Beran Rudolf K F, Serebrov Victor, Pyle Anna Marie
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA.
J Biol Chem. 2007 Nov 30;282(48):34913-20. doi: 10.1074/jbc.M707165200. Epub 2007 Oct 5.
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.
非结构（NS）蛋白3是一种DEXH/D-box解旋酶蛋白，是丙型肝炎病毒（HCV）复制复合体的重要组成部分。全长NS3蛋白包含两个功能模块，这两个模块在HCV的生命周期中均至关重要：N端的丝氨酸蛋白酶结构域和C端的ATP酶/解旋酶结构域（NS3hel）。截短的NS3hel构建体已得到广泛研究；其ATP酶、核酸结合和解旋酶活性已被检测，并且NS3hel已被用作抗病毒药物开发的靶点。然而，尚未对NS3和NS3hel的活性进行全面比较，因此尚不清楚蛋白酶结构域在NS3解旋酶功能中是否起关键作用。鉴于许多DEXH/D-box蛋白在与辅因子蛋白相互作用时会被激活，确定蛋白酶结构域是否作为刺激NS3解旋酶功能的辅因子很重要。在此我们表明，蛋白酶结构域极大地增强了RNA与NS3的直接结合和功能结合。虽然静电作用在此过程中起重要作用，但NS3hel与蛋白酶结构域之间的相互作用界面存在特定的变构作用。最重要的是，我们确定蛋白酶结构域是NS3解开RNA所必需的。我们的结果表明，除了其在切割宿主和病毒蛋白中的作用外，NS3蛋白酶结构域对于病毒RNA复制过程至关重要，并且鉴于其对RNA结合的静电作用，它还可能有助于病毒RNA的包装。
