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

甲型病毒包括重要的和新出现的人类病原体，如脑炎病毒和 致关节炎的基孔肯雅、罗斯河和马亚罗病毒。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制定抗病毒战略。 甲型病毒是具有高度有序结构的小囊膜RNA病毒。核衣壳核心是 由包裹在衣壳蛋白外壳中的正义RNA基因组组成，并被病毒包围 包膜，包含有组织的E2和E1跨膜蛋白晶格的膜。的确有 加深对甲型病毒结构、进入和复制以及宿主蛋白功能的了解 这些过程。相比之下，对宿主蛋白在细胞生长后期的作用知之甚少。 甲型病毒退出途径，包括核衣壳组装/转运和病毒在血浆中的萌发 薄膜。这项拨款旨在利用蛋白质组学来识别与病毒衣壳相互作用的宿主蛋白。 蛋白质，并确定它们在病毒退出中的作用。这种方法一直很困难，因为衣壳蛋白是 高度碱性，在检索过程中与宿主蛋白非特异性相互作用。标记衣壳 具有相对较大标签的蛋白质，如荧光蛋白，会导致有组织衣壳的主要扭曲 格子和病毒颗粒。相反，我们开发了一种基于插入13个残基生物素的方法 受体多肽进入衣壳上的允许位置。表达Bira生物素化酶的细胞感染 导致有效的生物素标记细胞和病毒颗粒中的衣壳和核衣壳。我们的初步数据 已经建立了使用链霉亲和素磁珠高效和特异地回收生物素化衣壳的方法。我们 将使用这个系统来识别感染细胞中与衣壳相关的细胞蛋白。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识别参与 甲型病毒退出。我们将使用我们优化的方法进行化学交联，特别是以生物素为基础的 衣壳相关细胞蛋白的回收和洗脱。蛋白质将通过最先进的质量来鉴定 基于统计和生物信息学分析的光谱方法和进一步研究的优先事项 样本和对照，以及蛋白质细胞位置、途径和功能。 2.明确宿主蛋白在甲型病毒退出中的作用。我们将测试一组按优先级排列的候选主机 在甲型病毒退出途径中起作用的蛋白质。宿主蛋白将被siRNA靶向和作用耗尽 对甲型病毒的初次感染和病毒产生情况进行了测定。对蛋白质表现出最特异的影响 利用我们建立的电池，甲型病毒退出将在敲除和过度表达细胞中进一步表征 病毒退出过程中定义的步骤的分析。 这些研究的结果将提供影响甲型病毒的宿主蛋白的基本信息。 组装和萌芽，以及潜在的抗病毒策略的新靶点。

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.