Biswas Avik, Treadaway Jason, Tellinghuisen Timothy L
The Scripps Research Institute, Scripps Florida, Jupiter, Florida, USA.
The Scripps Research Institute, Scripps Florida, Jupiter, Florida, USA
J Virol. 2016 Jul 27;90(16):7205-7218. doi: 10.1128/JVI.00037-16. Print 2016 Aug 15.
The hepatitis C virus NS5A protein is tethered to cellular membranes via an amphipathic amino-terminal helix that is inserted in-plane into the outer endoplasmic reticulum (ER)-derived membrane leaflet. The charged face of the helix faces the cytoplasm and may contribute to interactions involved in replicase assembly and function. Using an aggressive charge flip mutagenesis strategy, we identified a number of essential residues for replication on the charged face of the NS5A anchor and identified a double charge face mutant that is lethal for RNA replication but generates suppressor mutations in the carboxy-terminal helix of the NS4B protein. This suppressor restores RNA replication of the NS5A helix double flip mutant (D1979K/D1982K) and, interestingly, seems to function by restoring the proper localization of NS5A to the viral replicase. These data add to our understanding of the complex organization and assembly of the viral replicase via NS4B-NS5A interactions.
Information about the functional role of the cytosolic face of the NS5A anchoring helix remains obscure. In this study, we show that while the hydrophobic face of the NS5A anchor helix mediates membrane association, the polar cytosolic face of the helix plays a key role during hepatitis C virus (HCV) replication by mediating the interaction of NS5A with other HCV nonstructural proteins via NS4B. Such an interaction determines the subcellular localization of NS5A by engaging NS5A in the HCV replication process during the formation of a functional HCV replication complex. Thus, collectively, it can be stated that the findings in the present study provide further information about the interactions between the HCV nonstructural proteins during HCV RNA replication and provide a platform to gain more insights about the molecular architecture of HCV replication complexes.
丙型肝炎病毒NS5A蛋白通过一个两亲性氨基末端螺旋与细胞膜相连，该螺旋平面内插入到源自内质网（ER）的外膜小叶中。螺旋的带电荷面朝向细胞质，可能有助于参与复制酶组装和功能的相互作用。我们采用激进的电荷翻转诱变策略，在NS5A锚定的带电荷面上鉴定出许多复制必需的残基，并鉴定出一个双电荷面突变体，该突变体对RNA复制具有致死性，但会在NS4B蛋白的羧基末端螺旋中产生抑制突变。这种抑制作用恢复了NS5A螺旋双翻转突变体（D1979K/D1982K）的RNA复制，有趣的是，它似乎通过将NS5A正确定位到病毒复制酶上来发挥作用。这些数据加深了我们对通过NS4B-NS5A相互作用形成的病毒复制酶复杂组织和组装的理解。
关于NS5A锚定螺旋胞质面功能作用的信息仍然不清楚。在本研究中，我们表明，虽然NS5A锚定螺旋的疏水面向介导膜结合，但螺旋的极性胞质面在丙型肝炎病毒（HCV）复制过程中通过介导NS5A与其他HCV非结构蛋白通过NS4B的相互作用发挥关键作用。这种相互作用通过在功能性HCV复制复合体形成过程中使NS5A参与HCV复制过程来决定NS5A的亚细胞定位。因此，总的来说，可以说本研究中的发现提供了关于HCV RNA复制过程中HCV非结构蛋白之间相互作用的进一步信息，并提供了一个平台，以更深入地了解HCV复制复合体的分子结构。
