Identification and characterization of host proteins involved in the alphavirus exit pathway

甲病毒包括重要的和新出现的人类病原体，如脑炎病毒和乙型肝炎病毒。 基孔肯雅病毒、罗斯河病毒和马亚罗病毒。目前还没有获得许可的抗病毒疗法 或疫苗，需要新的方法和信息来开发抗病毒策略。 甲病毒是具有高度有序结构的小包膜RNA病毒。核衣壳核心是 由正义RNA基因组组成，包在衣壳蛋白壳中，并被病毒包围 包膜是一种含有E2和E1跨膜蛋白的有组织晶格的膜。有 增加对甲病毒结构、进入和复制以及宿主蛋白质功能的了解， 这些过程。相比之下，对于宿主蛋白质在蛋白质合成的后期步骤中的作用， 甲病毒出口途径，包括核衣壳组装/转运和病毒在血浆中出芽 膜的这项资助旨在利用蛋白质组学来鉴定与病毒衣壳相互作用的宿主蛋白质 蛋白质，并确定它们在病毒退出中的作用。这种方法一直是困难的，因为衣壳蛋白是 本质上是高度碱性的，并且在回收过程中与宿主蛋白质非特异性相互作用。标记衣壳 具有相对较大标记的蛋白质如荧光蛋白导致有组织的衣壳中的主要变形 晶格和病毒粒子。相反，我们开发了一种基于插入13个残基生物素的方法， 受体肽插入衣壳上的允许位点。表达BirA生物素化酶的细胞的感染 导致细胞和病毒颗粒中衣壳和核衣壳的有效生物素标记。我们的初步数据 已经建立了使用链霉亲和素磁珠的生物素化衣壳的有效和特异性回收。我们 将使用这个系统来识别感染细胞中与captain相关的细胞蛋白。具体目标是： 1.使用一种新的基于生物素的衣壳下拉策略来鉴定参与 甲病毒退出。我们将使用我们优化的方法进行化学交联，特定的生物素为基础的 回收和洗脱captain相关的细胞蛋白。将通过最新技术质量鉴定蛋白质 光谱分析方法，并优先进行进一步研究的基础上，统计和生物信息学分析 样品和对照，以及蛋白质细胞位置、途径和功能。 2.定义宿主蛋白在甲病毒退出中的作用。我们将测试一组经过优先级排序的候选主机 蛋白质在甲病毒退出途径中的作用。宿主蛋白将被siRNA靶向和效应耗尽 对甲病毒初次感染和病毒产量的影响。蛋白质显示最具体的影响， 甲病毒的退出将进一步表征敲除和过表达细胞使用我们建立的电池 用于病毒退出中规定步骤的检测。 这些研究的结果将提供影响甲病毒的宿主蛋白的基本信息 组装和出芽，以及抗病毒策略的潜在新靶点。

Alphaviruses include important and emerging human pathogens such as encephalitic viruses and the arthritogenic Chikungunya, Ross River, and Mayaro viruses. There are currently no licensed antiviral therapies or vaccines, and new approaches and information are needed to develop antiviral strategies. Alphaviruses are small enveloped RNA viruses with highly ordered structures. The nucleocapsid core is composed of the plus-sense RNA genome enclosed in a capsid protein shell, and is surrounded by the virus envelope, a membrane containing an organized lattice of the E2 and E1 transmembrane proteins. There is increasing understanding of alphavirus structure, entry, and replication, and the functions of host proteins in these processes. In contrast, much less is known about the roles of host proteins in the late steps of the alphavirus exit pathway, including nucleocapsid assembly/transport and virus budding at the plasma membrane. This grant aims to use proteomics to identify host proteins that interact with the virus capsid protein, and determine their roles in virus exit. Such an approach has been difficult because capsid protein is highly basic in nature and interacts non-specifically with host proteins during retrieval. Tagging the capsid protein with relatively large tags such as fluorescent proteins leads to major distortions in the organized capsid lattice and the viral particle. Instead, we have developed an approach based on insertion of a 13 residue biotin acceptor peptide into a permissive site on capsid. Infection of cells expressing the BirA biotinylation enzyme leads to efficient biotin labeling of capsid and nucleocapsid in cells and virus particles. Our preliminary data have established efficient and specific retrieval of biotinylated capsid using Streptavidin magnetic beads. We will use this system to identify capsid-associated cellular proteins in infected cells. The specific aims are: 1. Use a novel biotin-based capsid pull-down strategy to identify host proteins involved in alphavirus exit. We will use our optimized methods to perform chemical crosslinking, specific biotin-based retrieval, and elution of capsid-associated cellular proteins. Proteins will be identified by state of the art mass spectrometry methods and prioritized for further study based on statistical and bioinformatics analyses of samples and controls, and on protein cellular locations, pathways, and functions. 2. Define the role of host proteins in alphavirus exit. We will test a prioritized set of candidate host proteins for roles in the alphavirus exit pathway. Host proteins will be depleted by siRNA targeting and effects on alphavirus primary infection and virus production determined. Proteins showing the most specific effects on alphavirus exit will be further characterized in knock-out and overexpressing cells using our established battery of assays for defined steps in virus exit. The results of these studies will provide fundamental information on host proteins that affect alphavirus assembly and budding, and potential new targets for antiviral strategies.