Structure and Function of the Hepatitis C Virus Genome

Summary

Principal Investigator: Peter Sarnow
Abstract: DESCRIPTION (provided by applicant): Approximately 80% of hepatocellular carcinoma, a leading cause of cancer-related deaths, is caused by infection with either hepatitis B or hepatitis C virus (HCV). The burden of HCV infection worldwide is very large, with most of the personal and economic burden yet to come, as cirrhosis and cancer take years to develop. Approximately 170 million people are infected worldwide with HCV. Most infected individuals do not clear the disease, but develop chronic infections that often lead to end-stage liver disease. Current treatment is limited to co-treatment with ribavirin and interferon 1, a therapy that is expensive and ineffective in 50% of infected individuals. Therefore, there is an urgent need to identify new and accessible viral and cellular targets for therapies against HCV. This application will study two important HCV RNA-protein interactions that regulate HCV gene amplification. A highly-structured RNA element is located in the viral 5'noncoding region that functions as an internal ribosome entry site (IRES) that can directly recruit 40S ribosomal subunits to the viral genome. Specifically, we will employ biochemical, NMR and single-molecule fluorescence approaches to explore the timing and control of subunit joining on IRES-initiated mRNAs. In addition, we will examine roles for eukaryotic initiation factors eIF5B and eIF2 in causing conformational changes in the ribosomal subunits. Using FRET and optical tweezer approaches, we will measure stabilities of IRES-ribosomal complexes and rearrangements of these complexes on mRNAs. Proposed long-range communication between 5'and 3'ends in HCV viral RNAs will be studied using NMR. Secondly, we will study the interaction of RNA helicase RIG-I with viral and host RNA ligands. Specifically, a cell-based crosslinking-immunoprecipitation (CLIP) assay will be employed to identify HCV and cellular RNA ligands for RIG-I. Using an infectious HCV cell-based system, we will determine whether RIG-I targets the viral RNA in replicating or translating RNA molecules. Using siRNA-mediated gene knock down, biochemical and cell-biological assays, we will characterize roles, 5'end modifications and localization of identified RIG-I ligands. Using biochemical assays, NMR and FRET analyses, biophysical and structural parameters for RIG-I ligand recognition and enzymatic kinetics that are activated by the RNAs will be examined and a structural framework for these activities will be established. Structural, dynamic and mechanistic views of HCV non-coding region structures, that modulate translation, and that of specific RNA ligands associated with RIG-I, that are part of innate immune responses, will be merged with approaches that probe function, and potential inhibition by therapeutics. PUBLIC HEALTH RELEVANCE: An estimated 170 million people worldwide and 4 million in the United States are infected with hepatitis C virus (HCV). The majority of patients do not resolve the infection and become chronic carriers, ultimately needing expensive liver transplants. There is no vaccine for HCV, and current treatments, which include ribavirin and interferon 1, are expensive and relatively ineffective. It was discovered that HCV binds ribosomes by a novel, unprecedented mechanism of internal ribosome binding, and that a key defense protein against viruses, helicase RIG-I, interacts with the viral genome. This proposal explores whether the mechanisms by which ribosome and RIG-I bind to the viral RNA genome present an Achilles heel that can be used for antiviral intervention. To do this, novel biochemical, structural and genetic approaches will be employed to study protein-HCV RNA interactions.
Funding Period: 1999-09-15 - 2014-12-31
more information: NIH RePORT

