MOLECULAR BASIS OF REOVIRUS PATHOGENESIS

Summary

Principal Investigator: Terence S Dermody
Abstract: Abstract The goal of tfie proposed research is to determine mechanisnfis of viral pathogenesis using mammalian reovirus as an experimental system. Discrete steps in virus-host interaction often depend on selective recognition of cellular receptors and viral replication proteins that operate at distinct sites. However, the precise molecular mechanisms that govern viral dissemination and target cell selection are not well understood for most pathogenic viruses. This gap in knowledge has impeded the rational design of antiviral agents and vaccines that act at the earliest stages of infection. The proposed research uses reovirus, a highly tractable experimental model that shows promise for oncolytic and vaccine applications, to define how a virus interacts with its host at specific steps of the virus-host encounter. Reovirus infects a variety of mammalian species, including humans, but disease is restricted to the very young. Following peroral inoculation of newborn mice, reovirus infects intestinal mucosa, disseminates via hematogenous or neural routes, and targets the heart, liver, and central nervous system (CNS) to cause disease. Reovirus attachment protein a^ Influences the pathway of viral spread in the host and tropism for target tissues. The al protein is a filamentous trimer that binds to junctional adhesion molecule-A (JAM-A) and cell-surface sialic acid. In the previous funding period, we defined mechanisms by which a1 engages JAM-A and sialic acid, discovered that JAM-A-binding influences hematogenous spread and sialic acid-binding influences neural spread, and elucidated an essential function for nonstructural protein ols in reovirus dissemination. Our progress was facilitated by the development of a fully plasmid-based reverse genetics system for reovirus, the first of its kind for any mammalian double-stranded RNA virus. Based on these new discoveries, we hypothesize that reovirus spread to target tissues requires sequential recognition of discrete host-cell receptors by al and is dependent on ols. We propose three integrated specific aims in the extension period of this grant to test this hypothesis. In Specific Aim 1, mechanisms by which reovirus binding to JAM-A promotes infection of endothelial cells and hematogenous dissemination will be determined. We hypothesize that reovirus binding to JAM-A perturbs tight junction physiology in a manner to promote viral entry. To test this hypothesis, we will define the intracellular distribution of JAM-A and JAM-A-associated proteins following reovirus infection of polarized endothelial cells using confocal microscopy and subcellular fractionation. The phosphorylation status of JAM-A following reovirus attachment will be determined using wild-type and point-mutant JAM-A derivatives. Transgenic mice in which JAM-A is either expressed or ablated solely in endothelial or hematopoietic cells will be infected with reovirus and monitored for survival, viral load, and pathologic injury. These experiments will elucidate how a virus exploits a junction-associated receptor to initiate infection and clarify how a broadly expressed viral receptor dictates an exquisitely specific step in viral pathogenesis. In Specific Aim 2, receptors engaged by reovirus to infect the murine CNS will be identified. Although JAM-A is required for reovirus growth in endothelial cells, it is dispensable for reovirus growth in the brain. We hypothesize that reovirus uses unique receptors to infect the CNS. To test this hypothesis, we will identify neural receptors for reovirus using two complementary approaches. In the first, membrane preparations from JAM-A-null cortical neurons will be screened for candidate reovirus receptors using virus-overlay-protein- binding assays. In the second, a cDNA library will be generated from JAM-A-null brain tissue and screened for cDNAs that encode reovirus-binding molecules. Candidate receptors will be validated using receptor-specific antibodies, receptor-deficient cell lines, and assays of virus-receptor binding. The function of newly identified neural receptors in reovirus pathogenesis will be defined using receptor-null mice. These studies will reveal mechanisms by which viruses selectively target specific cells in the CNS. In Specific Aim 3, mechanisms by which the G1S protein influences reovirus pathogenesis will be elucidated. The als protein is required for reovirus dissemination and mediates cell-cycle arrest and apoptosis induction. We hypothesize that a1s-induced cell cycle arrest leads to apoptosis of target cells that are in turn engulfed by phagocytic ceils to initiate systemic dissemination. To test this hypothesis, we will identify sequences in a1s that influence reovirus cell cycle arrest, apoptosis induction, and systemic dissemination using mutant viruses engineered by reverse genetics. Targets of reovirus infection in vivo will be monitored for apoptotic cell death following inoculation with wild-type and als-mutant viruses to define the function of a1s- induced apoptosis in viral transit in the infected host. Cellular proteins engaged by a1s will be identified using tandem-affinity purification. Cells genetically deficient in candidate als-interacting proteins will be tested for reovirus infection, cell-cycle arrest, and apoptosis. These experiments will illuminate how a virus induces cell cycle arrest and apoptosis and the means by which these events lead to viral dissemination in the host. Results of the proposed experiments will contribute broadly to an understanding of mechanisms used by viruses to breach mucosal surfaces, disseminate systemically, and target specific host tissues to cause disease, The molecular blueprint of reovirus pathogenesis enabled by this work will identify key steps in virus- host interaction amenable to antiviral intervention and accelerate progress in the development of reovirus as a vector for oncolytic and vaccine applications.
Funding Period: 1996-09-30 - 2016-05-31
more information: NIH RePORT

