单正链RNA病毒包括一大类对人类健康产生威胁的病毒，其复制策略高度保守－在宿主膜表面组装复制酶，是好的抗病毒靶点。丙型肝炎病毒(HCV) NS5A在复制酶组装中起重要作用。一般认为其膜定位由N段的两性a螺旋(AH)介导，我们最新研究发现此AH不参与NS5A膜定位，提示其新功能。我们前期研究发现HCV直接抗病毒药物DCV可能靶向NS5A的近膜定位相关区域(membrane proximal region)，阻止复制酶的组装。我们假设NS5A AH作为近膜定位相关区域重要组成，调控复制酶的组装。我们将以HCV为模型，解析NS5A AH调控复制酶组装的具体环节；利用生物正交反应（bioorthogonal）解析NS5A AH参与调控的HCV复制酶的近膜定位相关区域的相互作用。该研究将揭示近膜定位相关区域组分调控病毒复制酶组装的新机制；为针对这一病毒复制酶共有结构设计新的直接抗病毒药物提供新思路。

The positive-strand RNA viruses are the largest class of viruses and include many medical and economically important pathogens, including hepatitis C virus (HCV); Picornavirueses; and Flaviviruses, such as zika virus (ZIKV). Assembling a viral replicase on host intracellular membranes is a common strategy for almost all of the positive-strand RNA viruses. Understanding how the key modules of the replicase is involved in the replicase assembly may provide insights into the pathway of the replicase assembly. HCV assembles the replicase in the endoplasmic reticulum (ER) to, inducing protrusion of the ER to form a double membrane vesicle (DMV). Overexpression of the HCV polyprotein encompassing NS3 to NS5B could induce the formation of the similar DMV structure as seen in the virally infected cells. Expression of NS5A alone can also induce DMVs, suggesting a critical role of NS5A in the replication complex assembly. NS5A contains an N-terminal amphipathic helix (AH), followed domain I, domain II and domain III. Amphipathic helices (AHs) are widely distributed in proteome. It is characterized by the segregation of the hydrophobic and polar residues between the two opposite faces of the α-helix. This unique property gives the functional diversities of the AH, such as membrane deformation, membrane curvature sensing, specific lipid recognition. The NS5A AH was previously shown to mediate its membrane association. However, our very recent studies show that the AH is dispensable for membrane association of NS5A, arguing novel roles of AH in the viral life cycle. In addition, we recently report that a direct anti-viral (DAA), HCV NS5A inhibitor daclatasvir, probably by inducing a position change of NS5A, allosterically impairs NS4B-involved protein–protein interactions within the viral replicase and disrupts the replicase quaternary structure in a replicase assembly surrogate system. This study points a model that DCV, by targeting the membrane proximal region of the viral replicase, interferes with the replicase assemble. The NS5A AH is located in the membrane proximal region of the viral replicase and given the central role of HCV NS5A in the viral replicase assembly, dissecting the molecular mechanisms of the NS5A AH in the viral replicase assembly may deepen our understanding of the pathways of viral replicase assembly and how the membrane proximal region of the viral replicase regulates the replicase assembly. These studies may shed light on designing novel DAAs targeting the membrane proximal region of the viral replicase. In this project, using a viral replicase assembly surrogate system, we will dissect the roles of the NS5A AH in the membrane anchoring of NS5A, the viral polyprotein cleavage, and the viral replicase assembly. In addition, by employing a bioorthogonal strategy, for the first time, we will dissect the NS5A AH- and NS4B-involved interactomes within membrane proximal region of the viral replicase.