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).

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI069000-07
Application #
8206508
Study Section
Special Emphasis Panel (ZRG1-IDM-R (03))
Program Officer
Koshy, Rajen
Project Start
2006-02-15
Project End
2016-01-31
Budget Start
2012-02-01
Budget End
2013-01-31
Support Year
7
Fiscal Year
2012
Total Cost
$391,459
Indirect Cost
$141,459
Name
Stanford University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
Sedano, Cecilia D; Sarnow, Peter (2014) Hepatitis C virus subverts liver-specific miR-122 to protect the viral genome from exoribonuclease Xrn2. Cell Host Microbe 16:257-64
Pager, Cara T; Schutz, Sylvia; Abraham, Teresa M et al. (2013) Modulation of hepatitis C virus RNA abundance and virus release by dispersion of processing bodies and enrichment of stress granules. Virology 435:472-84
Cox, Erica Machlin; Sagan, Selena M; Mortimer, Stefanie A W et al. (2013) Enhancement of hepatitis C viral RNA abundance by precursor miR-122 molecules. RNA 19:1825-32
Sagan, Selena M; Sarnow, Peter; Wilson, Joyce A (2013) Modulation of GB virus B RNA abundance by microRNA-122: dependence on and escape from microRNA-122 restriction. J Virol 87:7338-47
Szabo, Gyongyi; Sarnow, Peter; Bala, Shashi (2012) MicroRNA silencing and the development of novel therapies for liver disease. J Hepatol 57:462-6
Machlin, Erica S; Sarnow, Peter; Sagan, Selena M (2011) Masking the 5' terminal nucleotides of the hepatitis C virus genome by an unconventional microRNA-target RNA complex. Proc Natl Acad Sci U S A 108:3193-8
Wehner, Karen A; Schutz, Sylvia; Sarnow, Peter (2010) OGFOD1, a novel modulator of eukaryotic translation initiation factor 2alpha phosphorylation and the cellular response to stress. Mol Cell Biol 30:2006-16
Parameswaran, Poornima; Sklan, Ella; Wilkins, Courtney et al. (2010) Six RNA viruses and forty-one hosts: viral small RNAs and modulation of small RNA repertoires in vertebrate and invertebrate systems. PLoS Pathog 6:e1000764
Jopling, Catherine L; Schutz, Sylvia; Sarnow, Peter (2008) Position-dependent function for a tandem microRNA miR-122-binding site located in the hepatitis C virus RNA genome. Cell Host Microbe 4:77-85