Establishment of Assay Systems for HCV NS3 Protease in Vivo

• Main Authors: Li, Chi-Hui, 黎琪慧
• Other Authors: Szecheng J. Lo
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/11042154291707150331

碩士 === 國立陽明大學 === 微生物暨免疫學研究所 === 89 === Hepatitis C virus, a major pathogen of post-transfusional non-A, non-B hepatitis, is a single positive sense RNA virus. After long-term treatment of interferonα-2b alone or combination with ribavirin, the usual therapy to RNA virus, only few patients (10~15%) were curable. With worldwide distribution (1~2 %) and high frequency to become chronic infection, which often progresses to liver cirrhosis and hepatocellular carcinoma, it is urgent to establish assay systems for screening of inhibitors of HCV. The HCV encoded a polyprotein with the structure proteins at the N-terminus and the non-structure proteins at the C-terminus, which has to be processed by the host signal pepetidase and viral NS3 protease, respectively. This suggests that NS3 protease is a suitable target for anti-HCV drug development. To establish an assay system for HCV protease in vivo, three main plasmids were constructed: (i) pδ-HCV5A/B-GFP; (ii) pFBD-HCV5AB-AD; (iii) pFNS34-SEAP. The first one used the HDV delta protein, which has an NLS that will target the δ-HCV5A/B-GFP fusion protein to the nucleoli. If the HCV5A/B junction is cleaved by the NS3 protease, GFP will disperse through the cytoplasm rather than targeting to the nucleoli. But our results showed that no cleavage by protease was observed by western blotting with the anti-δ antibody. The strategy for creating pBD-HCV5AB-AD is using the GAL4 transcription activator inserted with an NS3 protease cleavage site between the DNA binding domain (BD) and the transcription activation domain (AD). The GAL4 activated expression of reporter gene, GFP, will diminish if GAL4 fusion protein is cleaved by HCV NS3 protease. However, no positive result was obtained in this system. The third plasmid pFNS34-SEAP was constructed with the 4A/4B cleavage site inserted between NS34 and SEAP (secreted alkaline phophotase). The SEAP will be secreted out once the NS3 auto-cleaves the immediately downstream 4A/4B junction and expose the signal sequence of SEAP. By the chemiluminescent method, the activity of SEAP in culture medium was measured. Positive results were obtained and this system allowed us to screen potential inhibitors. Treating transiently NS34-SEAP expressing cells with a NS4A/4B junction deriving inhibitor, the SEAP activity of cell medium decreased apparently when the concentration of the inhibitor was used in 2.5mM. Three systems were tested in this study, only one succeeded. It leaded to a conclusion that the flanking sequence may change the protein conformation and is important for protein function.