Hepatitis C virus (HCV) infection and the associated chronic hepatitis, cirrhosis and hepatocellular carcinoma are the leading cause of liver transplantation in the United States. There are approximately 4 million HCV carriers in the U.S. population. Estimates place the total number of HCV infected individuals at 170 million worldwide, representing nearly 3% of the population. During the course of HCV replication in infected individuals, the NS5a protein, a key component of the viral replicase complex, is phosphorylated by an unknown cellular kinase. This phosphorylation has been shown to also occur in cell lines carrying HCV replicons, where a link between replication efficiency and phosphorylation has been observed. The studies in this proposal are designed to identity and characterize the cellular kinase or kinases responsible for the phosphorylation of the NS5a protein of HCV by the use of conventional chromatographic separations and 2D gel electrophoresis/MALDI-TOF mass spectrometry. The effects of the observed phosphorylation of viral RNA replication will be examined using a series of replicon mutants that exhibit differing levels of NS5a phosphorylation and replication competence. The region of the NS5a protein required for the interaction with the NS5a associated kinase and the HCV replicase complex components will be identified using a battery of NS5a deletions and mutants. In addition, identification of the components of the replication complex associated with the NS5a protein and NS5a kinase will be examined and may provide some insight into the largely uncharacterized requirements for HCV viral RNA replication.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
1F32AI051820-01
Application #
6487088
Study Section
Special Emphasis Panel (ZRG1-F08 (20))
Program Officer
Johnson, Leslye D
Project Start
2002-07-01
Project End
Budget Start
2002-07-01
Budget End
2003-06-30
Support Year
1
Fiscal Year
2002
Total Cost
$38,320
Indirect Cost
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
Tellinghuisen, Timothy L; Foss, Katie L; Treadaway, Jason C et al. (2008) Identification of residues required for RNA replication in domains II and III of the hepatitis C virus NS5A protein. J Virol 82:1073-83
Randall, Glenn; Panis, Maryline; Cooper, Jacob D et al. (2007) Cellular cofactors affecting hepatitis C virus infection and replication. Proc Natl Acad Sci U S A 104:12884-9
Tellinghuisen, Timothy L; Paulson, Matthew S; Rice, Charles M (2006) The NS5A protein of bovine viral diarrhea virus contains an essential zinc-binding site similar to that of the hepatitis C virus NS5A protein. J Virol 80:7450-8
Lindenbach, Brett D; Evans, Matthew J; Syder, Andrew J et al. (2005) Complete replication of hepatitis C virus in cell culture. Science 309:623-6
Tellinghuisen, Timothy L; Marcotrigiano, Joseph; Gorbalenya, Alexander E et al. (2004) The NS5A protein of hepatitis C virus is a zinc metalloprotein. J Biol Chem 279:48576-87