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

甲病毒退出途径中宿主蛋白的鉴定和表征

基本信息

批准号：
10352876
负责人：
MARGARET KIELIAN
金额：
$ 25.2万
依托单位：
ALBERT EINSTEIN COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-24 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10352876
关键词：
Affect Alphavirus Antiviral Agents Antiviral Therapy Arthritogenic Bioinformatics Biological Biological Assay Biotin Biotinylation Capsid Capsid Proteins Cell membrane Cells Chemicals Chikungunya virus Collaborations Crosslinker Cytoplasm Data Disease Enzymes Evaluation Flow Cytometry Genome Grant Human Infection Integral Membrane Protein Integration Host Factors Knock-in Knock-out Label Ligase Lipid Bilayers Location Mass Spectrum Analysis Mayaro virus Mediating Medical Membrane Membrane Fusion Methods Nature Nucleocapsid Pathway interactions Peptides Permeability Plasma Cells Primary Infection Process Production Protein Analysis Protein Biosynthesis Proteins Proteome Proteomics Publishing RNA RNA Viruses Reporter Retrieval Rivers Role Ross river virus Sampling Semliki forest virus Sindbis Virus Site Small Interfering RNA Statistical Data Interpretation Streptavidin Structural Protein Structure System Testing Trypsin Vaccines Venezuelan Equine Encephalitis Virus Vesicular stomatitis Indiana virus Viral Virion Virus Virus-like particle base biodefense chikungunya crosslink emerging human pathogen human pathogen magnetic beads mutant novel novel strategies overexpression particle protein crosslink secondary infection uptake viral resistance virus envelope

项目摘要

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.

α病毒包括重要的和新兴的人类病原体，例如脑病病毒和关节型基孔肯雅，罗斯河和玛雅罗病毒。目前没有执照的抗病毒疗法或疫苗，需要新的方法和信息来制定抗病毒策略。 α病毒是具有高度有序结构的小包膜RNA病毒。 Nucleocapsid核心是由封闭在衣壳蛋白壳中的加分般的RNA基因组组成，并被病毒包围包膜，一种膜，该膜包含E2和E1跨膜蛋白的有组织晶格。有对α病毒结构，进入和复制以及宿主蛋白在这些过程。相比之下，关于宿主蛋白在晚期的晚期中的作用知之甚少 α病毒出口途径，包括等离子体处的核皮质组件/传输和病毒膜。该赠款旨在使用蛋白质组学来识别与病毒capsid相互作用的宿主蛋白蛋白质，并确定其在病毒出口中的作用。这种方法很困难，因为衣壳蛋白是在检索过程中，性质高度基础，并且与宿主蛋白非特异性相互作用。标记capsid 具有相对较大标签（例如荧光蛋白）的蛋白质会导致有组织的衣壳发生重大扭曲晶格和病毒颗粒。相反，我们已经开发了一种基于插入13个残基生物素的方法受体肽进入CAPSID上的宽松部位。表达BIRA生物素化酶的细胞感染导致细胞和病毒颗粒中衣壳和核素的有效生物素标记。我们的初步数据使用链霉亲和素磁珠确定了生物素化衣壳的有效和特异性检索。我们将使用该系统鉴定受感染细胞中与衣壳相关的细胞蛋白。具体目的是： 1。使用新型的基于生物素的Capsid下拉策略来识别涉及的宿主蛋白 α病毒出口。我们将使用优化的方法执行化学交联，特定的基于生物素取回胶囊相关的细胞蛋白的检索和洗脱。蛋白质将通过最先进的质量确定光谱法和根据统计和生物信息学分析的进一步研究优先研究样品和对照，以及蛋白质细胞位置，途径和功能。 2。定义宿主蛋白在α病毒出口中的作用。我们将测试优先的一组候选人主机蛋白质在α病毒出口途径中的作用。宿主蛋白将被siRNA靶向和效应耗尽确定原发性感染和病毒的产生。蛋白质显示出最具体的影响使用我们已建立的电池，α病毒出口将进一步表征在淘汰和过表达电池中关于病毒出口的定义步骤的测定。这些研究的结果将提供有关影响α病毒的宿主蛋白的基本信息组装和萌芽，以及抗病毒策略的潜在新目标。