Mak P, Palant O, Labonté P, Plotch S
Molecular Biology and Virology Section, Wyeth-Ayerst Research, 401 N. Middletown Road, Pearl River, NY 10965, USA.
FEBS Lett. 2001 Aug 10;503(1):13-8. doi: 10.1016/s0014-5793(01)02678-3.
The hepatitis C virus (HCV) protease genes (NS2/3 and NS3) were expressed in yeast with their natural substrates fused to a ligand-dependent transcriptional activator, the retinoic acid receptor (RARbeta). RARbeta can activate transcription in yeast cells in response to retinoic acids. We hypothesized that cis-cleavage at the NS2-3 or NS3-4A junctions by the appropriate HCV proteases would release RARbeta, thereby activating transcription of a reporter gene. Our results from Western blot analyses and reporter gene activation indicate that the wild-type NS2/3 and NS3 enzymes are catalytically active in yeast cells, whereas mutations in the catalytic domain of NS2(C993V) and NS3(S1165A) lead to inactive enzymes. We conclude that HCV NS2/3 and NS3 protease activities can be reconstituted in yeast.
丙型肝炎病毒（HCV）蛋白酶基因（NS2/3和NS3）在酵母中表达，其天然底物与配体依赖性转录激活因子维甲酸受体（RARβ）融合。RARβ可响应维甲酸在酵母细胞中激活转录。我们推测，适当的HCV蛋白酶在NS2-3或NS3-4A连接处的顺式切割会释放RARβ，从而激活报告基因的转录。我们的蛋白质印迹分析和报告基因激活结果表明，野生型NS2/3和NS3酶在酵母细胞中具有催化活性，而NS2（C993V）和NS3（S1165A）催化结构域中的突变导致酶无活性。我们得出结论，HCV NS2/3和NS3蛋白酶活性可在酵母中重建。
