Grakoui A, McCourt D W, Wychowski C, Feinstone S M, Rice C M
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110-1093.
Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10583-7. doi: 10.1073/pnas.90.22.10583.
Host and viral proteinases are believed to be required for the production of at least nine hepatitis C virus (HCV)-specific polyprotein cleavage products. Although several cleavages appear to be catalyzed by host signal peptidase or the HCV NS3 serine proteinase, the enzyme responsible for cleavage at the 2/3 site has not been identified. In this report, we have defined the 2/3 cleavage site and obtained evidence which suggests that this cleavage is mediated by a second HCV-encoded proteinase, located between aa 827 and 1207. This region encompasses the C-terminal portion of the 23-kDa NS2 protein, the 2/3 cleavage site, and the serine proteinase domain of NS3. Efficient processing at the 2/3 site was observed in mammalian cells, Escherichia coli, and in plant or animal cell-free translation systems in the absence of microsomal membranes. Cleavage at the 2/3 site was abolished by alanine substitutions for NS2 residues His-952 or Cys-993 but was unaffected by several other substitution mutations, including those that inactivate NS3 serine proteinase function. Mutations abolishing cleavage at the 2/3 site did not block cleavage at other sites in the HCV polyprotein. Cotransfection experiments indicate that the 2/3 site can be cleaved in trans, which should facilitate purification and further characterization of this enzyme.
宿主和病毒蛋白酶被认为是产生至少九种丙型肝炎病毒（HCV）特异性多蛋白切割产物所必需的。尽管几种切割似乎是由宿主信号肽酶或HCV NS3丝氨酸蛋白酶催化的，但负责在2/3位点切割的酶尚未确定。在本报告中，我们确定了2/3切割位点，并获得了证据，表明这种切割是由位于第827至1207位氨基酸之间的第二种HCV编码蛋白酶介导的。该区域包括23 kDa NS2蛋白的C末端部分、2/3切割位点以及NS3的丝氨酸蛋白酶结构域。在不存在微粒体膜的情况下，在哺乳动物细胞、大肠杆菌以及植物或动物无细胞翻译系统中均观察到了在2/3位点的有效加工。用丙氨酸替代NS2残基His-952或Cys-993可消除在2/3位点的切割，但不受其他几种替代突变的影响，包括那些使NS3丝氨酸蛋白酶功能失活的突变。消除在2/3位点切割的突变不会阻断HCV多蛋白中其他位点的切割。共转染实验表明，2/3位点可以反式切割，这将有助于该酶的纯化和进一步表征。
