Roles for microRNA-122 in hepatitis C virus RNA amplification

Summary

Principal Investigator: Peter Sarnow
Abstract: DESCRIPTION (provided by applicant): An estimated 170 million people worldwide and 4 million people in the United States are infected with hepatitis C virus (HCV). The majorities of patients do not resolve the infection and develop chronic infections that often lead to end-stage liver disease and hepatocellular carcinoma. Current treatment is limited to a combination therapy of ribavirin and interferon 1. This therapy is expensive and ineffective in 50% of infected individuals. Thus, there is an urgent need to identify viral or cellular molecules that can be used as novel targets in antiviral therapy. It was discovered that HCV binds two molecules of a liver-specific microRNA, miR-122, resulting in a novel, unprecedented upregulation of the viral genome. Sequestration of miR-122 in HCV-infected cultured cells or in livers of infected chimpanzees leads to a dramatic loss of infectious virus without emergence of resistant virus. Therefore, the dependence of HCV on miR-122 presents an Achilles heel of the virus that can be explored for antiviral intervention. This application proposes to study the roles for miR-122 in the viral life cycle and in cholesterol biosynthesis using a novel class of antisense molecules, locked nucleic acids (LNAs) that can easily be delivered to the liver in animals where it sequesters miR-122 in an inactive small duplex RNA. In particular, the first aim will characterize the RNA-RNA interactions in the miR-122/HCV complex, using genetic and biochemical approaches. Aim 2 will test the hypothesis that miR-122 protects the 5'end sequences of the HCV RNA from degradation by ribonucleases or RNA modification enzymes, or aids in the avoidance of activation of double-stranded RNA sensors such as the retinoic acid inducible gene I. These studies will be performed in specific and genome-wide siRNA-mediated gene knockdown experiments. Aim 3 proposes to examine roles for the known isoforms of miR-122 that contain extra 3'terminal adenosine or guanosine residues, on HCV RNA abundance. Deep sequencing analysis and gene knockdown of suspected nucleotidyl transferases will aid in this analysis. The final aim will characterize in detail the mechanism by which miR-122 regulates the expression of Insig1, the major negative regulator of cholesterol and fatty acid metabolism in the liver. In particular, the miR-122-mediated down-regulation of a distinct polyadenylation/cleavage site in a specific Insig1 isoform mRNAs will be examined. Overall, this application will address fundamental aspects about the functions of miR-122 in the HCV life cycle and cholesterol metabolism. The outcomes from these studies will detail novel mechanisms of gene expression mediated by microRNAs in eukaryotic cells and will point to new venues for antiviral therapies. 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 two molecules of a liver- specific microRNA, miR-122, resulting in a novel, unprecedented upregulation of the viral genome. This proposal explores the mechanisms by which miR-122 protects HCV RNA in the liver. The dependence of HCV on miR-122 presents an Achilles heel of the virus that can be used for antiviral intervention. We will study the roles for miR-122 in the viral life cycle and in cholesterol biosynthesis using a novel class of antisense molecules (locked nucleic acids) that can easily bind and inactivate miR-122 in the liver of animals. This is a highly significant approach, because LNA-mediated sequestration of miR-122 in the liver of HCV-infected chimpanzees resulted in a 2.5 fold drop in viral load without any emergence of resistant virus (Lanford et al. 2010. Science: 327:198-201).
Funding Period: 2006-02-15 - 2016-01-31
more information: NIH RePORT

Top Publications

  1. 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
  2. pmc OGFOD1, a novel modulator of eukaryotic translation initiation factor 2alpha phosphorylation and the cellular response to stress
    Karen A Wehner
    Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USAA
    Mol Cell Biol 30:2006-16. 2010
  3. pmc Six RNA viruses and forty-one hosts: viral small RNAs and modulation of small RNA repertoires in vertebrate and invertebrate systems
    Poornima Parameswaran
    Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, United States of America
    PLoS Pathog 6:e1000764. 2010
  4. 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
  5. pmc MicroRNA silencing and the development of novel therapies for liver disease
    Gyongyi Szabo
    Department of Medicine, University of Massachusetts Medical School, MA 01605, United States
    J Hepatol 57:462-6. 2012
  6. 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
  7. 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
  8. 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

Research Grants

Detail Information

Publications9

  1. 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...
  2. pmc OGFOD1, a novel modulator of eukaryotic translation initiation factor 2alpha phosphorylation and the cellular response to stress
    Karen A Wehner
    Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USAA
    Mol Cell Biol 30:2006-16. 2010
    ..These findings argue that OGFOD1 plays important proapoptotic roles in the regulation of translation and HRI-mediated phosphorylation of eIF2alpha in cells subjected to arsenite-induced stress...
  3. pmc Six RNA viruses and forty-one hosts: viral small RNAs and modulation of small RNA repertoires in vertebrate and invertebrate systems
    Poornima Parameswaran
    Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, United States of America
    PLoS Pathog 6:e1000764. 2010
    ..These experiments define vsRNAs as one possible component of the interplay between animal viruses and their hosts...
  4. 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...
  5. pmc MicroRNA silencing and the development of novel therapies for liver disease
    Gyongyi Szabo
    Department of Medicine, University of Massachusetts Medical School, MA 01605, United States
    J Hepatol 57:462-6. 2012
    ..In this review, we highlight the latest advances in the field of microRNA biology and their potential as emerging therapeutic targets in liver disease...
  6. 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...
  7. 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...
  8. 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...

Research Grants30