Cho Y G, Moon H S, Sung Y C
Department of Life Science, School of Environmental Engineering, Pohang University of Science and Technology, South Korea.
J Virol Methods. 1997 May;65(2):201-7. doi: 10.1016/s0166-0934(97)02183-6.
An in vivo assay system was developed for the serine protease of hepatitis C virus (HCV) using the sindbis (SIN) viral replication system in which HCV serine protease activity is essential for the replication of the HCV-SIN chimeric virus. Two chimeric viral cDNA clones were constructed by inserting the NS3/4A region and NS3/4A region with the putative helicase deleted, into the N-terminal region of SIN core protein. The constructs were named Tpro CT and Tpro T, respectively. BHK-21 cells transfected with the in vitro transcribed RNAs from Tpro CT and Tpro T showed specific cytopathic morphology and produced chimeric viruses, Vpro CT and Vpro T. In contrast, in vitro transcribed RNAs from Tpro CTI and Tpro TI, in which serine of catalytic triad of HCV protease was changed to alanine, were not infectious. When the chimeric viruses were passaged in BHK-21 cells at about 0.1 multiplicity of infection (MOI), Vpro T, but not Vpro CT, stably expressed HCV protease for up to five passages. Surprisingly, the cell culture media of BHK-21 cells infected with Vpro T, compared to wild-type sindbis virus, showed rapid pH changes by more than 0.8 pH degree at 72 h post-infection. HCV-SIN hybrid viruses could be used in screening the HCV protease-inhibitor in cell culture systems.
利用辛德毕斯（SIN）病毒复制系统开发了一种用于丙型肝炎病毒（HCV）丝氨酸蛋白酶的体内检测系统，在该系统中，HCV丝氨酸蛋白酶活性对于HCV-SIN嵌合病毒的复制至关重要。通过将NS3/4A区域和缺失推定解旋酶的NS3/4A区域插入SIN核心蛋白的N端区域，构建了两个嵌合病毒cDNA克隆。构建体分别命名为Tpro CT和Tpro T。用来自Tpro CT和Tpro T的体外转录RNA转染的BHK-21细胞呈现出特异性细胞病变形态并产生嵌合病毒Vpro CT和Vpro T。相比之下，来自Tpro CTI和Tpro TI的体外转录RNA（其中HCV蛋白酶催化三联体的丝氨酸被替换为丙氨酸）没有感染性。当嵌合病毒在BHK-21细胞中以约0.1感染复数（MOI）传代时，Vpro T而非Vpro CT可稳定表达HCV蛋白酶达五代。令人惊讶的是，与野生型辛德毕斯病毒相比，感染Vpro T的BHK-21细胞的细胞培养基在感染后72小时显示pH值快速变化超过0.8个pH单位。HCV-SIN杂交病毒可用于在细胞培养系统中筛选HCV蛋白酶抑制剂。
