Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Mount Preston Street, Leeds LS2 9JT, United Kingdom.
J Virol. 2011 Jul;85(14):7460-4. doi: 10.1128/JVI.00393-11. Epub 2011 May 18.
NS5A plays a critical, yet poorly defined, role in hepatitis C virus genome replication. The protein consists of three domains, each of which is able to bind independently to the 3' untranslated region (UTR) of the viral positive strand genomic RNA. The peptidyl-prolyl isomerase cyclophilin A (CypA) binds to domain II, catalyzing cis-trans isomerization. CypA inhibitors such as cyclosporine (CsA) have been shown to inhibit hepatitis C virus (HCV) replication. We show here that CypA stimulated domain II RNA binding activity, and this stimulation was abrogated by CsA. An isomerase mutant of CypA (H126Q) failed to bind to domain II and did not stimulate RNA binding. Finally, we demonstrate that the RNA binding of two domain II mutants, the D316E and D316E/Y317N mutants, previously shown to exhibit CypA independence for RNA replication, was unaffected by CypA. This study provides an insight into the molecular mechanism of CypA activity during HCV replication and further validates the use of CypA inhibitors in HCV therapy.
NS5A 在丙型肝炎病毒基因组复制中发挥着关键但定义不明确的作用。该蛋白由三个结构域组成，每个结构域都能够独立地与病毒正链基因组 RNA 的 3'非翻译区（UTR）结合。肽基脯氨酰顺反异构酶亲环素 A（CypA）结合到结构域 II 上，催化顺反异构化。已表明亲环素 A 抑制剂（如环孢菌素 A（CsA））能够抑制丙型肝炎病毒（HCV）的复制。我们在这里表明 CypA 刺激了结构域 II RNA 结合活性，而 CsA 则阻断了这种刺激。CypA 的一个异构酶突变体（H126Q）无法与结构域 II 结合，也不能刺激 RNA 结合。最后，我们证明了两个结构域 II 突变体（先前证明对 RNA 复制具有 CypA 独立性的 D316E 和 D316E/Y317N 突变体）的 RNA 结合不受 CypA 的影响。这项研究提供了对 CypA 在 HCV 复制过程中活性的分子机制的深入了解，并进一步验证了使用 CypA 抑制剂治疗 HCV 的有效性。
