The cellular proteins that regulate hepatitis C virus RNA replication

The main purpose of this study was to investigate the cellular proteins that may modulate hepatitis C virus (HCV) RNA replication by interacting with HCV RNA-dependent RNA polymerase. Using yeast two-hybrid screening and various biochemical assays, I identified two cellular proteins that bound HCV polymerase NS5B protein.;In this dissertation, I demonstrated that a ubiquitin-like protein hPLIC1 [human homolog 1 of p&barbelow;rotein l&barbelow;inking i&barbelow;ntergrin-associated protein (IAP) and c&barbelow;ytoskeleton], which physically associates with two E3 ubiquitin protein ligases as well as proteasomes, interacted with NS5B via its C-terminal ubiquitin-associated domain. The pulse-chase labeling experiment revealed that overexpression of hPLIC1, but not the mutant lacking the NS5B-binding domain, significantly shortened the half-life of NS5B and enhanced the polyubiquitination of NS5B. Furthermore, in the Huh7 cells that express an HCV subgenomic replicon, the amounts of both NS5B and the replicon RNA were reduced by over-expression of hPLIC1. Thus, hPLIC1 may be a regulator of HCV RNA replication by modulating the stability of NS5B.;I also found that NS5A and NS5B bind to a human vesicle-associated membrane protein (VAMP)-associated protein of 33 kDa (hVAP-33): NS5A binds to the C-terminus, whereas NS5B binds to the N-terminus of hVAP-33. hVAP-33 contains a coiled-coil domain followed by a membrane-spanning domain at its C-terminus. I further showed that the N-terminal half of hVAP-33 is critical for the membrane protein transport and that NS5B blocks this transport at a post-Golgi step by binding to this domain.;I discovered that hVAP-33 is partially associated with detergent-resistant membrane fraction (probably representing lipid rafts) using membrane flotation analysis. In a human hepatocyte cell line harboring an HCV RNA replicon, the over-expression of the dominant negative mutants and small-interfering RNA of hVAP-33 significantly relocated the nonstructural proteins from detergent-resistant membrane fractions to detergent-sensitive membrane fractions and reduced the amounts of both HCV replicon RNA and protein. Therefore, hVAP-33 is critical for the formation of the HCV replication complex on detergent-resistant membranes.;This dissertation thus identifies a potential negative regulator and a potential positive regulator of HCV replication. Whether these proteins regulate viral replication in HCV-infected patients are is to be investigated.