School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK.
J Gen Virol. 2013 May;94(Pt 5):1009-1018. doi: 10.1099/vir.0.050633-0. Epub 2013 Jan 16.
The NS5A protein of hepatitis C virus (HCV) plays roles in both virus genome replication and the assembly of infectious virus particles. NS5A comprises three domains, separated by low-complexity sequences. Whilst the function of domain I appears to be predominantly involved with genome replication, the roles of domains II and III are less well defined. It has been reported previously that a deletion spanning the majority of domain II but retaining the C-terminal 35 residues had no effect on virus production; however, deletion of the entire domain II eliminated genome replication, pointing to a key role for the C terminus of this domain. Recent work has also highlighted this region as the potential binding site of the host factor cyclophilin A (CypA). To define this requirement for replication in more detail, and to investigate the involvement of CypA, we conducted a mutagenic study of the C-terminal 30 residues of domain II within the context of both the infectious JFH-1 virus and a JFH-1-derived subgenomic replicon. We showed that 12 of these residues were absolutely required for virus genome replication, whilst mutations of the remainder either had no phenotype or exhibited a partial reduction in genome replication. There was an absolute correlation between the datasets for virus and subgenomic replicon, indicating that this region is involved solely in the process of genome replication. Comparison of our data with a previously published analysis of the same region in genotype 1b revealed some important differences between the two genotypes of HCV.
丙型肝炎病毒 (HCV) 的 NS5A 蛋白在病毒基因组复制和感染性病毒颗粒组装中都发挥作用。NS5A 由三个结构域组成，由低复杂度序列分隔。虽然结构域 I 的功能似乎主要与基因组复制有关，但结构域 II 和 III 的作用尚未明确界定。先前已有报道称，跨越大部分结构域 II 但保留 C 末端 35 个残基的缺失对病毒产生没有影响；然而，整个结构域 II 的缺失消除了基因组复制，这表明该结构域的 C 末端起着关键作用。最近的工作还强调了该区域是宿主因子亲环素 A (CypA) 的潜在结合位点。为了更详细地定义该区域在复制中的要求，并研究 CypA 的参与，我们在感染性 JFH-1 病毒和 JFH-1 衍生的亚基因组复制子的背景下，对结构域 II 的 C 末端 30 个残基进行了诱变研究。我们表明，这些残基中有 12 个是病毒基因组复制所必需的，而其余残基的突变要么没有表型，要么表现出基因组复制的部分减少。病毒和亚基因组复制子的数据集之间存在绝对相关性，表明该区域仅参与基因组复制过程。我们的数据与之前对基因型 1b 同一区域的分析进行比较，发现 HCV 的两种基因型之间存在一些重要差异。
