SLC35A2 mutant clonal cells uncover a previously unappreciated differential contribution of gangliosides to intoxication by CTx.
View details for DOI 10.1083/jcb.201108103
View details for Web of Science ID 000297819900007
View details for PubMedID 22123862
Lipolysis-stimulated lipoprotein receptor (LSR) is the host receptor for the binary toxin Clostridium difficile transferase (CDT)
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2011; 108 (39): 16422-16427
Clostridium difficile infection (CDI) causes antibiotic-associated diarrhea and pseudomembranous colitis. Hypervirulent strains of the pathogen, which are responsible for increased morbidity and mortality of CDI, produce the binary actin-ADP ribosylating toxin Clostridium difficile transferase (CDT) in addition to the Rho-glucosylating toxins A and B. CDT depolymerizes the actin cytoskeleton, increases adherence and colonization of Clostridia by induction of microtubule-based cell protrusions and, eventually, causes death of target cells. Using a haploid genetic screen, we identified the lipolysis-stimulated lipoprotein receptor as the membrane receptor for CDT uptake by target cells. Moreover, we show that Clostridium perfringens iota toxin, which is a related binary actin-ADP ribosylating toxin, enters target cells via the lipolysis-stimulated lipoprotein receptor. Identification of the toxin receptors is essential for understanding of the toxin uptake and provides a most valuable basis for antitoxin strategies.
View details for DOI 10.1073/pnas.1109772108
View details for Web of Science ID 000295255300059
View details for PubMedID 21930894
A haploid genetic screen identifies the major facilitator domain containing 2A (MFSD2A) transporter as a key mediator in the response to tunicamycin
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2011; 108 (29): 11756-11765
Tunicamycin (TM) inhibits eukaryotic asparagine-linked glycosylation, protein palmitoylation, ganglioside production, proteoglycan synthesis, 3-hydroxy-3-methylglutaryl coenzyme-A reductase activity, and cell wall biosynthesis in bacteria. Treatment of cells with TM elicits endoplasmic reticulum stress and activates the unfolded protein response. Although widely used in laboratory settings for many years, it is unknown how TM enters cells. Here, we identify in an unbiased genetic screen a transporter of the major facilitator superfamily, major facilitator domain containing 2A (MFSD2A), as a critical mediator of TM toxicity. Cells without MFSD2A are TM-resistant, whereas MFSD2A-overexpressing cells are hypersensitive. Hypersensitivity is associated with increased cellular TM uptake concomitant with an enhanced endoplasmic reticulum stress response. Furthermore, MFSD2A mutant analysis reveals an important function of the C terminus for correct intracellular localization and protein stability, and it identifies transmembrane helical amino acid residues essential for mediating TM sensitivity. Overall, our data uncover a critical role for MFSD2A by acting as a putative TM transporter at the plasma membrane.
View details for DOI 10.1073/pnas.1018098108
View details for Web of Science ID 000292876900017
View details for PubMedID 21677192
Global gene disruption in human cells to assign genes to phenotypes by deep sequencing
2011; 29 (6): 542-U108
Insertional mutagenesis in a haploid background can disrupt gene function. We extend our earlier work by using a retroviral gene-trap vector to generate insertions in >98% of the genes expressed in a human cancer cell line that is haploid for all but one of its chromosomes. We apply phenotypic interrogation via tag sequencing (PhITSeq) to examine millions of mutant alleles through selection and parallel sequencing. Analysis of pools of cells, rather than individual clones enables rapid assessment of the spectrum of genes involved in the phenotypes under study. This facilitates comparative screens as illustrated here for the family of cytolethal distending toxins (CDTs). CDTs are virulence factors secreted by a variety of pathogenic Gram-negative bacteria responsible for tissue damage at distinct anatomical sites. We identify 743 mutations distributed over 12 human genes important for intoxication by four different CDTs. Although related CDTs may share host factors, they also exploit unique host factors to yield a profile characteristic for each CDT.
