Department of Molecular Genetics & Microbiology, Duke University Medical Center, Durham, NC, United States of America.
Department of Medicine, Duke University Medical Center, Durham, NC, United States of America.
PLoS Pathog. 2019 Feb 7;15(2):e1007163. doi: 10.1371/journal.ppat.1007163. eCollection 2019 Feb.
Hepatitis C virus (HCV) assembly and envelopment are coordinated by a complex protein interaction network that includes most of the viral structural and nonstructural proteins. While the nonstructural protein 4A (NS4A) is known to be important for viral particle production, the specific function of NS4A in this process is not well understood. We performed mutagenesis of the C-terminal acidic domain of NS4A and found that mutation of several of these amino acids prevented the formation of the viral envelope, and therefore the production of infectious virions, without affecting viral RNA replication. In an overexpression system, we found that NS4A interacted with several viral proteins known to coordinate envelopment, including the viral E1 glycoprotein. One of the NS4A C-terminal mutations, Y45F, disrupted the interaction of NS4A with E1. Specifically, NS4A interacted with the first hydrophobic region of E1, a region previously described as regulating viral particle production. Indeed, we found that an E1 mutation in this region, D72A, also disrupted the interaction of NS4A with E1. Supernatants from HCV NS4A Y45F transfected cells had significantly reduced levels of HCV RNA, however they contained equivalent levels of Core protein. Interestingly, the Core protein secreted from these cells formed high order oligomers with a density matching the infectious virus secreted from wild-type cells. These results suggest that this Y45F mutation in NS4A causes secretion of low-density Core particles lacking genomic HCV RNA. These results corroborate previous findings showing that the E1 D72A mutation also causes secretion of Core complexes lacking genomic HCV RNA, and therefore suggest that the interaction between NS4A and E1 is involved in the incorporation of viral RNA into infectious HCV particles. Our findings define a new role for NS4A in the HCV lifecycle and help elucidate the protein interactions necessary for production of infectious virus.
丙型肝炎病毒（HCV）的组装和包膜是由一个复杂的蛋白质相互作用网络协调的，该网络包括大多数病毒的结构和非结构蛋白。虽然非结构蛋白 4A（NS4A）已知对病毒粒子的产生很重要，但 NS4A 在这个过程中的具体功能还不是很清楚。我们对 NS4A 的 C 端酸性结构域进行了突变，发现这些氨基酸中的几个突变阻止了病毒包膜的形成，因此也阻止了传染性病毒粒子的产生，而不影响病毒 RNA 的复制。在一个过表达系统中，我们发现 NS4A 与几个已知协调包膜的病毒蛋白相互作用，包括病毒 E1 糖蛋白。NS4A 的 C 端突变之一 Y45F 破坏了 NS4A 与 E1 的相互作用。具体来说，NS4A 与 E1 的第一个疏水区相互作用，该区域以前被描述为调节病毒粒子的产生。事实上，我们发现该区域的 E1 突变 D72A 也破坏了 NS4A 与 E1 的相互作用。从转染 HCV NS4A Y45F 的细胞的上清液中，HCV RNA 的水平显著降低，但 Core 蛋白的水平相当。有趣的是，从这些细胞分泌的 Core 蛋白形成了与从野生型细胞分泌的传染性病毒相匹配的高密度三聚体。这些结果表明，NS4A 的 Y45F 突变导致缺乏基因组 HCV RNA 的低密度 Core 粒子的分泌。这些结果与之前的发现相吻合，即 E1 D72A 突变也导致缺乏基因组 HCV RNA 的 Core 复合物的分泌，因此表明 NS4A 和 E1 之间的相互作用参与了将病毒 RNA 掺入传染性 HCV 粒子中。我们的研究结果确定了 NS4A 在 HCV 生命周期中的一个新作用，并有助于阐明产生传染性病毒所需的蛋白质相互作用。