Top Publications

  1. ncbi Reovirus binding determinants in junctional adhesion molecule-A
    Kristen M Guglielmi
    Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA
    J Biol Chem 282:17930-40. 2007
  2. pmc Focal adhesion kinase is a component of antiviral RIG-I-like receptor signaling
    Rebecca A Bozym
    Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA 15219, USA
    Cell Host Microbe 11:153-66. 2012
  3. pmc Comparative analysis of Reoviridae reverse genetics methods
    Shane D Trask
    Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 8026, USA
    Methods 59:199-206. 2013
  4. pmc Utilization of sialylated glycans as coreceptors enhances the neurovirulence of serotype 3 reovirus
    Johnna M Frierson
    Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
    J Virol 86:13164-73. 2012
  5. pmc Directional release of reovirus from the apical surface of polarized endothelial cells
    Caroline M Lai
    Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
    MBio 4:e00049-13. 2013
  6. pmc Genetic determinants of reovirus pathogenesis in a murine model of respiratory infection
    Rachel M Nygaard
    Department of Microbiology, University of Minnesota, Minneapolis, Minnesota, USA
    J Virol 87:9279-89. 2013
  7. ncbi Mechanisms of reovirus bloodstream dissemination
    Karl W Boehme
    Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
    Adv Virus Res 87:1-35. 2013
  8. ncbi Reovirus receptors, cell entry, and proapoptotic signaling
    Pranav Danthi
    Department of Biology, Indiana University, Bloomington, IN, USA
    Adv Exp Med Biol 790:42-71. 2013
  9. pmc JAM-A associates with ZO-2, afadin, and PDZ-GEF1 to activate Rap2c and regulate epithelial barrier function
    Ana C Monteiro
    Department of Pathology, Emory University School of Medicine, Atlanta, GA 30306 Department of Pharmacology, Emory University School of Medicine, Atlanta, GA 30306 Emory Rollins School of Public Health, Atlanta, GA 30306 Interfaculty Institute of Biochemistry, University of Tubingen, D 72076 Tubingen, Germany Department of Pediatrics and Pathology, Vanderbilt University School of Medicine, Nashville, TN 37230 Departments of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37230 Elizabeth B Lamb Center for Pediatric Research, Vanderbilt University School of Medicine, Nashville, TN 37230
    Mol Biol Cell 24:2849-60. 2013
  10. pmc Nonstructural protein σ1s mediates reovirus-induced cell cycle arrest and apoptosis
    Karl W Boehme
    Department of Microbiology and Immunology
    J Virol 87:12967-79. 2013