View details for DOI 10.1038/nbt.1857
View details for Web of Science ID 000291342100021
View details for PubMedID 21623355
Objective determination of the oncolytic potency of conditionally-replicating adenoviruses using mathematical modeling
JOURNAL OF GENE MEDICINE
2010; 12 (7): 564-571
Conditionally-replicating adenoviruses (CRAds) infect and replicate in tumor cells, releasing viral progeny upon lysis of the cell. This is a dynamic and inherently exponential process and, thus, the assessment of CRAds should incorporate these dynamics. In vitro experiments are therefore prone to subjective assessment because no validated assay exists that truly appreciates the dynamics of the process. An objective assay could simplify experiments and reduce the number of CRAd variants required to enter a full preclinical evaluation.We developed a simple and practical mathematical model incorporating easily obtainable parameters of the interaction between replicating viruses and growing tumor cells in vitro and, in the present study, validate this model by fitting the predicted values to experimentally-derived values.From the exponential curves of cellular growth and the viral propagation rate in glioma cells, we derive the four parameters needed in this model and show a robust fit to experimental data. Because the initial infection conditions appear to significantly influence the final outcome of CRAd experiments, these conditions are determined using the same cells and correlated with the expression of the primary adenovirus receptor CAR (coxsackie and adenovirus receptor).The results obtained shed light upon the method of action of CRAds and provide an objective and practical model and assay for determining and predicting CRAd activity in tumor cells.
View details for DOI 10.1002/jgm.1468
View details for Web of Science ID 000280451500003
View details for PubMedID 20603863
Generation of iPSCs from cultured human malignant cells
2010; 115 (20): 4039-4042
Induced pluripotent stem cells (iPSCs) can be generated from various differentiated cell types by the expression of a set of defined transcription factors. So far, iPSCs have been generated from primary cells, but it is unclear whether human cancer cell lines can be reprogrammed. Here we describe the generation and characterization of iPSCs derived from human chronic myeloid leukemia cells. We show that, despite the presence of oncogenic mutations, these cells acquired pluripotency by the expression of 4 transcription factors and underwent differentiation into cell types derived of all 3 germ layers during teratoma formation. Interestingly, although the parental cell line was strictly dependent on continuous signaling of the BCR-ABL oncogene, also termed oncogene addiction, reprogrammed cells lost this dependency and became resistant to the BCR-ABL inhibitor imatinib. This finding indicates that the therapeutic agent imatinib targets cells in a specific epigenetic differentiated cell state, and this may contribute to its inability to fully eradicate disease in chronic myeloid leukemia patients.
View details for DOI 10.1182/blood-2009-07-231845
View details for Web of Science ID 000277923600008
View details for PubMedID 20233975
Replacement of native adenovirus receptor-binding sites with a new attachment moiety diminishes hepatic tropism and enhances bioavailability in mice
HUMAN GENE THERAPY
2008; 19 (8): 783-794
The in vivo efficacy of adenoviral vectors (AdVs) in gene delivery strategies is hampered by the broad tissue tropism of the virus and its efficient binding to human erythrocytes. To circumvent these limitations, we developed a prototype AdV lacking native binding sites. We replaced the adenoviral fiber with a chimeric molecule consisting of the fiber tail domain, the reovirus sigma1 oligomerization domain, and a polyhistidine tag as model targeting moiety. We also abolished the integrin-binding motif in the penton base protein. The chimeric attachment molecule was efficiently incorporated onto AdV capsids, allowed efficient propagation of AdV without requirement for complementing fiber and conferred highly specific tropism to the AdV. Importantly, the targeted AdV exhibited markedly reduced tropism for liver cells. In comparison with control AdV with native tropism, the targeted AdV showed 1000-fold reduced transduction of HepG2 cells and 10,000-fold reduced transduction of mouse liver cells in freshly isolated liver slices. After intravenous inoculation of C57BL/6 mice, the targeted AdV exhibited delayed clearance in comparison with the native AdV, leaving approximately 10-fold greater levels in the blood 2 hr after inoculation. For all tissues analyzed, the targeted AdV displayed significantly reduced in vivo transduction in comparison with the native vector. Furthermore, in contrast to the native AdV, the targeted AdV did not bind human erythrocytes. Together, our findings suggest that the targeted AdV design described here provides a promising platform for systemic in vivo gene delivery.
