Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Heidelberg, Germany.
PLoS Pathog. 2010 Dec 16;6(12):e1001233. doi: 10.1371/journal.ppat.1001233.
Non-structural protein 2 (NS2) plays an important role in hepatitis C virus (HCV) assembly, but neither the exact contribution of this protein to the assembly process nor its complete structure are known. In this study we used a combination of genetic, biochemical and structural methods to decipher the role of NS2 in infectious virus particle formation. A large panel of NS2 mutations targeting the N-terminal membrane binding region was generated. They were selected based on a membrane topology model that we established by determining the NMR structures of N-terminal NS2 transmembrane segments. Mutants affected in virion assembly, but not RNA replication, were selected for pseudoreversion in cell culture. Rescue mutations restoring virus assembly to various degrees emerged in E2, p7, NS3 and NS2 itself arguing for an interaction between these proteins. To confirm this assumption we developed a fully functional JFH1 genome expressing an N-terminally tagged NS2 demonstrating efficient pull-down of NS2 with p7, E2 and NS3 and, to a lower extent, NS5A. Several of the mutations blocking virus assembly disrupted some of these interactions that were restored to various degrees by those pseudoreversions that also restored assembly. Immunofluorescence analyses revealed a time-dependent NS2 colocalization with E2 at sites close to lipid droplets (LDs) together with NS3 and NS5A. Importantly, NS2 of a mutant defective in assembly abrogates NS2 colocalization around LDs with E2 and NS3, which is restored by a pseudoreversion in p7, whereas NS5A is recruited to LDs in an NS2-independent manner. In conclusion, our results suggest that NS2 orchestrates HCV particle formation by participation in multiple protein-protein interactions required for their recruitment to assembly sites in close proximity of LDs.
非结构蛋白 2（NS2）在丙型肝炎病毒（HCV）组装中发挥重要作用，但该蛋白对组装过程的确切贡献及其完整结构尚不清楚。在这项研究中，我们使用遗传、生化和结构方法的组合来破译 NS2 在感染性病毒颗粒形成中的作用。我们针对靶向 N 端膜结合区的 NS2 生成了一个大型突变面板。这些突变体是根据我们通过确定 N 端 NS2 跨膜片段的 NMR 结构建立的膜拓扑模型选择的。选择那些影响病毒体组装但不影响 RNA 复制的突变体在细胞培养中进行假回复。在 E2、p7、NS3 和 NS2 本身中出现了各种程度恢复病毒组装的拯救突变，这表明这些蛋白之间存在相互作用。为了证实这一假设，我们开发了一种完全功能性的 JFH1 基因组，表达了一个 N 端标记的 NS2，该 NS2 可有效地与 p7、E2 和 NS3 进行下拉，并且在较低程度上与 NS5A 进行下拉。一些阻断病毒组装的突变体破坏了其中一些相互作用，这些相互作用在各种程度上被那些也恢复组装的假回复所恢复。免疫荧光分析显示，NS2 与 E2 在接近脂滴（LDs）的位置上随时间发生共定位，同时还有 NS3 和 NS5A。重要的是，在组装缺陷的突变体中，NS2 会破坏 NS2 与 E2 和 NS3 在 LDs 周围的共定位，而 p7 的假回复则会恢复这种共定位，而 NS5A 则以 NS2 独立的方式被募集到 LDs 上。总之，我们的结果表明，NS2 通过参与多个蛋白-蛋白相互作用来协调 HCV 颗粒的形成，这些相互作用对于它们在接近 LDs 的组装部位的募集是必需的。
