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HCV-Host Cell Interactions: Identification of Cellular Factors and their Implications on HCV Replication

by
Zhengbin Zhang
Biochemistry and Molecular Biology

B.M.D., 1994, Medical School, Wuhan University, China
M.S., 1997, Medical School, Wuhan University, China

Thesis Advisor: Virendra N. Pandey, Ph.D.
Associate Professor
Department of Biochemistry and Molecular Biology

Department of Biochemistry and Molecular Biology
MSB E609B

Friday, June 6, 2008
12:00 p.m.


Abstract

Chronic infection by Hepatitis C virus (HCV) is the leading cause of severe hepatitis that often develops into liver cirrhosis and hepatocellular carcinoma. The molecular mechanisms underlying HCV replication and pathogenesis are poorly understood. Similarly, the role(s) of host factors in the replication of HCV remain largely undefined. Based on our knowledge of other RNA viruses, it is likely that a number of cellular factors may be involved in facilitating HCV replication. It has been demonstrated that elements within the 3’ nontranslated region (3’NTR) of the (+) strand HCV genome are essential for initiation of (-) strand synthesis. The RNA signals within the highly conserved 3’ NTR may be the site for recruiting cellular factors which mediate virus replication/pathogenesis. However, the identities of putative cellular factors interacting with these RNA signals remain unknown. In this study, we demonstrate that an RNA affinity capture system developed in our lab used in conjunction with LC/MS/MS mass spectrometry has allowed us to positively identify more than 70 cellular proteins that interact with the 3’NTR (+) of HCV. Binding of these cellular proteins was not competed out by a 10-fold excess of nonspecific competitor RNA. With few exceptions, all of the identified cellular proteins are RNA binding proteins whose reported cellular functions provide unique insights into host cell-virus interactions and possible mechanisms influencing HCV replication and HCV-associated pathogenesis. SiRNA mediated silencing of selected 3’ NTR-binding proteins (FBP, HuR, DDX5 and NF) in an HCV replicon cell line reduced replicon RNA to undetectable levels, suggesting important roles for these cellular factors in HCV replication.
Far-upstream element (FUSE) binding protein (FBP) is a cellular factor that we have identified as a binder of HCV 3’NTR. Mapping of the binding site showed that FBP specifically interacts with the poly(U) tract within the poly(U/UC) region of the 3’NTR. Silencing of FBP expression by siRNA in cells carrying HCV subgenomic replicons severely reduced viral replication, while overexpression of FBP significantly enhanced the replication. We confirmed these observations by an in-vitro HCV replication assay in the cell-free replicative lysate, which suggested that there is a direct correlation between the cellular FBP level and HCV replication. FBP Immunoprecipitation co-precipitated HCV NS5A indicating interaction of FBP with HCV NS5A, which is known to function as a link between HCV translation and replication. Although FBP is mainly localized in the nucleus, we found that in MH14 cells a significant level of this protein is co-localized with NS5A in the cytosol, a site of HCV replication. While the mechanism of FBP involvement in HCV replication is yet to be delineated, our findings suggest that it may be an important regulatory component that is essential for efficient replication of HCV.
This study provides new insights on HCV-Host interaction by identifying a number of cellular factors that can bind to the 3’NTR of HCV genome. Specifically, we revealed FBP as an important regulatory cellular factor for HCV replication. The mechanism identified here will help to further characterize life cycle and pathogenesis of HCV. Furthermore, the cellular factors may be used as new potential targets of antiviral treatment.


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