Department of Biological Science, Florida State University, Tallahassee, Florida, USA.
J Virol. 2012 Dec;86(23):12616-24. doi: 10.1128/JVI.01351-12. Epub 2012 Sep 12.
Nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) is an indispensable component of the HCV replication and assembly machineries. Although its precise mechanism of action is not yet clear, current evidence indicates that its structure and function are regulated by the cellular peptidylprolyl isomerase cyclophilin A (CyPA). CyPA binds to proline residues in the C-terminal half of NS5A, in a distributed fashion, and modulates the structure of the disordered domains II and III. Cyclophilin inhibitors (CPIs), including cyclosporine (CsA) and its nonimmunosuppressive derivatives, inhibit HCV infection of diverse genotypes, both in vitro and in vivo. Here we report a mechanism by which CPIs inhibit HCV infection and demonstrate that CPIs can suppress HCV assembly in addition to their well-documented inhibitory effect on RNA replication. Although the interaction between NS5A and other viral proteins is not affected by CPIs, RNA binding by NS5A in cell culture-based HCV (HCVcc)-infected cells is significantly inhibited by CPI treatment, and sensitivity of RNA binding is correlated with previously characterized CyPA dependence or CsA sensitivity of HCV mutants. Furthermore, the difference in CyPA dependence between a subgenomic and a full-length replicon of JFH-1 was due, at least in part, to an additional role that CyPA plays in HCV assembly, a conclusion that is supported by experiments with the clinical CPI alisporivir. The host-directed nature and the ability to interfere with more than one step in the HCV life cycle may result in a higher genetic barrier to resistance for this class of HCV inhibitors.
丙型肝炎病毒（HCV）的非结构蛋白 5A（NS5A）是 HCV 复制和组装机制中不可或缺的组成部分。尽管其确切的作用机制尚不清楚，但目前的证据表明，其结构和功能受细胞肽脯氨酰顺反异构酶 cyclophilin A（CyPA）的调节。CyPA 以分布式的方式结合 NS5A C 端半部分的脯氨酸残基，并调节无规结构域 II 和 III 的结构。环孢菌素抑制剂（CPIs），包括环孢菌素（CsA）及其非免疫抑制剂衍生物，在体外和体内均能抑制多种基因型的 HCV 感染。在这里，我们报告了 CPI 抑制 HCV 感染的一种机制，并证明 CPI 除了对 RNA 复制具有已被充分证实的抑制作用外，还可以抑制 HCV 组装。尽管 NS5A 与其他病毒蛋白之间的相互作用不受 CPI 影响，但在基于细胞培养的 HCV（HCVcc）感染细胞中，NS5A 的 RNA 结合被 CPI 处理显著抑制，并且 RNA 结合的敏感性与先前表征的 CyPA 依赖性或 CsA 敏感性 HCV 突变体相关。此外，JFH-1 的亚基因组和全长复制子之间的 CyPA 依赖性差异至少部分归因于 CyPA 在 HCV 组装中的额外作用，这一结论得到了临床 CPI alisporivir 实验的支持。该类 HCV 抑制剂具有宿主定向性和能够干扰 HCV 生命周期中的多个步骤的能力，可能导致其对耐药性的遗传障碍更高。
