Hepatitis C virus (HCV) is the etiologic agent of parenterally transmitted non-A, non-B viral hepatitis. Chronic infection puts individuals at risk for the development of cirrhosis, hepatocellular carcinoma, and liver failure, making chronic hepatitis C the leading indication for liver transplantation. While HCV-specific protease and polymerase inhibitors are showing promise in early clinical development, rapid emergence of resistance indicates that additional viral targets and combinations of antivirals will be needed for effective control. We propose to investigate the structure and function of the replicase complex, the central holoenzyme of HCV replication. Increased understanding of how the components of this complex come together, as well as clarifying the functions and mechanisms of each constituent, will facilitate the development of novel antiviral drugs. We will use genetic analyses complemented with biochemical studies to investigate protein-protein interactions within the replication complex. These studies will provide a context in which to build a structural understanding of the replicase. We then aim to elucidate the roles of two important but enigmatic viral proteins, the NS3 helicase and NS5A. These proteins, both potentially extremely valuable drug targets, have as-yet-unknown roles in RNA replication. We will investigate the mechanism of NS3 helicase activity and the roles of this enzyme in the viral life cycle using X-ray crystallography and complementary genetic approaches. We will pursue an atomic-resolution structure of full-length NS5A, as well as investigate its structure in complex with an RNA substrate. We will also attempt to develop cell-free replication assays for HCV;the current lack of such systems is a major roadblock to studies of this virus. Availability of a cell-free assay would allow us to relate structural, biochemical, and genetic data to specific steps of RNA synthesis. Through these studies, we hope to begin to understand mechanisms of HCV RNA replication and to uncover novel avenues for therapeutic intervention.

Public Health Relevance

Hepatitis C virus is a leading cause of liver disease including cancer. This proposal aims to study the structure and function of the protein complex that replicates the virus genome. The resulting findings should aid development of more effective treatments aimed at eradicating this deadly viral disease.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA057973-20
Application #
8447379
Study Section
Virology - A Study Section (VIRA)
Program Officer
Read-Connole, Elizabeth Lee
Project Start
1992-08-01
Project End
2014-02-28
Budget Start
2013-03-01
Budget End
2014-02-28
Support Year
20
Fiscal Year
2013
Total Cost
$353,659
Indirect Cost
$144,393
Name
Rockefeller University
Department
Microbiology/Immun/Virology
Type
Other Domestic Higher Education
DUNS #
071037113
City
New York
State
NY
Country
United States
Zip Code
10065
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Chung, Hyo-Young; Gu, Meigang; Buehler, Eugen et al. (2014) Seed sequence-matched controls reveal limitations of small interfering RNA knockdown in functional and structural studies of hepatitis C virus NS5A-MOBKL1B interaction. J Virol 88:11022-33
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Kim, Hee-Young; Li, Xiaolan; Jones, Christopher T et al. (2013) Development of a multiplex phenotypic cell-based high throughput screening assay to identify novel hepatitis C virus antivirals. Antiviral Res 99:6-11
Kapoor, Amit; Simmonds, Peter; Cullen, John M et al. (2013) Identification of a pegivirus (GB virus-like virus) that infects horses. J Virol 87:7185-90
Lindenbach, Brett D; Rice, Charles M (2013) The ins and outs of hepatitis C virus entry and assembly. Nat Rev Microbiol 11:688-700
Gu, Meigang; Rice, Charles M (2013) Structures of hepatitis C virus nonstructural proteins required for replicase assembly and function. Curr Opin Virol 3:129-36

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