Sindbis nonstructural protein nsP2: Interaction with cellular proteins

描述(申请人提供)：这个项目代表了我们对甲型病毒复制和与细胞相互作用机制的持续研究。辛德比斯病毒(SINV)是在分子和细胞水平上研究与甲型病毒复制和发病相关的基本问题的最有价值的工具。然而，尽管几个研究小组做出了巨大努力，我们对这些过程的了解还远远不完整。SINV非结构蛋白nsP2不仅是复制复合体(RCS)的组成部分，而且也分布在RCS外的细胞质中，并在感染细胞的细胞核中高浓度存在。根据我们的初步数据，我们推测SINV nsP2不仅在病毒非结构蛋白的加工和病毒特异性RNA的合成中发挥关键作用，而且还通过抑制细胞基因的转录来控制病毒诱导的应激反应的发展。因此，该蛋白是SINV复制过程中细胞病变效应和抗病毒细胞反应下调的关键病毒因子之一。我们的具体目标是：1.鉴定SINV nsP2在不同细胞内形成的蛋白质复合体。2.研究nsP2结合细胞蛋白的生物学功能。3.确定在病毒-宿主细胞相互作用中起关键作用的SINV nsP2的结构域和序列。这一应用的结果将提供关于SINV nsP2与不同细胞隔间中的细胞蛋白相互作用的详细信息。我们将产生关于SINV复制中细胞蛋白功能的新数据，并阐明SINV复制干扰细胞转录的机制。使用多种方法，我们将分析这些数据的生物学意义，并确定在病毒与宿主细胞相互作用中nsP2功能至关重要的nsP2结构域和序列。这些信息将促进我们在细胞水平上对SINV致病机制的理解，它将不仅适用于SINV，而且也适用于其他旧大陆甲型病毒。已鉴定的与SINV nsP2相互作用并参与SINV RC形成和抑制细胞转录的宿主蛋白可能成为抗病毒治疗的靶点。 甲型病毒是一组广泛分布的人类和动物病原体；在美国，它们中的一些会导致高度衰弱的疾病，并代表着严重的公共卫生治疗。这项应用的目的是阐明病毒非结构蛋白之一在抑制抗病毒反应中的作用。了解病毒特异性干扰抗病毒反应的机制将导致新的抗病毒治疗策略的发展。

DESCRIPTION (provided by applicant): This project represents our continuing investigation into the mechanism of alphavirus replication and interaction with cells. Sindbis virus (SINV) is the most valuable tool for examining fundamental issues pertaining to alphavirus replication and pathogenesis on the molecular and cellular levels. However, despite great efforts by several research groups, our knowledge of these processes is far from complete. SINV nonstructural protein nsP2 is not only a component of the replicative complexes (RCs), but is also distributed in the cytoplasm outside of the RCs and is present at high concentrations in the nuclei of infected cells. We hypothesize, on the basis of our preliminary data, that SINV nsP2 plays critical roles not only in the processing of viral nonstructural proteins and synthesis of virus-specific RNAs, but also in controlling the development of the virus-induced stress response by inhibiting transcription of cellular genes. Thus, this protein is one of the viral factors critically involved in development of cytopathic effect and downregulation of the antiviral cell response during SINV replication. Our specific aims are: 1. To identify protein complexes formed by SINV nsP2 in different cellular compartments. 2. To study biological functions of the nsP2-binding cellular proteins. 3. To identify domains and sequences in SINV nsP2 playing critical roles in virus-host cell interactions. The results of this application will provide detailed information about the interaction of SINV nsP2 with cellular proteins in different cellular compartments. We will generate new data about the functions of cellular proteins in SINV replication and elucidate the mechanism of interference of SINV replication with cellular transcription. Using a variety of approaches, we will analyze the biological significance of these data and identify the nsP2 domains and sequences that are critical in the functioning of nsP2 in virus-host cell interactions. This information will advance our understanding of SINV pathogenesis on cellular level, and it will be mostly applicable, not only to SINV, but also to other Old World alphaviruses. The identified host proteins interacting with SINV nsP2 and involved in SINV RC formation and inhibition of cellular transcription might be used as targets for the antiviral therapy. Alphaviruses is a group of widely distributed human and animal pathogens; some of them induce highly debilitating diseases and represent a serious public health treat in the US. The goal of this application is to elucidate the role of one of the viral nonstructural protein in suppression of the antiviral response. Understanding of the virus-specific mechanisms of interference with antiviral response will lead to development of new antiviral therapeutic strategies.