I. C型肝炎病毒非結構性蛋白質NS5A對Ras-ERK訊息傳遞路徑及下游分子的影響II. C型肝炎病毒非結構性蛋白質NS4A與真核細胞轉譯延伸因子的相互作用關係

Abstract

Hepatitis C virus (HCV) is a positive, single-stranded RNA virus. Human infected by HCV causes type C hepatitis. Approximately 85% of persons infected with HCV develop chronic hepatitis and may lead to hepatocellular carcinoma. Because there is no efficient cell culture system for HCV infection and replication, the molecular mechanism of HCV pathogenesis is not well understood. NS5A is a non-structural protein of HCV. It participates in interferon resistance. NS5A has been shown to interact with a variety of cellular signaling proteins and perturb the signal transduction pathways such as MAPK and TNF-a signaling pathways. Ras-ERK is a MAPK signaling pathway involved in regulation of cell proliferation and cell growth. In this study, doxycycline-regulated HeLa-tetoff-NS5A and HepG2-teton-NS5A cell lines were used to examine the effect of NS5A on Ras-ERK signaling pathway and its downstream molecules. The induction of NS5A in HepG2-teton NS5A cells markedly reduced the level of phosphorylated ERK1/2, but no significant difference was detected on the level of phosphorylated MEK1/2 and the expression of MKP1. Flow cytometry analyses indicate that the NS5A has no significant effect on the regulation of cell cycle. Therefore, the mechanisms of NS5A involved in the down-regulation of ERK pathway and cell growths remain to be elucidated. The HCV non-structural protein NS4A interacts with NS3 and is a cofactor of NS3 serine-type protease essential for the proteolytic processing of the viral polyprotein. By performing GST pull down assay, our laboratory has previously identified eEF1A that specifically interacted with GST-NS4A. In combination with in vitro translation system the C-terminal domain of eEF1A was identified to be involved in the interaction. NS4A was recently reported to inhibit protein synthesis, but the mechanism remains unclear. In this study, the interaction between NS4A and eEF1A was confirmed by immunoprecipitation assay following cotransfection of plasmids ecoding NS4A and eEF1A into culture cells. NS4A interacted with full length eEF1A, but not the N-terminal (amino acid 1-240) or C-terminal (amino acid 201-462) eEF1A. Furthermore, a mutation at amino acid Arg-28 disrupts the interaction between NS4A and eEF1A. Whether NS4A interferes with protein synthesis by forming complex with eEF1A and what’s the role of eEF1A on HCV replication remain to be elucidated.