Top Publications

  1. ncbi Interaction of viruses with the mammalian RNA interference pathway
    Sylvia Schütz
    Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA
    Virology 344:151-7. 2006
  2. pmc Modulation of GB virus B RNA abundance by microRNA-122: dependence on and escape from microRNA-122 restriction
    Selena M Sagan
    Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA
    J Virol 87:7338-47. 2013
  3. pmc Modulation of hepatitis C virus RNA abundance and virus release by dispersion of processing bodies and enrichment of stress granules
    Cara T Pager
    Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305 5124, United States
    Virology 435:472-84. 2013
  4. ncbi Combating hepatitis C virus by targeting microRNA-122 using locked nucleic acids
    Erica S Machlin
    Department of Microbiology and Immunology, Stanford University School of Medicine, CA 94305, USA
    Curr Gene Ther 12:301-6. 2012
  5. pmc Masking the 5' terminal nucleotides of the hepatitis C virus genome by an unconventional microRNA-target RNA complex
    Erica S Machlin
    Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA
    Proc Natl Acad Sci U S A 108:3193-8. 2011
  6. ncbi The anti-hepatitis C agent nitazoxanide induces phosphorylation of eukaryotic initiation factor 2alpha via protein kinase activated by double-stranded RNA activation
    Menashe Elazar
    Division of Gastroenterology and Hepatology, Department of Medicine, Stanford University School of Medicine, Palo Alto, California, USA
    Gastroenterology 137:1827-35. 2009
  7. ncbi Translational insensitivity to potent activation of PKR by HCV IRES RNA
    Takashi Shimoike
    Department of Virology II, National Institute of Infectious Diseases, Musashi Murayama, Tokyo 208 0011, Japan
    Antiviral Res 83:228-37. 2009
  8. pmc Position-dependent function for a tandem microRNA miR-122-binding site located in the hepatitis C virus RNA genome
    Catherine L Jopling
    Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, UK Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA
    Cell Host Microbe 4:77-85. 2008
  9. pmc Viral dsRNA inhibitors prevent self-association and autophosphorylation of PKR
    Sean A McKenna
    Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305 5126, USA
    J Mol Biol 372:103-13. 2007
  10. ncbi Molecular framework for the activation of RNA-dependent protein kinase
    Sean A McKenna
    Department of Structural Biology and Stanford Magnetic Resonance Laboratory, Stanford University School of Medicine, Stanford, California 94305 5126, USA
    J Biol Chem 282:11474-86. 2007

