Modulation of the hepatitis C virus RNA-dependent RNA polymerase activity by the non-structural (NS) 3 helicase and the NS4B membrane protein
Modulation of the hepatitis C virus RNA-dependent RNA polymerase activity by the non-structural (NS) 3 helicase and the NS4B membrane protein
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DOI：
10.1074/jbc.m204124200
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发表时间：
2002-11-22
影响因子：
4.8
通讯作者：
McCarthy, JEG
中科院分区：
文献类型：
--
作者：
Piccininni, S;Varaklioti, A;McCarthy, JEG
丙型肝炎病毒（HCV）非结构蛋白5 B（NS 5 B）被认为是负责HCV复制的中心催化酶，但关于其活性如何控制有许多未回答的问题。在这项研究中，我们发现，其他两个HCV蛋白，NS 3（蛋白酶/解旋酶）和NS 4 B（未知功能的疏水蛋白），物理和功能相互作用的NS 5 B聚合酶。我们描述了一种新的程序，用于产生高纯度的NS 4 B，并使用这种蛋白质的生化研究与NS 5 B和NS 3。为了研究蛋白质-蛋白质相互作用的功能效应，我们已经开发了一种体外复制测定法，该测定法使用HCV基因组的相应的阳性（（+）-3 '-非翻译区）和阴性（（-）-3'-末端区）RNA链的天然非编码3'区。我们的研究表明，NS 3显着调节NS 5 B的模板识别，并改变这种酶产生的合成产物。使用NS 3的NTPase-deficient突变体形式证明了这种蛋白质的NTR酶活性（以及因此的解旋酶活性）是这些作用所特别需要的。此外，发现NS 4 B是NS 3-NS 5 B复制复合物的负调节因子。总体而言，这些结果表明，NS 3，NS 4 B和NS 5 B可以相互作用，形成一个调节复合物，可能在HCV复制过程中发挥作用。
The hepatitis C virus (HCV) nonstructural protein 5B (NS5B) is believed to be the central catalytic enzyme responsible for HCV replication but there are many unanswered questions about how its activity is controlled. In this study we reveal that two other HCV proteins, NS3 (a protease/helicase) and NS4B (a hydrophobic protein of unknown function), physically and functionally interact with the NS5B polymerase. We describe a new procedure for generating highly pure NS4B, and use this protein in biochemical studies together with NS5B and NS3. To study the functional effects of the protein-protein interactions, we have developed an in vitro replication assay using the natural noncoding 3' regions of the respective positive ((+)-3'-untranslated region) and negative ((-)-3'-terminal region) RNA strands of the HCV genome. Our studies show that NS3 dramatically modulates template recognition by NS5B and changes the synthetic products generated by this enzyme. The use of an NTPase-deficient mutant form of NS3 demonstrates that the NTPase activity (and thus helicase activity) of this protein is specifically required for these effects. Moreover, NS4B is found to be a negative regulator of the NS3-NS5B replication complex. Overall, these results reveal that NS3, NS4B, and NS5B can interact to form a regulatory complex that could feature in the process of HCV replication.