View details for DOI 10.1089/hum.2007.133
View details for Web of Science ID 000259034300003
View details for PubMedID 18627267
Enhanced tumor cell kill by combined treatment with a small-molecule antagonist of mouse double minute 2 and adenoviruses encoding p53
MOLECULAR CANCER THERAPEUTICS
2007; 6 (5): 1552-1561
Strategies to treat cancer by restoring p53 tumor suppressor functions are being actively investigated. These approaches range from expressing an exogenous p53 gene in p53 mutant cancers to antagonizing a p53 inhibitor in p53 wild-type (WT) cancer cells. In addition, exogenous p53 is used to strengthen the anticancer efficacy of oncolytic adenoviruses. Many cancers express high levels of the major negative regulator of p53, mouse double minute 2 (MDM2) protein. Recently, a novel class of highly potent and specific MDM2 antagonists, the Nutlins, was identified. We envisioned that Nutlins could protect both endogenous and exogenous p53 from MDM2-mediated inactivation. We therefore investigated treating human cancer cells with a combination of adenovirus-mediated p53 gene therapy and Nutlin. Combination treatment resulted in broadly effective cell kill of p53 WT and p53-negative cancer cells. Cytotoxicity was associated with profound cell cycle checkpoint activation and apoptosis induction. We also tested Nutlin in combination with oncolytic adenoviruses. Nutlin treatment accelerated viral progeny burst from oncolytic adenovirus-infected cancer cells and caused an estimated 10- to 1,000-fold augmented eradication of p53 WT cancer cells. These findings suggest that Nutlins are promising compounds to be combined with p53 gene therapy and oncolytic virotherapy for cancer.
View details for DOI 10.1158/1535-7163.MCT-06-0631
View details for Web of Science ID 000246626900012
View details for PubMedID 17513604
A conditionally replicating adenovirus with strict selectivity in killing cells expressing epidermal growth factor receptor
2007; 361 (1): 56-67
Virotherapy of cancer using oncolytic adenoviruses has shown promise in both preclinical and clinical settings. One important challenge to reach the full therapeutic potential of oncolytic adenoviruses is accomplishing efficient infection of cancer cells and avoiding uptake by normal tissue through tropism modification. Towards this goal, we constructed and characterized an oncolytic adenovirus, carrying mutated capsid proteins to abolish the promiscuous adenovirus native tropism and encoding a bispecific adapter molecule to target the virus to the epidermal growth factor receptor (EGFR). The new virus displayed a highly selective targeting profile, with reduced infection of EGFR-negative cells and efficient killing of EGFR-positive cancer cells including primary EGFR-positive osteosarcoma cells that are refractory to infection by conventional adenoviruses. Our method to modify adenovirus tropism might thus be useful to design new oncolytic adenoviruses for more effective treatment of cancer.
View details for DOI 10.1016/j.virol.2006.11.011
View details for Web of Science ID 000246042300007
View details for PubMedID 17184803
Cyclophosphamide increases transgene expression mediated by an oncolytic adenovirus in glioma-bearing mice monitored by bioluminescence imaging
2006; 14 (6): 779-788
Approaches to improve the oncolytic potency of replication-competent adenoviruses include the insertion of therapeutic transgenes into the viral genome. Little is known about the levels and duration of in vivo transgene expression by cells infected with such "armed" viruses. Using a tumor-selective adenovirus encoding firefly luciferase (AdDelta24CMV-Luc) we investigated these questions in an intracranial mouse model for malignant glioma. Luciferase expression was detected by bioluminescence imaging, and the effect of the immunosuppressive agent cyclophosphamide (CPA) on transgene expression was assessed. Intratumoral AdDelta24CMV-Luc injection led to a localized dose-dependent expression of luciferase. Surprisingly, this expression decreased rapidly during the course of 14 days. In contrast, mice injected with nonreplicating Ad.CMV-Luc demonstrated stable transgene expression. Treatment of mice with CPA in combination with AdDelta24CMV-Luc retarded the loss of transgene expression. Staining of mouse brains for inflammatory cells demonstrated decreased tumor infiltration by immune cells in CPA-treated mice. Moreover, in immunodeficient NOD/SCID mice loss of transgene expression was less rapid and not prevented by CPA treatment. Together, our data demonstrate that transgene expression and viral replication decrease rapidly after intratumoral injection of oncolytic adenovirus in mouse brains and that treatment with the immunomodulator CPA prolongs viral-mediated gene expression.
