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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：
10495264
负责人：
MARGARET KIELIAN
金额：
$ 21万
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 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10495264
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，如脑炎病毒和甲型h1n1流感病毒