宿主AAA＋ATPase p97/VCP作为复制复合体重要组成参与单正链RNA病毒复制共同机制的研究

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, which cause hand-foot and mouth disease; and Flaviviruses, such as the west nile virus (WNV) and the zika virus (ZIKV). Positive-strand RNA viruses share a conserved replication mechanism, in which viral proteins induce host membrane modification to assemble membrane-associated viral replication complexes (RCs). Viruses hijack host factors to facilitate this energy unfavorable process. The formation of the RC represents a load-and-choke point of the viral life cycle and may serves as an attractive anti-viral target. In a previous study, using HCV as a model, by affinity purification of the viral replicase, we identified the viral replicase associated host factors and uncovered an ATPases associated with diverse cellular activities (AAA+ATPase) family member, Valosin-containing protein (VCP) as a pivotal host factor that is required for viral replication, depending on its ATPase activity. VCP is recruited to and co-localizes with HCV nonstructural protein clustering sites and inhibition of VCP alters HCV NS5A distribution as judged by fluorescence microscopy in an HCV replicase assembly surrogate system, suggesting a role of VCP in HCV replicase assembly. Very recently, it has been reported that VCP plays a role in west nile virus (WNV) replication and a human genome-wide RNAi screening also identifies VCP to be involved in enterovirus 71 (EV71) replication. While the molecular mechanisms of VCP in these virus replication remains elusive. The AAA+ proteins use their ATPase activity to catalyze conformational changes in diverse substrate proteins. While VCP barely exhibits substrate specificity, it employs a large number of cofactors for temporal and spatial regulation. In this study, we will use VCP pharmacological inhibitors and shRNA-mediated knockdown to inhibit VCP function and then dissect the molecular mechanisms of VCP in HCV viral replicase assembly, to identify the VCP viral substrate and the involving host cofactors for VCP. Meanwhile, we will take polio virus (PV) and yellow fever virus (YFV) as examples, to investigate the roles of VCP in these viruses replication. Understanding the molecular mechanism of VCP regulating viral replication complex may facilitate developing a pan-antiviral strategy by targeting the most conserved viral replication steps.

许多单正链RNA病毒如寨卡病毒 (Zika virus)、肠道病毒（Enterovirus）及丙型肝炎病毒（HCV）等对人类健康产生重大威胁。其复制策略高度保守即在特定的宿主膜表面组装病毒的复制复合体，是有效的抗病毒靶点。前期研究我们发现VCP作为HCV复制复合体结合蛋白可能参与病毒复制复合体组装。近期有报道VCP可能调控肠道病毒及黄病毒属病毒成员复制，但机制未知。VCP属于一种AAA＋ATPase，通过水解ATP催化底物构想变化。本研究拟解析VCP调控HCV复制复合体组装的分子机制，鉴定其作用的病毒底物及参与其作用的宿主辅助因子；在此基础上进一步探讨VCP是否以相似机制调控脊髓灰质炎病毒（PV）及黄热病病毒（YFV）复制，并鉴定其作用于PV及YFV的病毒靶点。本研究以期找到VCP作用于一类单正链RNA病毒复制调控的可能的共同机制，为靶向VCP及其作用底物作为广谱抗病毒策略提供理论依据。

我们前期研究发现宿主蛋白AAA＋ATPase p97/VCP作为重要宿主因子参与丙型肝炎病毒（HCV）复制酶复合物（replicase）组装，以HCV为模型，本研究提示了NS5A的聚集机制：NS5A在体内通过特定的二聚化形式结合病毒的基因组，在病毒病毒基因RNA上串联的NS5A二聚体形成NS5A array，此NS5A array有非正常聚集倾向，VCP作用于NS5A，利用其AAA＋ ATPase活性，解聚NS5A array中形成的非正常聚集，保证病毒replicase组装顺利进行。VCP活性的缺失导致NS5A array中NS5A非正常聚集的出现，进而影响NS5A的超磷酸化以及replicase各组分间的蛋白相互作用，抑制病毒replicase的组装。通过shRNA介导的Knockdown以及VCP的化学抑制剂，发现VCP抑制剂或VCP的Knockdown显著抑制PV及YFV的感染，进一步发现VCP在YFV的进入以及复制过程中均发挥作用；利用PV作为模型，首先我们我们测试了VCP化学抑制剂对病毒蛋白的聚集效应，未发现同HCV类似的非正常聚集效应。以Enterovirus A病毒为研究模型，发现VCP及其相互作用蛋白UFD1和Nucleolin在病毒进入宿主细胞中发挥作用。由此，VCP参与单正链RNA病毒的作用机制包括病毒进入以及病毒replicase组装。由VCP调控NS5A的聚集出发，联想NS5A抑制剂DCV作用机制的特殊性－在极低的作用浓度下发挥高效抗病毒效应，推测DCV是否通过影响NS5A的聚集发挥抗病毒效应。研究发现DCV不同于VCP抑制剂，不影响NS5A的聚集。发现DCV的全新的作用模式：DCV结合于NS5A domain I与宿主膜形成的近膜定位相关区域（membrane proximal region）；DCV的结合导致NS5A相对于膜的位置的变化；此位置的变化通过NS5A/NS3的相互作用传递给NS3，进而影响replicase各组分的相互作用，抑制replicase 组装。本研究提示了HCV replicase的与宿主膜形成的近膜定位相关区域的蛋白可作为抗病毒药物的潜在靶点，为其他单正链RNA的药物开发提供新的思路。