Detail Information

Publications31

  1. ncbi Reovirus binding determinants in junctional adhesion molecule-A
    Kristen M Guglielmi
    Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA
    J Biol Chem 282:17930-40. 2007
    ....
  2. pmc Focal adhesion kinase is a component of antiviral RIG-I-like receptor signaling
    Rebecca A Bozym
    Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA 15219, USA
    Cell Host Microbe 11:153-66. 2012
    ..These findings suggest that FAK serves as a link between cytoskeletal perturbations that occur during virus infection and activation of innate immune signaling...
  3. pmc Comparative analysis of Reoviridae reverse genetics methods
    Shane D Trask
    Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 8026, USA
    Methods 59:199-206. 2013
    ..Analysis and comparison of each method suggest several key lines of research that might lead to a reverse genetics system for RV, analogous to those used for MRV and BTV...
  4. pmc Utilization of sialylated glycans as coreceptors enhances the neurovirulence of serotype 3 reovirus
    Johnna M Frierson
    Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
    J Virol 86:13164-73. 2012
    ..These results suggest that SA binding enhances the kinetics of reovirus replication in neural tissues and highlight a functional role for sialylated glycans as reovirus coreceptors in the CNS...
  5. pmc Directional release of reovirus from the apical surface of polarized endothelial cells
    Caroline M Lai
    Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
    MBio 4:e00049-13. 2013
    ..Understanding how viruses invade the bloodstream may aid in the development of therapeutics that block this step in viral pathogenesis...
  6. pmc Genetic determinants of reovirus pathogenesis in a murine model of respiratory infection
    Rachel M Nygaard
    Department of Microbiology, University of Minnesota, Minneapolis, Minnesota, USA
    J Virol 87:9279-89. 2013
    ..These findings provide new insights into mechanisms underlying reovirus replication in the respiratory tract and systemic spread from the lung. ..
  7. ncbi Mechanisms of reovirus bloodstream dissemination
    Karl W Boehme
    Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
    Adv Virus Res 87:1-35. 2013
    ..Understanding mechanisms of reovirus bloodborne spread may shed light on how microbial pathogens invade the bloodstream to disseminate and cause disease in infected hosts. ..
  8. ncbi Reovirus receptors, cell entry, and proapoptotic signaling
    Pranav Danthi
    Department of Biology, Indiana University, Bloomington, IN, USA
    Adv Exp Med Biol 790:42-71. 2013
    ..This chapter will focus on the mechanisms by which reovirus attachment and disassembly activate NF-κB and stimulate the cellular proapoptotic machinery. ..
  9. pmc JAM-A associates with ZO-2, afadin, and PDZ-GEF1 to activate Rap2c and regulate epithelial barrier function
    Ana C Monteiro
    Department of Pathology, Emory University School of Medicine, Atlanta, GA 30306 Department of Pharmacology, Emory University School of Medicine, Atlanta, GA 30306 Emory Rollins School of Public Health, Atlanta, GA 30306 Interfaculty Institute of Biochemistry, University of Tubingen, D 72076 Tubingen, Germany Department of Pediatrics and Pathology, Vanderbilt University School of Medicine, Nashville, TN 37230 Departments of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37230 Elizabeth B Lamb Center for Pediatric Research, Vanderbilt University School of Medicine, Nashville, TN 37230
    Mol Biol Cell 24:2849-60. 2013
    ..These results suggest that JAM-A regulates epithelial permeability via association with ZO-2, afadin, and PDZ-GEF1 to activate Rap2c and control contraction of the apical cytoskeleton. ..
  10. pmc Nonstructural protein σ1s mediates reovirus-induced cell cycle arrest and apoptosis
    Karl W Boehme
    Department of Microbiology and Immunology
    J Virol 87:12967-79. 2013
    ..Collectively, these findings provide evidence that the σ1s-mediated properties are genetically linked and suggest that these effects are mechanistically related. ..
  11. pmc The invasive chytrid fungus of amphibians paralyzes lymphocyte responses
    J Scott Fites
    Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235, USA
    Science 342:366-9. 2013
    ..Their production was absent in zoospores and reduced by nikkomycin Z, suggesting that they may be components of the cell wall. Evasion of host immunity may explain why this pathogen has devastated amphibian populations worldwide. ..
  12. pmc An ITAM in a nonenveloped virus regulates activation of NF-κB, induction of beta interferon, and viral spread
    Rachael E Stebbing
    Department of Molecular Biomedical Sciences and Center for Comparative Medicine and Translational Research, North Carolina State University, Raleigh, North Carolina, USA
    J Virol 88:2572-83. 2014
    ..This first demonstration of a functional ITAM in a nonenveloped virus presents a new mechanism for viral ITAM-mediated signaling with likely organ-specific consequences in the host. ..
  13. pmc Trans-dimerization of JAM-A regulates Rap2 and is mediated by a domain that is distinct from the cis-dimerization interface
    Ana C Monteiro
    Epithelial Pathobiology and Mucosal Inflammation Research Unit, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322
    Mol Biol Cell 25:1574-85. 2014
    ..Trans-dimerization of JAM-A may thus act as a barrier-inducing molecular switch that is activated when cells become confluent. ..
  14. pmc Reovirus-mediated induction of ADAR1 (p150) minimally alters RNA editing patterns in discrete brain regions
    Jennifer L Hood
    Vanderbilt Brain Institute, Vanderbilt University School of Medicine, Nashville, TN, United States
    Mol Cell Neurosci 61:97-109. 2014
    ....
  15. pmc A single-amino-acid polymorphism in reovirus protein μ2 determines repression of interferon signaling and modulates myocarditis
    Susan C Irvin
    Department of Molecular Biomedical Sciences, North Carolina State University, Raleigh, North Carolina, USA
    J Virol 86:2302-11. 2012
    ..These results demonstrate that a single amino acid difference between viruses can dictate virus strain-specific differences in suppression of the host IFN-β response and, consequently, damage to the heart...