View details for DOI 10.1016/j.ymthe.2006.08.008
View details for Web of Science ID 000242723300004
View details for PubMedID 16996314
Genetic targeting of adenovirus vectors using a reovirus sigma 1-based attachment protein
2006; 13 (5): 997-1005
Targeting adenovirus vectors (AdV's) for selective transduction of specific cell types requires ablation of native adenovirus tropism and introduction of a unique target-binding moiety. To bring these requirements within reach, we developed a novel strategy to target AdV's genetically that relies on replacement of the entire adenovirus fiber protein with a fusion molecule comprising the virion-anchoring domain of fiber and the oligomerization domain of reovirus attachment protein sigma1. The chimeric molecule forms trimers, is transported to the nucleus, and assembles onto the adenovirus capsid. In contrast to previously reported genetically targeted vectors, the AdV presented herein propagates efficiently without a requirement for complementing fiber. Due to ablation of the native adenovirus tropism, the infectivity of this AdV was at least 35-fold reduced on 293 cells. Importantly, a His tag incorporated into the chimeric attachment protein conferred His-tag-dependent tropism to the AdV, which resulted in a 12- to 40-fold greater transduction efficiency on two different cell lines expressing a His-tag-binding receptor. In addition, the infection efficiency was strongly reduced by preincubation with a His-tag-specific Ab. Thus, this sigma1-based chimeric attachment molecule provides a promising new platform for the generation of truly targeted AdV's.
View details for DOI 10.1016/j.ymthe.2005.11.019
View details for Web of Science ID 000237523000022
View details for PubMedID 16515889
Tissue inhibitor of metalloproteinase-3 expression from an oncolytic adenovirus inhibits matrix metalloproteinase activity In vivo without affecting antitumor efficacy in malignant glioma
2005; 65 (20): 9398-9405
Oncolytic adenoviruses exhibiting tumor-selective replication are promising anticancer agents. Insertion and expression of a transgene encoding tissue inhibitor of metalloproteinase-3 (TIMP-3), which has been reported to inhibit angiogenesis and tumor cell infiltration and induce apoptosis, may improve the antitumor activity of these agents. To assess the effects of TIMP-3 gene transfer to glioma cells, a replication-defective adenovirus encoding TIMP-3 (Ad.TIMP-3) was employed. Ad.TIMP-3 infection of a panel of glioma cell cultures decreased the proliferative capacity of these cells and induced morphologic changes characteristic for apoptosis. Next, a conditionally replicating adenovirus encoding TIMP-3 was constructed by inserting the TIMP-3 expression cassette into the E3 region of the adenoviral backbone containing a 24-bp deletion in E1A. This novel oncolytic adenovirus, AdDelta24TIMP-3, showed enhanced oncolytic activity on a panel of primary cell cultures and two glioma cell lines compared with the control oncolytic virus AdDelta24Luc. In vivo inhibition of matrix metalloproteinase (MMP) activity by AdDelta24TIMP-3 was shown in s.c. glioma xenografts. The functional activity of TIMP-3 was imaged noninvasively using a near-IR fluorescent MMP-2-activated probe. Tumoral MMP-2 activity was significantly reduced by 58% in the AdDelta24TIMP-3-treated tumors 24 hours after infection. A study into the therapeutic effects of combined oncolytic and antiproteolytic therapy was done in both a s.c. and an intracranial model for malignant glioma. Treatment of s.c. (U-87MG) or intracranial (U-87deltaEGFR) tumors with AdDelta24TIMP-3 and AdDelta24Luc both significantly inhibited tumor growth and prolonged survival compared with PBS-treated controls. However, expression of TIMP-3 in the context of AdDelta24 did not significantly affect the antitumor efficacy of this oncolytic agent.
View details for DOI 10.1158/0008-5472.CAN-04-4264
View details for Web of Science ID 000232566800042
View details for PubMedID 16230403
Replication-dependent transgene expression from a conditionally replicating adenovirus via alternative splicing to a heterologous splice-acceptor site
JOURNAL OF GENE MEDICINE
2005; 7 (8): 1053-1062
Oncolytic viruses are promising anticancer agents because they selectively kill cancer cells and multiply within a tumor. Their oncolytic potency might be improved by expressing a therapeutic gene from the virus genome. In this regard, proper kinetics and level of transgene expression are important. In addition, expression of cytotoxic transgene products should be confined to cancer cells. Here, we developed oncolytic adenoviruses that provide transgene expression dependent on viral replication.We constructed an oncolytic adenovirus that expresses luciferase under regulation of the endogenous major late promoter (MLP) via alternative splicing to an inserted splice-acceptor site analogous to that of the adenovirus serotype 40 long fiber gene. Splicing of the luciferase transcript was studied by RT-PCR analysis. Expression was measured in the presence and absence of the flavonoid apigenin, an inhibitor of viral replication.The inserted splice-acceptor site was properly recognized by the adenoviral splicing machinery. Luciferase expression levels were markedly higher than levels obtained with the cytomegalovirus (CMV) promoter, especially at late stages of infection. Inhibiting adenovirus replication reduced luciferase expression levels dramatically by 4 to 5 logs, whereas expression levels with the CMV-luciferase adenovirus were only moderately affected (2 logs).Transgene delivery using the endogenous late gene expression machinery resulted in an expression pattern distinct from expression driven by the conventional CMV promoter. The high expression levels and strict coupling of expression to viral replication should be useful for adequate monitoring of replication and might provide a platform for the design of armed conditionally replicating adenoviruses (CRAds) with enhanced oncolytic potency.
