Université de Lyon, Lyon, France.
PLoS Pathog. 2011 Jul;7(7):e1002144. doi: 10.1371/journal.ppat.1002144. Epub 2011 Jul 21.
Hepatitis C virus (HCV) assembly remains a poorly understood process. Lipid droplets (LDs) are thought to act as platforms for the assembly of viral components. The JFH1 HCV strain replicates and assembles in association with LD-associated membranes, around which viral core protein is predominantly detected. In contrast, despite its intrinsic capacity to localize to LDs when expressed individually, we found that the core protein of the high-titer Jc1 recombinant virus was hardly detected on LDs of cell culture-grown HCV (HCVcc)-infected cells, but was mainly localized at endoplasmic reticulum (ER) membranes where it colocalized with the HCV envelope glycoproteins. Furthermore, high-titer cell culture-adapted JFH1 virus, obtained after long-term culture in Huh7.5 cells, exhibited an ER-localized core in contrast to non-adapted JFH1 virus, strengthening the hypothesis that ER localization of core is required for efficient HCV assembly. Our results further indicate that p7 and NS2 are HCV strain-specific factors that govern the recruitment of core protein from LDs to ER assembly sites. Indeed, using expression constructs and HCVcc recombinant genomes, we found that p7 is sufficient to induce core localization at the ER, independently of its ion-channel activity. Importantly, the combined expression of JFH1 or Jc1 p7 and NS2 induced the same differential core subcellular localization detected in JFH1- vs. Jc1-infected cells. Finally, results obtained by expressing p7-NS2 chimeras between either virus type indicated that compatibilities between the p7 and the first NS2 trans-membrane domains is required to induce core-ER localization and assembly of extra- and intra-cellular infectious viral particles. In conclusion, we identified p7 and NS2 as key determinants governing the subcellular localization of HCV core to LDs vs. ER and required for initiation of the early steps of virus assembly.
丙型肝炎病毒（HCV）的组装过程仍不为人知。人们认为脂滴（LDs）可以作为病毒成分组装的平台。JFH1 HCV 株与 LD 相关膜一起复制和组装，病毒核心蛋白主要在这些膜周围检测到。相比之下，尽管其单独表达时固有地定位于 LDs，但我们发现高滴度 Jc1 重组病毒的核心蛋白在细胞培养感染 HCV（HCVcc）的细胞中的 LDs 上几乎检测不到，但主要定位于内质网（ER）膜，在那里它与 HCV 包膜糖蛋白共定位。此外，在 Huh7.5 细胞中经过长期培养获得的高滴度细胞培养适应 JFH1 病毒与非适应 JFH1 病毒相反，表现出 ER 定位的核心，这加强了核心 ER 定位对于 HCV 组装的有效性的假说。我们的结果进一步表明，p7 和 NS2 是 HCV 株特异性因子，它们控制核心蛋白从 LDs 募集到 ER 组装部位。事实上，使用表达构建体和 HCVcc 重组基因组，我们发现 p7 足以独立于其离子通道活性诱导核心在 ER 中的定位。重要的是，JFH1 或 Jc1 的 p7 和 NS2 的联合表达诱导了在 JFH1-与 Jc1 感染细胞中检测到的相同的核心亚细胞定位差异。最后，通过表达两种病毒类型之间的 p7-NS2 嵌合体获得的结果表明，p7 和第一 NS2 跨膜结构域之间的兼容性对于诱导核心-ER 定位和细胞内外感染性病毒颗粒的组装是必需的。总之，我们确定了 p7 和 NS2 是控制 HCV 核心向 LDs 与 ER 亚细胞定位的关键决定因素，并且是病毒组装早期步骤启动所必需的。
