Infectious Diseases Department, Vertex Pharmaceuticals Incorporated, Boston, Massachusetts, USA
Infectious Diseases Department, Vertex Pharmaceuticals Incorporated, Boston, Massachusetts, USA.
J Virol. 2015 Jan;89(1):165-80. doi: 10.1128/JVI.01677-14. Epub 2014 Oct 15.
The precise role(s) and topological organization of different factors in the hepatitis C virus (HCV) RNA replication complex are not well understood. In order to elucidate the role of viral and host proteins in HCV replication, we have developed a novel in vitro replication system that utilizes a rolling-circle RNA template. Under close-to-physiological salt conditions, HCV NS5BΔ21, an RNA-dependent RNA polymerase, has poor affinity for the RNA template. Human replication protein A (RPA) and HCV NS5A recruit NS5BΔ21 to the template. Subsequently, NS3 is recruited to the replication complex by NS5BΔ21, resulting in RNA synthesis stimulation by helicase. Both RPA and NS5A(S25-C447), but not NS5A(S25-K215), enabled the NS5BΔ21-NS3 helicase complex to be stably associated with the template and synthesize RNA product in a highly processive manner in vitro. This new in vitro HCV replication system is a useful tool that may facilitate the study of other replication factors and aid in the discovery of novel inhibitors of HCV replication.
The molecular mechanism of hepatitis C virus (HCV) replication is not fully understood, but viral and host proteins collaborate in this process. Using a rolling-circle RNA template, we have reconstituted an in vitro HCV replication system that allows us to interrogate the role of viral and host proteins in HCV replication and delineate the molecular interactions. We showed that HCV NS5A(S25-C447) and cellular replication protein A (RPA) functionally cooperate as a processivity factor to stimulate HCV replication by HCV NS5BΔ21 polymerase and NS3 helicase. This system paves the way to test other proteins and may be used as an assay for discovery of HCV inhibitors.
丙型肝炎病毒（HCV）RNA 复制复合物中不同因素的确切作用和拓扑结构尚不清楚。为了阐明病毒和宿主蛋白在 HCV 复制中的作用，我们开发了一种利用滚环 RNA 模板的新型体外复制系统。在接近生理盐条件下，HCV NS5BΔ21（一种 RNA 依赖性 RNA 聚合酶）与 RNA 模板亲和力差。人复制蛋白 A（RPA）和 HCV NS5A 将 NS5BΔ21 募集到模板上。随后，NS3 通过 NS5BΔ21 被募集到复制复合物中，导致解旋酶刺激 RNA 合成。RPA 和 HCV NS5A（S25-C447），而不是 NS5A（S25-K215），使 NS5BΔ21-NS3 解旋酶复合物能够稳定地与模板结合，并以高度连续性的方式在体外合成 RNA 产物。这种新的 HCV 体外复制系统是一种有用的工具，可能有助于研究其他复制因子，并有助于发现 HCV 复制的新型抑制剂。
丙型肝炎病毒（HCV）复制的分子机制尚未完全阐明，但病毒和宿主蛋白在这个过程中协同作用。我们使用滚环 RNA 模板，重新构建了一种体外 HCV 复制系统，使我们能够研究病毒和宿主蛋白在 HCV 复制中的作用，并阐明分子相互作用。我们发现 HCV NS5A（S25-C447）和细胞复制蛋白 A（RPA）作为一个延伸因子，通过 HCV NS5BΔ21 聚合酶和 NS3 解旋酶，共同发挥功能刺激 HCV 复制。该系统为测试其他蛋白铺平了道路，并可用于发现 HCV 抑制剂的测定。
