The serine protease domain of hepatitis C viral NS3 activates RNA helicase activity by promoting the binding of RNA substrate
The serine protease domain of hepatitis C viral NS3 activates RNA helicase activity by promoting the binding of RNA substrate
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DOI：
10.1074/jbc.m707165200
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发表时间：
2007-11-30
影响因子：
4.8
通讯作者：
Pyle, Anna Marie
中科院分区：
文献类型：
--
作者：
Beran, Rudolf K. F.;Serebrov, Victor;Pyle, Anna Marie
非结构蛋白（NS）3是一种DEXH/D-box马达蛋白，是丙型肝炎病毒（HCV）复制复合体的重要组成部分。全长NS 3蛋白含有两个功能模块，这两个模块在HCV的生命周期中是必不可少的：N末端的丝氨酸蛋白酶结构域和C末端的ATP酶/解旋酶结构域（NS 3 hel）。截短的NS 3 hel构建体已经被广泛研究;已经检查了ATP酶、核酸结合和解旋酶活性，并且NS 3 hel已经被用作抗病毒药物开发中的靶标。然而，还没有进行NS 3和NS 3 hel活性的全面比较，因此仍不清楚蛋白酶结构域是否在NS 3解旋酶功能中起重要作用。鉴于许多DEXH/D-box蛋白在与辅因子蛋白相互作用后被激活，重要的是确定蛋白酶结构域是否充当刺激NS 3解旋酶功能的辅因子。在这里，我们表明，蛋白酶结构域大大增强了直接和功能性结合的RNA NS 3。尽管静电在该过程中起重要作用，但NS 3 hel和蛋白酶结构域之间的相互作用界面具有特定的变构贡献。最重要的是，我们确定蛋白酶结构域是NS 3 RNA解旋所必需的。我们的研究结果表明，除了其在切割宿主和病毒蛋白质中的作用外，NS 3蛋白酶结构域对于病毒RNA复制的过程是必不可少的，并且鉴于其对RNA结合的静电贡献，它也可能有助于病毒RNA的包装。
Nonstructural (NS) protein 3 is a DEXH/D-box motor protein that is an essential component of the hepatitis C viral (HCV) replicative complex. The full-length NS3 protein contains two functional modules, both of which are essential in the life cycle of HCV: a serine protease domain at the N terminus and an ATPase/helicase domain (NS3hel) at the C terminus. Truncated NS3hel constructs have been studied extensively; the ATPase, nucleic acid binding, and helicase activities have been examined and NS3hel has been used as a target in the development of antivirals. However, a comprehensive comparison of NS3 and NS3hel activities has not been performed, so it remains unclear whether the protease domain plays a vital role in NS3 helicase function. Given that many DEXH/D-box proteins are activated upon interaction with cofactor proteins, it is important to establish if the protease domain acts as the cofactor for stimulating NS3 helicase function. Here we show that the protease domain greatly enhances both the direct and functional binding of RNA to NS3. Whereas electrostatics plays an important role in this process, there is a specific allosteric contribution from the interaction interface between NS3hel and the protease domain. Most importantly, we establish that the protease domain is required for RNA unwinding by NS3. Our results suggest that, in addition to its role in cleavage of host and viral proteins, the NS3 protease domain is essential for the process of viral RNA replication and, given its electrostatic contribution to RNA binding, it may also assist in packaging of the viral RNA.