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.

甲病毒包括重要的和新出现的人类病原体，例如脑炎病毒和致关节炎的基孔肯雅病毒、罗斯河病毒和马亚罗病毒。目前没有获得许可的抗病毒疗法或疫苗，并且需要新的方法和信息来制定抗病毒策略。甲病毒是具有高度有序结构的小包膜RNA病毒。核衣壳核心是由包裹在衣壳蛋白壳中的正链RNA基因组组成，并被病毒包围包膜，一种含有 E2 和 E1 跨膜蛋白组织晶格的膜。有增加对甲病毒结构、进入和复制以及宿主蛋白功能的了解这些过程。相比之下，人们对宿主蛋白在最后步骤中的作用知之甚少。甲病毒退出途径，包括核衣壳组装/运输和病毒在血浆中出芽膜。该资助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。这种方法很困难，因为衣壳蛋白是本质上是高度碱性的，并且在修复过程中与宿主蛋白发生非特异性相互作用。标记衣壳具有相对较大标签的蛋白质（例如荧光蛋白）会导致有组织的衣壳发生严重扭曲晶格和病毒颗粒。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肽进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对生物素化衣壳进行高效且特异性的检索。我们将使用该系统来识别受感染细胞中的衣壳相关细胞蛋白。具体目标是： 1. 使用一种新型的基于生物素的衣壳下拉策略来识别参与的宿主蛋白甲病毒退出。我们将使用我们优化的方法进行化学交联，特定的生物素基衣壳相关细胞蛋白的检索和洗脱。蛋白质将通过最先进的质量进行鉴定光谱测定方法，并根据统计和生物信息学分析优先进行进一步研究样品和对照，以及蛋白质细胞位置、途径和功能。 2. 定义宿主蛋白在甲病毒退出中的作用。我们将测试一组优先的候选主机在甲病毒退出途径中发挥作用的蛋白质。宿主蛋白将被 siRNA 靶向和效应耗尽对甲病毒的初次感染和病毒产生进行了测定。显示出最具体效果的蛋白质甲病毒退出将使用我们建立的电池在敲除和过度表达细胞中进一步表征病毒退出过程中确定步骤的检测。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萌芽，以及抗病毒策略的潜在新目标。