View details for DOI 10.1002/jgm.754
View details for Web of Science ID 000231477900006
View details for PubMedID 15756711
Gene-directed enzyme prodrug therapy with carboxylesterase enhances the anticancer efficacy of the conditionally replicating adenovirus Ad Delta 24
2005; 12 (12): 1011-1018
Conditionally replicating adenoviruses (CRAds) selectively replicate in and thereby kill cancer cells. The CRAd AdDelta24 with pRb-binding-deficient E1A kills cancer cells efficiently. Arming CRAds with genes encoding prodrug-converting enzymes could allow for enhanced anticancer efficacy by the combined effects of oncolytic replication and local prodrug activation. Here, we investigated combination treatment of human colon cancer cell lines with AdDelta24-type CRAds and gene-directed enzyme prodrug therapy (GDEPT) using two different enzyme/prodrug systems, that is, thymidine kinase/ganciclovir (TK/GCV) and carboxylesterase (CE)/CPT-11. On all three cell lines tested, GDEPT with TK/GCV made CRAd treatment less efficacious. In contrast, expression of a secreted form of CE (sCE2) combined with CPT-11 treatment markedly enhanced the efficacy of AdDelta24 virotherapy. Based on this observation, we constructed an AdDelta24 variant expressing sCE2. In the absence of CPT-11, this new CRAd Ad5-Delta24.E3-sCE2 was similarly effective as its parent in killing human colon cancer cells. Low concentrations of CPT-11 inhibited Ad5-Delta24.E3-sCE2 propagation. Nevertheless, CPT-11 specifically augmented the cytotoxicity of Ad5-Delta24.E3-sCE2 against all three-colon cancer cell lines. Hence, the positive contribution of sCE2/CPT-11 GDEPT to colon cancer cytotoxicity outweighed its negative influence on CRAd propagation. Therefore, CRAd-sCE2/CPT-11 combination therapy appears useful for more effective treatment of colon cancer.
View details for DOI 10.1038/sj.gt.3302492
View details for Web of Science ID 000229578300011
View details for PubMedID 15729367
- Coxsackievirus and adenovirus receptor expression on primary osteosarcoma specimens and implications for gene therapy with recombinant adenoviruses CLINICAL CANCER RESEARCH 2005; 11 (6): 2445-2447
Conditionally replicating adenoviruses expressing short hairpin RNAs silence the expression of a target gene in cancer cells
2004; 64 (8): 2663-2667
RNA interference (RNAi) is a posttranscriptional silencing mechanism triggered by double-stranded RNA that was recently shown to function in mammalian cells. Expression of cancer-associated genes was knocked down by expressing short hairpin RNAs (shRNAs) in cancer cells. By virtue of its excellent target specificity, RNAi may be used as a new therapeutic modality for cancer. The success of this approach will largely depend on efficient delivery of shRNAs to tumor cells. Tumor-selective replication competent viruses are especially suited to efficiently deliver anticancer genes to tumors. In addition, their intrinsic capacity to kill cancer cells makes these viruses promising anticancer agents per se. In this study, conditionally replicating adenoviruses were constructed encoding shRNAs targeted against firefly luciferase. These replicating viruses were shown to specifically silence the expression of the target gene in human cancer cells down to 30% relative to control virus. This finding offers the promise of using RNAi in the context of cancer gene therapy with oncolytic viruses.
