The NS4A Protein of Hepatitis C Virus Promotes RNA-Coupled ATP Hydrolysis by the NS3 Helicase
The NS4A Protein of Hepatitis C Virus Promotes RNA-Coupled ATP Hydrolysis by the NS3 Helicase
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DOI：
10.1128/jvi.01849-08
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发表时间：
2009-04-01
影响因子：
5.4
通讯作者：
Pyle, Anna Marie
中科院分区：
文献类型：
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作者：
Beran, Rudolf K. F.;Lindenbach, Brett D.;Pyle, Anna Marie
非结构蛋白3 （NS3）是丙型肝炎病毒（HCV）的重要复制成分，也是DExH/D-box蛋白家族的一员。NS3的c端区域（NS3hel）表现出RNA刺激的NTPase和解旋酶活性，而NS3的n端丝氨酸蛋白酶结构域增强了NS3hel对RNA的结合和解绕。非结构蛋白4A （NS4A）结合到NS3蛋白酶结构域，并作为NS3丝氨酸蛋白酶活性的专性辅因子。鉴于其在刺激蛋白酶活性方面的作用，我们试图确定NS4A是否也影响NS3hel的活性。在这里，我们发现NS4A增强了NS3hel在ATP存在下结合RNA的能力，从而作为解旋酶活性的辅助因子。这种作用是由NS4A c端酸性区域的氨基酸介导的。当这些残基发生突变时，人们观察到atp偶联RNA结合和NS3双解绕的急剧减少。这些相同的突变在HCV复制子中是致死性的，从而在体外和体内建立了NS4A在NS3解旋酶机制及其复制功能中的重要作用。
Nonstructural protein 3 (NS3) is an essential replicative component of the hepatitis C virus (HCV) and a member of the DExH/D-box family of proteins. The C-terminal region of NS3 (NS3hel) exhibits RNA-stimulated NTPase and helicase activity, while the N-terminal serine protease domain of NS3 enhances RNA binding and unwinding by NS3hel. The nonstructural protein 4A (NS4A) binds to the NS3 protease domain and serves as an obligate cofactor for NS3 serine protease activity. Given its role in stimulating protease activity, we sought to determine whether NS4A also influences the activity of NS3hel. Here we show that NS4A enhances the ability of NS3hel to bind RNA in the presence of ATP, thereby acting as a cofactor for helicase activity. This effect is mediated by amino acids in the C-terminal acidic domain of NS4A. When these residues are mutated, one observes drastic reductions in ATP-coupled RNA binding and duplex unwinding by NS3. These same mutations are lethal in HCV replicons, thereby establishing in vitro and in vivo that NS4A plays an important role in the helicase mechanism of NS3 and its function in replication.