Wardell A D, Errington W, Ciaramella G, Merson J, McGarvey M J
J Gen Virol. 1999 Mar;80 ( Pt 3):701-709. doi: 10.1099/0022-1317-80-3-701.
The non-structural protein 3 (NS3) of hepatitis C virus (HCV) possesses three activities which are likely to be essential for virus replication; a serine protease located in the N terminus and helicase and NTPase activities located in the C terminus. Sequence analysis of the helicase/NTPase domain has identified motifs indicative of the DEAD-box family of helicases. Here we present the characterization of the helicase and NTPase activities of full-length NS3, expressed as a His-tagged fusion protein in E. coli, and make comparisons with published data of NS3 helicase domain alone. The helicase and NTPase activities of full-length NS3 have been demonstrated and we have characterized the effects of amino acid substitutions on conserved motifs of NS3 helicase. Helicase and NTPase activities were dependent on Mg2+ and ATP and inhibited by monovalent cations. NS3 was able to hydrolyse all four NTPs and dNTPs to drive DNA duplex unwinding but with differing abilities. NTPase activity was stimulated by all polynucleotides tested, with poly(U) having the greatest effect. Mutational analysis of conserved motifs of NS3 helicase showed all conserved residues to be required for optimal activity. These results are in accord with a recently proposed model for NS3 helicase activity.
丙型肝炎病毒（HCV）的非结构蛋白3（NS3）具有三种可能对病毒复制至关重要的活性；位于N端的丝氨酸蛋白酶以及位于C端的解旋酶和NTPase活性。对解旋酶/NTPase结构域的序列分析已确定了指示解旋酶DEAD-box家族的基序。在此，我们展示了在大肠杆菌中作为His标签融合蛋白表达的全长NS3的解旋酶和NTPase活性的特征，并与单独的NS3解旋酶结构域的已发表数据进行比较。已证实全长NS3的解旋酶和NTPase活性，并且我们已表征了氨基酸取代对NS3解旋酶保守基序的影响。解旋酶和NTPase活性依赖于Mg2+和ATP，并受到单价阳离子的抑制。NS3能够水解所有四种NTP和dNTP以驱动DNA双链解旋，但能力不同。所有测试的多核苷酸均刺激NTPase活性，其中聚（U）的影响最大。对NS3解旋酶保守基序的突变分析表明，所有保守残基都是最佳活性所必需的。这些结果与最近提出的NS3解旋酶活性模型一致。
