Functional analysis of Hepatitis C Virus replicase NS5B and a regulatory factor NS5A

根除HCV持续感染可以预防或延缓肝细胞癌的发生。HCV NS5B是一种RdRP，是根除HCV复制的强有力靶点。为了寻找新的策略，我们通过引入聚簇丙氨酸取代（Cm）和点丙氨酸取代（point alanine substitution），确定了5个对RdRP活性至关重要的NS5B残基。本项目旨在进一步了解RdRP酶的结构和功能，表征NS5B相互作用伙伴、NS5A和宿主因子核蛋白，并最终利用HCV RNA复制子系统评估这些相互作用和NS5B突变对HCV RNA复制的影响。以下是我们得到的结果。1)观察了NS5B的低聚物形成，通过扫描Cm和Pm文库确定了两个对低聚物形成至关重要的残基。有趣的是，这两个残基是RdRP活性不可或缺的残基之一，这强烈表明低聚物相互作用可能是RdRP活性的先决条件。发现NS5B与NS5A之间的相互作用更为明显。3)我们发现在c端截断的NS5B在核仁中富集。Nucleolin被定义为与NS5B相互作用的因子。NS5B结合片段对RdRP活性影响不大，但相互作用会影响NS5B的亚细胞定位。4)我们利用MlLE HCV RNA复制子评估了RdRP的5个关键残基在体外HCV复制中是否不可或缺。实际上所有的残基都是丙型肝炎病毒复制所必需的。5)利用携带NS5A突变的HCV复制子，我们评估了NS5A- ns5b相互作用在HCV复制中的作用。在ns5b结合所需区域内的Cm突变体和一些Pm突变体不能支持HCV复制，这强烈表明相互作用的重要性。6)我们评估了核仁蛋白和NS5B在HCV RNA复制子系统中的相互作用。NS5B核蛋白结合缺陷的Cm突变体不能支持高效的HCV复制。用siRNA瞬间敲低核仁蛋白大大减少了g418形成的焦点，这强烈表明核仁蛋白和NS5B的相互作用对HCV复制至关重要。少

Eradication of HCV persistent infection may prevent or delay incidence of hepatocellular carcinoma. HCV NS5B is RdRP that is a strong target to eradicate HCV replication. In hope to find novel strategies, we had identified 5 residues of NS5B critical for RdRP activity by introducing clustered alanine substitution (Cm) and point alanine substitution. This grant aims to get further insight of structure and function of RdRP enzyme, to characterize NS5B-interacting partners, NS5A and a host factor nucleolin, and finally to evaluate these interactions and mutations of NS5B on HCV RNA replication using a HCV RNA replicon system. Followings are the results we obtained.1)Oligomer formation of NS5B was observed and two residues were defined by scanning the Cm and Pm libraries to be critical for oligomer formation. Interestingly the two residues are among the residues indispensable for RdRP activity, strongly suggesting that oligomeric interaction might be prerequisite for RdRP activity. 2)Speci … More fic interaction between NS5B and NS5A was found. The critical regions of the two factors for the interaction were mapped, 3)We found NS5B truncated at C-terminal was enriched in nucleoli. Nucleolin was defined to the interacting factor with NS5B. The NS5B-binding fragments did not affected much RdRP activity, although the interaction affected the subcellular localization of NS5B. 4)We evaluated whether the critical 5 residues for RdRP in vitro are indispensable for HCV replication using MlLE HCV RNA replicon. Actually all the residues are absolutely required for HCV replication. 5)Using HCV replicon harboring mutations of NS5A that are defective in NS5B-binding, we evaluated the role of the NS5A-NS5B interaction in HCV replication. Cm mutants and some Pm mutants within the regions necessary for NS5B-binding could not support HCV replication, strongly suggesting the importance of the inter action. 6)We evaluated the interaction of nucleolin and NS5B in the HCV RNA replicon system. Cm mutant of NS5B defective in nucleolin-binding could not support efficient HCV replication. Transient knock-down of nucleolin with siRNA greatly reduced the G418-forming foci, strongly suggesting that the interaction of nucleolin and NS5B is critical for HCV replication. Less