Beran Rudolf K F, Pyle Anna Marie
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA.
J Biol Chem. 2008 Oct 31;283(44):29929-37. doi: 10.1074/jbc.M804065200. Epub 2008 Aug 22.
Non-structural protein 3 (NS3) is a multifunctional enzyme possessing serine protease, NTPase, and RNA unwinding activities that are required for hepatitis C viral (HCV) replication. HCV non-structural protein 4A (NS4A) binds to the N-terminal NS3 protease domain to stimulate NS3 serine protease activity. In addition, the NS3 protease domain enhances the RNA binding, ATPase, and RNA unwinding activities of the C-terminal NS3 helicase domain (NS3hel). To determine whether NS3hel enhances the NS3 serine protease activity, we purified truncated and full-length NS3-4A complexes and examined their serine protease activities under a variety of salt and pH conditions. Our results indicate that the helicase domain enhances serine protease activity, just as the protease domain enhances helicase activity. Thus, the two enzymatic domains of NS3-4A are highly interdependent. This is the first time that such a complete interdependence has been demonstrated for a multifunctional, single chain enzyme. NS3-4A domain interdependence has important implications for function during the viral lifecycle as well as for the design of inhibitor screens that target the NS3-4A protease.
非结构蛋白3（NS3）是一种多功能酶，具有丙型肝炎病毒（HCV）复制所需的丝氨酸蛋白酶、NTP酶和RNA解旋活性。HCV非结构蛋白4A（NS4A）与NS3蛋白酶结构域的N端结合，以刺激NS3丝氨酸蛋白酶活性。此外，NS3蛋白酶结构域增强了C端NS3解旋酶结构域（NS3hel）的RNA结合、ATP酶和RNA解旋活性。为了确定NS3hel是否增强NS3丝氨酸蛋白酶活性，我们纯化了截短的和全长的NS3-4A复合物，并在各种盐和pH条件下检测了它们的丝氨酸蛋白酶活性。我们的结果表明，解旋酶结构域增强了丝氨酸蛋白酶活性，就像蛋白酶结构域增强解旋酶活性一样。因此，NS3-4A的两个酶结构域高度相互依赖。这是首次针对一种多功能单链酶证明了这种完全的相互依赖性。NS3-4A结构域的相互依赖性对病毒生命周期中的功能以及针对NS3-4A蛋白酶的抑制剂筛选设计具有重要意义。