Detail Information

Publications14

  1. ncbi Interaction of viruses with the mammalian RNA interference pathway
    Sylvia Schütz
    Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA
    Virology 344:151-7. 2006
    ..These findings argue that interactions between the RNAi pathway and viral genomes can profoundly affect the outcomes of the viral life cycles and contribute to the pathogenic signatures of the infectious agents...
  2. pmc Modulation of GB virus B RNA abundance by microRNA-122: dependence on and escape from microRNA-122 restriction
    Selena M Sagan
    Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA
    J Virol 87:7338-47. 2013
    ..This finding suggests that structural features at the end of the viral genome dictate whether miR-122 is required to aid in maintaining viral RNA abundance...
  3. pmc Modulation of hepatitis C virus RNA abundance and virus release by dispersion of processing bodies and enrichment of stress granules
    Cara T Pager
    Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305 5124, United States
    Virology 435:472-84. 2013
    ..These data argue that HCV subverts P-body and stress granule components to aid in viral gene expression at particular sites in the cytoplasm...
  4. ncbi Combating hepatitis C virus by targeting microRNA-122 using locked nucleic acids
    Erica S Machlin
    Department of Microbiology and Immunology, Stanford University School of Medicine, CA 94305, USA
    Curr Gene Ther 12:301-6. 2012
    ..This review summarizes the success of sequestration of liver-specific microRNA miR-122 by antisense locked nucleic acids and their use in combating hepatitis C virus in clinical trials...
  5. pmc Masking the 5' terminal nucleotides of the hepatitis C virus genome by an unconventional microRNA-target RNA complex
    Erica S Machlin
    Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA
    Proc Natl Acad Sci U S A 108:3193-8. 2011
    ..Finally, this remarkable microRNA-mRNA complex could be targeted with compounds that inactivate miR-122 or interfere with this unique RNA structure...
  6. ncbi The anti-hepatitis C agent nitazoxanide induces phosphorylation of eukaryotic initiation factor 2alpha via protein kinase activated by double-stranded RNA activation
    Menashe Elazar
    Division of Gastroenterology and Hepatology, Department of Medicine, Stanford University School of Medicine, Palo Alto, California, USA
    Gastroenterology 137:1827-35. 2009
    ..A pilot clinical study suggested that NTZ can augment the antiviral effect of interferon (IFN), although the molecular basis for its effect was unknown...
  7. ncbi Translational insensitivity to potent activation of PKR by HCV IRES RNA
    Takashi Shimoike
    Department of Virology II, National Institute of Infectious Diseases, Musashi Murayama, Tokyo 208 0011, Japan
    Antiviral Res 83:228-37. 2009
    ..Thus, HCV can use structured RNAs to its advantage in translation, while avoiding the deleterious effects of PKR activation...
  8. pmc Position-dependent function for a tandem microRNA miR-122-binding site located in the hepatitis C virus RNA genome
    Catherine L Jopling
    Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, UK Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA
    Cell Host Microbe 4:77-85. 2008
    ..These findings set a paradigm for dual, position-dependent functions of tandem microRNA-binding sites. Targeting an oligomeric microRNA complex offers potential as an antiviral-intervention strategy...
  9. pmc Viral dsRNA inhibitors prevent self-association and autophosphorylation of PKR
    Sean A McKenna
    Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305 5126, USA
    J Mol Biol 372:103-13. 2007
    ..These data indicate that inhibitory dsRNAs bind preferentially to the latent, dephosphorylated form of PKR and prevent dimerization that is required for trans-autophosphorylation...
  10. ncbi Molecular framework for the activation of RNA-dependent protein kinase
    Sean A McKenna
    Department of Structural Biology and Stanford Magnetic Resonance Laboratory, Stanford University School of Medicine, Stanford, California 94305 5126, USA
    J Biol Chem 282:11474-86. 2007
    ..We propose an updated model for PKR activation in which the communication between RNA binding, central linker, and kinase domains is critical in the propagation of the activation signal and for PKR dimerization...
  11. pmc Rapid purification of RNAs using fast performance liquid chromatography (FPLC)
    Insil Kim
    Department of Structural Biology, Stanford Magnetic Resonance Laboratory, School of Medicine, Stanford University, CA 94305, USA
    RNA 13:289-94. 2007
    ..This methodology allows simple and rapid purification of RNA oligonucleotides for structural and biophysical studies...
  12. ncbi Uncoupling of RNA binding and PKR kinase activation by viral inhibitor RNAs
    Sean A McKenna
    Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305 5126, USA
    J Mol Biol 358:1270-85. 2006
    ..Finally, we show that dsRNA binding and inactivation are non-equivalent; regions other than the dsRBD stem-loops of inhibitory RNA are required for inhibition...
  13. ncbi Specific recognition of HIV TAR RNA by the dsRNA binding domains (dsRBD1-dsRBD2) of PKR
    Insil Kim
    Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305 5126, USA
    J Mol Biol 358:430-42. 2006
    ..The results presented here provide the biophysical and spectroscopic basis for high-resolution structural studies, and show how local RNA structural features modulate recognition by dsRBDs...

Research Grants30

  1. Heat Shock Protein Synthesis Inhibitor Treatment of Hepatitis C Viral Infection
    SAMUEL WHEELER FRENCH; Fiscal Year: 2013
    ....
  2. STRUCTURE AND FUNCTION OF NUCLEIC ACIDS
    Ignacio Tinoco; Fiscal Year: 2013
    ..This information will lead to improved understanding of RNA structure, stability, and dynamics. It will help in understanding RNA function, and in controlling the role of RNA in human diseases. ..
  3. Roles for microRNA-122 in hepatitis C virus RNA amplification
    Peter Sarnow; Fiscal Year: 2013
    ..5 fold drop in viral load without any emergence of resistant virus (Lanford et al. 2010. Science: 327:198-201). ..