View details for Web of Science ID 000220810400005
View details for PubMedID 15087375
Cowpea mosaic virus: effects on host cell processes
MOLECULAR PLANT PATHOLOGY
2002; 3 (6): 411-418
SUMMARY Taxonomy: Cowpea mosaic virus (CPMV) is the type member of the Comoviridae and bears a strong resemblance to animal picornaviruses, both in gene organization and in the amino acid sequence of replication proteins. Little systematic work has been done to compare isolates of the virus from different parts of the world. Physical properties: Purified preparations of virus contain three centrifugal components; empty protein shells without RNA (T) and two nucleoprotein components (M and B), containing 24% and 34% RNA, respectively. The icosahedral particles have with a diameter of 28 nm, consist of 60 copies of two coat proteins, and are heat stable. Hosts: CPMV causes one of the most commonly reported virus diseases of cowpea (Vigna unguiculata), in which it produces chlorotic spots with diffuse borders in inoculated primary leaves. Trifoliate leaves develop a bright yellow or light green mosaic of increasing severity in younger leaves. The host range is rather limited, and few hosts are known outside the Leguminosae. The virus is transmitted by various beetles with biting mouthparts. Reported in Africa, the Philippines and Iran. Is apparently absent from North and South America. Useful website: http://mmtsb.scripps.edu/viper/1cpmv.html (structure); http://image.fs.uidaho.edu/vide/descr254.htm (general information).
View details for Web of Science ID 000179408500001
View details for PubMedID 20569348
Coalescence of the sites of cowpea mosaic virus RNA replication into a cytopathic structure
JOURNAL OF VIROLOGY
2002; 76 (12): 6235-6243
Cowpea mosaic virus (CPMV) replication induces an extensive proliferation of endoplasmic reticulum (ER) membranes, leading to the formation of small membranous vesicles where viral RNA replication takes place. Using fluorescent in situ hybridization, we found that early in the infection of cowpea protoplasts, CPMV plus-strand RNA accumulates at numerous distinct subcellular sites distributed randomly throughout the cytoplasm which rapidly coalesce into a large body located in the center of the cell, often near the nucleus. The combined use of immunostaining and a green fluorescent protein ER marker revealed that during the course of an infection, CPMV RNA colocalizes with the 110-kDa viral polymerase and other replication proteins and is always found in close association with proliferated ER membranes, indicating that these sites correspond to the membranous site of viral replication. Experiments with the cytoskeleton inhibitors oryzalin and latrunculin B point to a role of actin and not tubulin in establishing the large central structure. The induction of ER membrane proliferations in CPMV-infected protoplasts did not coincide with increased levels of BiP mRNA, indicating that the unfolded-protein response is not involved in this process.
View details for DOI 10.1128/JVI.76.12.6235-6243.2002
View details for Web of Science ID 000175912200041
View details for PubMedID 12021357
Cowpea mosaic virus 32- and 60-kilodalton replication proteins target and change the morphology of endoplasmic reticulum membranes
JOURNAL OF VIROLOGY
2002; 76 (12): 6293-6301
Cowpea mosaic virus (CPMV) replicates in close association with small membranous vesicles that are formed by rearrangements of intracellular membranes. To determine which of the viral proteins are responsible for the rearrangements of membranes and the attachment of the replication complex, we have expressed individual CPMV proteins encoded by RNA1 in cowpea protoplasts by transient expression and in Nicotiana benthamiana plants by using the tobacco rattle virus (TRV) expression vector. The 32-kDa protein (32K) and 60K, when expressed individually, accumulate in only low amounts but are found associated with membranes mainly derived from the endoplasmic reticulum (ER). 24K and 110K are freely soluble and accumulate to high levels. With the TRV vector, expression of 32K and 60K results in rearrangement of ER membranes. Besides, expression of 32K and 60K results in necrosis of the inoculated N. benthamiana leaves, suggesting that 32K and 60K are cytotoxic proteins. On the other hand, during CPMV infection 32K and 60K accumulate to high levels without causing necrosis.
View details for DOI 10.1128/JVI.76.12.6293-6301.2002
View details for Web of Science ID 000175912200046
View details for PubMedID 12021362
Characterization of plant proteins that interact with cowpea mosaic virus '6OK' protein in the yeast two-hybrid system
JOURNAL OF GENERAL VIROLOGY
2002; 83: 885-893
Cowpea mosaic virus (CPMV) replication occurs in close association with small membranous vesicles in the host cell. The CPMV RNA1-encoded 60 kDa nucleotide-binding protein ('60K') plays a role in the formation of these vesicles. In this study, five cellular proteins were identified that interacted with different domains of 60K using a yeast two-hybrid search of an Arabidopsis cDNA library. Two of these host proteins (termed VAP27-1 and VAP27-2), with high homology to the VAP33 family of SNARE-like proteins from animals, interacted specifically with the C-terminal domain of 60K and upon transient expression colocalized with 60K in CPMV-infected cowpea protoplasts. eEF1-beta, picked up using the central domain of 60K, was also found to colocalize with 60K. The possible role of these host proteins in the viral replicative cycle is discussed.
