Abstract

The primary objective of this proposal is to identify cellular genes that regulate hepatitis C virus (HCV) replication in the hepatocyte. The application is based on recently described liver gene expression profiles that characterize different time points during the course of HCV infection in experimentally infected chimpanzees. Three groups of genes were identified whose expression correlated with (a) the onset and duration of infection, (b) the magnitude of infection, and (c) the resolution of infection. Several genes involved in lipid metabolism were shown to be associated with the magnitude of infection in those studies. Experiments designed to validate the relevance of those observations revealed that replication of a subgenomic HCV replicon in Huh-7 cells was enhanced or suppressed by drugs that stimulate or inhibit cholesterol and fatty acid biosynthesis, respectively. In the current application, Huh-7 cells containing subgenomic and full length HCV-replicons will be used to determine if any of the genes identified in the infected chimpanzees can control HCV replication in hepatocytes. Expression of these genes in Huh-7 cells will be inhibited by RNA interference or enhanced by transfection, and the effect of those manipulations on HCV replication will be assessed.
Specific Aims 1 -3 will examine whether the three groups of liver genes identified in the chimpanzees are required either to maintain basal levels of HCV replication (Aim 1), to enhance HCV replication above basal levels (Aim 2); or to mediate the antiviral effects of interferon (Aim 3). In addition, in Aim 4, the specific step(s) in cellular cholesterol and fatty acid biosynthesis that regulate HCV replication will be identified, and the impact of cellular lipid metabolism on the intracellular localization and interactions of HCV proteins will be assessed. By elucidating the cellular regulatory mechanisms that control HCV replication, the studies described in this application may identify new targets for therapeutic antiviral intervention.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA108304-05
Application #
7274880
Study Section
Special Emphasis Panel (ZRG1-GMA-2 (50))
Program Officer
Read-Connole, Elizabeth Lee
Project Start
2003-09-19
Project End
2009-06-30
Budget Start
2007-07-01
Budget End
2009-06-30
Support Year
5
Fiscal Year
2007
Total Cost
$371,351
Indirect Cost

  Institution

Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037

  Publications

Gastaminza, Pablo; Pitram, Suresh M; Dreux, Marlene et al. (2011) Antiviral stilbene 1,2-diamines prevent initiation of hepatitis C virus RNA replication at the outset of infection. J Virol 85:5513-23
Gastaminza, Pablo; Whitten-Bauer, Christina; Chisari, Francis V (2010) Unbiased probing of the entire hepatitis C virus life cycle identifies clinical compounds that target multiple aspects of the infection. Proc Natl Acad Sci U S A 107:291-6
Gastaminza, Pablo; Dryden, Kelly A; Boyd, Bryan et al. (2010) Ultrastructural and biophysical characterization of hepatitis C virus particles produced in cell culture. J Virol 84:10999-1009
Takahashi, Ken; Asabe, Shinichi; Wieland, Stefan et al. (2010) Plasmacytoid dendritic cells sense hepatitis C virus-infected cells, produce interferon, and inhibit infection. Proc Natl Acad Sci U S A 107:7431-6
Garaigorta, Urtzi; Chisari, Francis V (2009) Hepatitis C virus blocks interferon effector function by inducing protein kinase R phosphorylation. Cell Host Microbe 6:513-22
Bobardt, Michael D; Cheng, Guofeng; de Witte, Lot et al. (2008) Hepatitis C virus NS5A anchor peptide disrupts human immunodeficiency virus. Proc Natl Acad Sci U S A 105:5525-30
Cheng, Guofeng; Montero, Ana; Gastaminza, Pablo et al. (2008) A virocidal amphipathic {alpha}-helical peptide that inhibits hepatitis C virus infection in vitro. Proc Natl Acad Sci U S A 105:3088-93
Gastaminza, Pablo; Cheng, Guofeng; Wieland, Stefan et al. (2008) Cellular determinants of hepatitis C virus assembly, maturation, degradation, and secretion. J Virol 82:2120-9
Cheng, Guofeng; Zhong, Jin; Chung, Josan et al. (2007) Double-stranded DNA and double-stranded RNA induce a common antiviral signaling pathway in human cells. Proc Natl Acad Sci U S A 104:9035-40
Kapadia, Sharookh B; Barth, Heidi; Baumert, Thomas et al. (2007) Initiation of hepatitis C virus infection is dependent on cholesterol and cooperativity between CD81 and scavenger receptor B type I. J Virol 81:374-83

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