| Summary: | 碩士 === 國立臺灣大學 === 微生物學研究所 === 94 === Hepatitis C virus (HCV) is the major infectious agent leading to non-A, non-B hepatitis. HCV belongs to the family Flaviviridae and is an enveloped, single-stranded RNA virus. Previously, a cDNA microarray analysis with HCV subgenomic replicon and patients parental Huh7 cells was performed in our lab to identify differentially expressed genes. According to the results and data from RT-PCR analysis of hepatocytes of HCC patients by another group, it is likely that HCV proteins are involved in the regulation of expression of several genes including SAA, DUSP1, GRB14, VEGF-A, integrin, FASTK, H-ras, HRG, and SULT2A1. In this study, cultured cells were transfected with expression plasmids of HCV nonstructural proteins. RT-PCR was applied to analyze the effects of the viral proteins on the expression of the cellular genes. NS4B down-regulated the expression of both FASTK and HRG mRNA. Nonstructural protein 4B (NS4B) is a relatively hydrophobic 27 kDa protein of unknown function. Nevertheless, NS4B was demonstrated to be important for the NS5A hyperphosphorylation and replication of HCV. In addition, NS4B has a GTPase activity. It inhibits translation of host cells, and causes malignant transformation in cooperation with the Ha-ras gene. Therefore, NS4B may play an important role in the pathogenicity of HCV. FASTK was shown to be a survival protein. To understand possible effects of the HCV NS4B protein on cellular survivability, western blot analysis was performed to detect the level of caspase3 in both anti-Fas antibody-treated and untreated 293 cells. Although NS4B seems to increase the severity of cell apoptosis, only limited effect was detected by nucleosome ELISA assay and flow cytometry. Apoptosis is one of gateways for viral particle spreads, the role of NS4B involved in the apoptosis and in the process of viral diffusion need to be further elucidated. On the other hand, HRG is an abundant plasma protein. It expresses mainly in the liver tissue. In this study, NS4B was found to down-regulate HRG both at the RNA and protein levels. When NS4B expression plasmid was cotransfected with a luciferase reporter carrying a 450 bp HRG promoter, no effect on the luciferase activity was detected. These results suggested that the regulatory effect of NS4B on HRG is not through a direct binding of the NS4B protein to the 450 bp promoter sequences.
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