Sindbis nonstructural protein nsP2: Interaction with cellular proteins

描述（由申请人提供）：该项目代表了我们对甲病毒复制和与细胞相互作用机制的持续研究。辛德毕斯病毒 (SINV) 是在分子和细胞水平上研究甲病毒复制和发病机制相关基本问题的最有价值的工具。然而，尽管几个研究小组付出了巨大的努力，我们对这些过程的了解还远远不够。 SINV 非结构蛋白 nsP2 不仅是复制复合体 (RC) 的组成部分，而且还分布在 RC 之外的细胞质中，并以高浓度存在于感染细胞的细胞核中。根据我们的初步数据，我们假设 SINV nsP2 不仅在病毒非结构蛋白的加工和病毒特异性 RNA 的合成中发挥着关键作用，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，该蛋白是与 SINV 复制过程中细胞病变效应的发展和抗病毒细胞反应下调关键相关的病毒因子之一。我们的具体目标是： 1. 鉴定 SINV nsP2 在不同细胞区室中形成的蛋白质复合物。 2. 研究nsP2结合细胞蛋白的生物学功能。 3. 鉴定 SINV nsP2 中在病毒-宿主细胞相互作用中发挥关键作用的结构域和序列。该应用的结果将提供有关 SINV nsP2 与不同细胞区室中的细胞蛋白相互作用的详细信息。我们将生成有关 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。我们将使用多种方法分析这些数据的生物学意义，并确定在病毒-宿主细胞相互作用中对 nsP2 功能至关重要的 nsP2 结构域和序列。这些信息将增进我们对细胞水平上的 SINV 发病机制的理解，并且它不仅适用于 SINV，而且也适用于其他旧世界甲病毒。已鉴定的宿主蛋白与 SINV nsP2 相互作用并参与 SINV RC 形成和细胞转录抑制，可用作抗病毒治疗的靶点。 甲病毒是一组广泛分布的人类和动物病原体；其中一些会诱发高度衰弱的疾病，在美国代表着严重的公共卫生问题。本申请的目的是阐明一种病毒非结构蛋白在抑制抗病毒反应中的作用。了解干扰抗病毒反应的病毒特异性机制将有助于开发新的抗病毒治疗策略。甲病毒是一组广泛分布的人类和动物病原体；其中一些诱发高度 使人衰弱的疾病，在美国是一种严重的公共卫生治疗。该提案的目标是 阐明一种病毒非结构蛋白在抑制抗病毒反应中的作用。 了解干扰抗病毒反应的病毒特异性机制将促进发展 新的抗病毒治疗策略。

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. 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 proposal 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.