View details for Web of Science ID 000174503100020
View details for PubMedID 11907339
Mutational analysis of the genome-linked protein of cowpea mosaic virus
2001; 290 (1): 21-29
In this study we have performed a mutational analysis of the cowpea mosaic comovirus (CPMV) genome-linked protein VPg to discern the structural requirements necessary for proper functioning of VPg. Either changing the serine residue linking VPg to RNA at a tyrosine or a threonine or changing the position of the serine from the N-terminal end to position 2 or 3 abolished virus infectivity. Some of the mutations affected the cleavage between the VPg and the 58K ATP-binding protein in vitro, which might have contributed to the lethal phenotype. RNA replication of some of the mutants designed to replace VPg with the related cowpea severe mosaic comovirus was completely abolished, whereas replication of others was not affected or only mildly affected, showing that amino acids that are not conserved between the comoviruses can be critical for the function of VPg. The replicative proteins of one of the mutants failed to accumulate in typical cytopathic structures and this might reflect the involvement of VPg in protein-protein interactions with the other replicative proteins.
View details for DOI 10.1006/viro.2001.1137
View details for Web of Science ID 000172463400003
View details for PubMedID 11883002
Alfalfa mosaic virus replicase proteins P1 and P2 interact and colocalize at the vacuolar membrane
JOURNAL OF VIROLOGY
2001; 75 (4): 1879-1887
Replication of Alfalfa mosaic virus (AMV) RNAs depends on the virus-encoded proteins P1 and P2. P1 contains methyltransferase- and helicase-like domains, and P2 contains a polymerase-like domain. Coimmunoprecipitation experiments revealed an interaction between in vitro translated-P1 and P2 and showed that these proteins are present together in fractions with RNA-dependent RNA polymerase activity. A deletion analysis in the yeast two-hybrid system showed that in P1 the C-terminal sequence of 509 amino acids with the helicase domain was necessary for the interaction. In P2, the sequence of the N-terminal 241 aa was required for the interaction. In infected protoplasts, P1 and P2 colocalized at a membrane structure that was identified as the tonoplast (i.e., the membrane that surrounds the vacuoles) by using a tonoplast intrinsic protein as a marker in immunofluorescence studies. While P1 was exclusively localized on the tonoplast, P2 was found both at the tonoplast and at other locations in the cell. As Brome mosaic virus replication complexes have been found to be associated with the endoplasmic reticulum (M. A. Restrepo-Hartwig and P. Ahlquist, J. Virol. 70:8908-8916, 1996), viruses in the family Bromoviridae apparently select different cellular membranes for the assembly of their replication complexes.
View details for Web of Science ID 000166697000032
View details for PubMedID 11160687
Cowpea mosaic virus infection induces a massive proliferation of endoplasmic reticulum but not Golgi membranes and is dependent on de novo membrane synthesis
JOURNAL OF VIROLOGY
2000; 74 (14): 6556-6563
Replication of cowpea mosaic virus (CPMV) is associated with small membranous vesicles that are induced upon infection. The effect of CPMV replication on the morphology and distribution of the endomembrane system in living plant cells was studied by expressing green fluorescent protein (GFP) targeted to the endoplasmic reticulum (ER) and the Golgi membranes. CPMV infection was found to induce an extensive proliferation of the ER, whereas the distribution and morphology of the Golgi stacks remained unaffected. Immunolocalization experiments using fluorescence confocal microscopy showed that the proliferated ER membranes were closely associated with the electron-dense structures that contain the replicative proteins encoded by RNA1. Replication of CPMV was strongly inhibited by cerulenin, an inhibitor of de novo lipid synthesis, at concentrations where the replication of the two unrelated viruses alfalfa mosaic virus and tobacco mosaic virus was largely unaffected. These results suggest that proliferating ER membranes produce the membranous vesicles formed during CPMV infection and that this process requires continuous lipid biosynthesis.
View details for Web of Science ID 000087817900038
View details for PubMedID 10864669