Filocamo G, Pacini L, Nardi C, Bartholomew L, Scaturro M, Delmastro P, Tramontano A, De Francesco R, Migliaccio G
Istituto di Ricerche di Biologia Molecolare P. Angeletti, 00040 Pomezia, Rome, Italy.
J Virol. 1999 Jan;73(1):561-75. doi: 10.1128/JVI.73.1.561-575.1999.
The NS3-NS4A serine protease of hepatitis C virus (HCV) mediates four specific cleavages of the viral polyprotein and its activity is considered essential for the biogenesis of the HCV replication machinery. Despite extensive biochemical and structural characterization, the analysis of natural variants of this enzyme has been limited by the lack of an efficient replication system for HCV in cultured cells. We have recently described the generation of chimeric HCV-Sindbis viruses whose propagation depends on the NS3-NS4A catalytic activity. NS3-NS4A gene sequences were fused to the gene coding for the Sindbis virus structural polyprotein in such a way that processing of the chimeric polyprotein, nucleocapsid assembly, and production of infectious viruses required NS3-NS4A-mediated proteolysis (G. Filocamo, L. Pacini, and G. Migliaccio, J. Virol. 71:1417-1427, 1997). Here we report the use of these chimeric viruses to select and characterize active variants of the NS3-NS4A protease. Our original chimeric viruses displayed a temperature-sensitive phenotype and formed lysis plaques much smaller than those formed by wild-type (wt) Sindbis virus. By serially passaging these chimeric viruses on BHK cells, we have selected virus variants which formed lysis plaques larger than those produced by their progenitors and produced NS3-NS4A proteins different in size and/or sequence from those of the original viruses. Characterization of the selected protease variants revealed that all of the mutated proteases still efficiently processed the chimeric polyprotein in infected cells and also cleaved an HCV substrate in vitro. One of the selected proteases was expressed in a bacterial system and showed a catalytic efficiency comparable to that of the wt recombinant protease.
丙型肝炎病毒（HCV）的NS3-NS4A丝氨酸蛋白酶介导病毒多聚蛋白的四种特异性切割，其活性被认为是HCV复制机制生物发生所必需的。尽管对该酶进行了广泛的生化和结构表征，但由于缺乏在培养细胞中高效的HCV复制系统，对该酶天然变体的分析受到限制。我们最近描述了嵌合HCV-辛德毕斯病毒的产生，其繁殖依赖于NS3-NS4A催化活性。NS3-NS4A基因序列以这样一种方式与编码辛德毕斯病毒结构多聚蛋白的基因融合，即嵌合多聚蛋白的加工、核衣壳组装以及感染性病毒的产生需要NS3-NS4A介导的蛋白水解作用（G.菲洛卡莫、L.帕奇尼和G.米利亚乔，《病毒学杂志》71:1417-1427，1997年）。在此，我们报告使用这些嵌合病毒来选择和表征NS3-NS4A蛋白酶的活性变体。我们最初的嵌合病毒表现出温度敏感表型，形成的裂解斑比野生型（wt）辛德毕斯病毒形成的裂解斑小得多。通过在BHK细胞上连续传代这些嵌合病毒，我们选择了形成比其亲本产生的裂解斑更大的病毒变体，并产生了大小和/或序列与原始病毒不同的NS3-NS4A蛋白。对所选蛋白酶变体的表征表明，所有突变的蛋白酶仍能在感染细胞中高效加工嵌合多聚蛋白，并且在体外也能切割HCV底物。所选的一种蛋白酶在细菌系统中表达，其催化效率与wt重组蛋白酶相当。