  16. pmc Intestinal microbiota promote enteric virus replication and systemic pathogenesis
    Sharon K Kuss
    Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
    Science 334:249-52. 2011
    ..These results suggest that antibiotic-mediated microbiota depletion diminishes enteric virus infection and that enteric viruses exploit intestinal microbes for replication and transmission...
  17. pmc The reovirus sigma1s protein is a determinant of hematogenous but not neural virus dissemination in mice
    Karl W Boehme
    Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
    J Virol 85:11781-90. 2011
    ..Together, these results indicate that σ1s is not required for reovirus spread by neural mechanisms. Instead, σ1s mediates hematogenous dissemination within the infected host, which is required for full reovirus neurovirulence...
  18. pmc A plasmid-based reverse genetics system for animal double-stranded RNA viruses
    Takeshi Kobayashi
    Department of Pediatrics, Elizabeth B Lamb Center for Pediatric Research, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
    Cell Host Microbe 1:147-57. 2007
    ..The plasmid-based reverse genetics approach described here can be exploited for studies of reovirus replication and pathogenesis and used to develop reovirus as a vaccine vector...
  19. pmc JAM-A regulates permeability and inflammation in the intestine in vivo
    Mike G Laukoetter
    Epithelial Pathobiology Research Unit, Department of Pathology, Emory University, Atlanta, GA 30322, USA
    J Exp Med 204:3067-76. 2007
    ..These findings demonstrate a complex role of JAM-A in intestinal homeostasis by regulating epithelial permeability, inflammation, and proliferation...
  20. pmc Reovirus preferentially infects the basolateral surface and is released from the apical surface of polarized human respiratory epithelial cells
    Katherine J D A Excoffon
    Department of Internal Medicine, University of Iowa, Iowa City, IA 52242, USA
    J Infect Dis 197:1189-97. 2008
    ..These results establish the existence of an infectious circuit for reovirus in polarized human respiratory epithelial cells...
  21. pmc Replacement of native adenovirus receptor-binding sites with a new attachment moiety diminishes hepatic tropism and enhances bioavailability in mice
    Frederik H E Schagen
    Department of Medical Oncology, VU University Medical Center, 1181 HV Amsterdam, The Netherlands
    Hum Gene Ther 19:783-94. 2008
    ..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...
  22. pmc Structure of reovirus sigma1 in complex with its receptor junctional adhesion molecule-A
    Eva Kirchner
    Interfaculty Institute for Biochemistry, University of Tuebingen, Tuebingen, Germany
    PLoS Pathog 4:e1000235. 2008
    ..These studies define biophysical mechanisms of reovirus cell attachment and provide a platform for manipulating reovirus tropism to enhance vector targeting...
  23. pmc Junctional adhesion molecule-A is required for hematogenous dissemination of reovirus
    Annukka A R Antar
    Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
    Cell Host Microbe 5:59-71. 2009
    ..These findings indicate that a broadly expressed IgSF viral receptor specifically mediates hematogenous dissemination in the host...
  24. pmc Reovirus nonstructural protein sigma1s is required for establishment of viremia and systemic dissemination
    Karl W Boehme
    Department of Pediatrics, and The Elizabeth B Lamb Center for Pediatric Research, D7235 Medical Center North, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
    Proc Natl Acad Sci U S A 106:19986-91. 2009
    ..These results suggest a key role for sigma1s in virus spread from intestinal lymphatics to the bloodstream, thereby allowing the establishment of viremia and dissemination to sites of secondary replication within the infected host...
  25. pmc An improved reverse genetics system for mammalian orthoreoviruses
    Takeshi Kobayashi
    Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
    Virology 398:194-200. 2010
    ..Improvements to the reovirus reverse genetics system enhance its applicability for studies of reovirus biology and clinical use...
  26. pmc Dual selection mechanisms drive efficient single-gene reverse genetics for rotavirus
    Shane D Trask
    Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 8026, USA
    Proc Natl Acad Sci U S A 107:18652-7. 2010
    ..Furthermore, application of a dual selection strategy to previously reported reverse genetics methods for RV may enhance the efficiency of recombinant virus recovery...
  27. pmc Chronic progressive deficits in neuron size, density and number in the trigeminal ganglia of mice latently infected with herpes simplex virus
    Sandor Dosa
    Department of Pathology, University of Illinois at Chicago, College of Medicine, Chicago, IL 60612, USA
    Brain Pathol 21:583-93. 2011
    ..These studies demonstrate that latent HSV infection is associated with progressive neuronal pathology and may lead to a better understanding of the role of HSV infections in chronic neurological diseases...
  28. pmc Reverse genetics for mammalian reovirus
    Karl W Boehme
    Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
    Methods 55:109-13. 2011
    ....
  29. pmc Crystal structure of reovirus attachment protein σ1 in complex with sialylated oligosaccharides
    Dirk M Reiter
    Interfaculty Institute of Biochemistry, University of Tuebingen, Tuebingen, Germany
    PLoS Pathog 7:e1002166. 2011
    ..They also establish a filamentous, trimeric carbohydrate-binding module that could potentially be used to endow other trimeric proteins with carbohydrate-binding properties...
  30. pmc The Nogo receptor NgR1 mediates infection by mammalian reovirus
    Jennifer L Konopka-Anstadt
    Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, TN 37232, USA Elizabeth B Lamb Center for Pediatric Research, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
    Cell Host Microbe 15:681-91. 2014
    ..These results suggest that reovirus uses different capsid components to bind distinct cell-surface molecules, engaging independent receptors to facilitate spread and tropism. ..

Research Grants30

  1. Structural Analysis of Reovirus Attachment Mechanisms
    Thilo Stehle; Fiscal Year: 2013
    ..These studies will enhance a basic understanding of mechanisms by which pathogenic viruses engage cellular receptors and accelerate the rational design of viral vectors for therapeutic purposes. ..
  2. Pacific NorthWest Regional Center of Excellence (PNWRCE)
    Jay A Nelson; Fiscal Year: 2013
    ..pseudomallei host pathogen response during both the septicemic as well as the intracellular phases of the disease. ..
  3. New England Regional Center of Excellence in Biodefense and Emerging Infectious D
    Dennis L Kasper; Fiscal Year: 2013
    ..NERCE will also continue its Developmental Projects program and Career Development in Biodefense program in an effort to initiate new research efforts and to attract new investigators to this field. ..
  4. Northeast Biodefense Center
    W Ian Lipkin; Fiscal Year: 2013
    ..As a Center based in a School of Public Health and a State Department of Health, the NBC has a firm commitment to and practical understanding of Emergency Preparedness. ..
  5. The Molecular Determinants of Virus Induced Biliary Atresia
    Gregory M Tiao; Fiscal Year: 2013
    ..In so doing develop we hope to develop new treatment strategies to alter the course of this challenging disease. This project is in complete accord with the NIH mission to reduce illness and disability. ..
  6. Semi-volatile PCBs: Sources, Exposures, Toxicities
    Larry W Robertson; Fiscal Year: 2013
    ..These data and dietary studies in the last Aim will provide a scientific basis for risk assessment and advice for stakeholders with the ultimate goal to protect highly-exposed individuals and populations. ..
  7. Pacific Southwest RCE for Biodefense &Emerging Infectious Diseases Research
    Alan G Barbour; Fiscal Year: 2013
    ..abstract_text> ..
  8. Rocky Mountain Regional Center of Excellence or Biodefense and Emerging Infectiou
    John T Belisle; Fiscal Year: 2013
    ..abstract_text> ..
  9. VACCINE INDUCED IMMUNITY IN THE YOUNG AND AGED
    Rafi Ahmed; Fiscal Year: 2013
    ....
  10. Gut Homing Cells in SIV infection
    Aftab A Ansari; Fiscal Year: 2013
    ....