DEFINING NOVEL, EVOLUTIONARILY CONSERVED HOST FACTORS CRITICAL FOR VIRUS INFECTION.

定义对病毒感染至关重要的新型、进化保守的宿主因子。

• 批准号: 10399465
• 负责人: DAVID WANG
• 金额: $ 39.38万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-02 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10399465
• 关键词: A549; ACK1 Gene; Actins; Affect; Animal Model; Astroviridae; Binding Proteins; Biology; Caenorhabditis elegans; Candidate Disease Gene; Caspase; Cell Culture Techniques; Cell Death; Cells; Chemicals; Coxsackie Viruses; Coxsackievirus Infections; Data; Dissection; Family; Family Picornaviridae; Gene Deletion; Genes; Genetic; Genetic Screening; Goals; Growth; Human; Human Cell Line; Human poliovirus; In Vitro; Infection; Integration Host Factors; Malignant Neoplasms; Mus; Mutagenesis; NCK adaptor protein 1; Nematoda; Organism; Orthologous Gene; Pathway interactions; Phenocopy; Phosphorylation; Phosphotransferases; Play; Proteins; RNA Interference; RNA Viruses; Role; Route; System; Tertiary Protein Structure; Testing; Viral; Viral Pathogenesis; Virus; Virus Diseases; Virus Replication; Wiskott-Aldrich Syndrome; antiviral drug development; design; experimental study; gene function; human pathogen; in vivo; insight; mutant; novel; reverse genetics; screening; viral RNA

SUMMARY The goal of this project is to identify and characterize evolutionarily conserved host genes that are important for virus infection in organisms from humans to nematodes by exploiting a unique C. elegans virus infection system. C. elegans has played a key role in many fundamental discoveries in biology, but its use in the study of viral pathogenesis has been limited by the lack of known viruses capable of naturally infecting C. elegans. The recent discovery of Orsay Virus (ORV), the first and to date only known virus of C. elegans, provide a novel route for genetic dissection of host factors essential for virus infection. In preliminary data, a chemical mutagenesis screen in C. elegans was developed that identified two host genes essential for ORV infection. The first gene, sid-3, encodes a non-receptor tyrosine kinase that is orthologous to the human “activated Cdc42 associated kinase” (ACK1/TNK2). The second gene, viro-2, encodes an ortholog of human Wiskott Aldrich syndrome proteins (N-WASP). ORV is a non-enveloped positive sense RNA virus; among viral families with human pathogens, ORV is most closely related to the Picorna-, Calici- and Astroviridae. Deletion of either TNK2 or N-WASP in human A549 cells led to ~1000-fold reduced titers of encephalomycarditis virus (EMCV). ~100-fold reduction in coxsackie virus B3 (CVB3) was also observed in TNK2 deleted cells. Strikingly, N-WASP is a known substrate for the kinase activity of TNK2, suggesting that these genes may comprise a key pathway utilized by viruses. In support of this possibility, a third gene in C. elegans, nck-1, which is the ortholog of the human NCK protein that binds to both TNK2 and N-WASP, was also found to be essential for ORV infection. TNK2 has not been previously implicated in virus infection and represents a novel mammalian pro-viral host factor discovered by genetic screening in C. elegans. The aims are: 1) Utilize C. elegans as a discovery engine to comprehensively identify and characterize host factors critical for ORV infection. 2) To gain mechanistic insight into TNK2 and N-WASP, the stage of EMCV and CVB3 infection impacted by these genes and their essential protein domains and substrates required for their pro-viral functions will be defined. In addition, among the C. elegans candidate genes identified in Aim 1 that have human orthologs, a subset that phenocopy sid-3 and viro-2, which are the most likely candidates to act in the same pathway, will be prioritized for similar analyses. 3) Finally, the effect of TNK2 deletion in mice on EMCV and CVB3 infection will be defined. This proposal will yield novel insights into fundamental host genes and pathways necessary for virus infection which could be novel targets for antiviral development.

总结 该项目的目标是鉴定和表征进化上保守的宿主基因， 重要的病毒感染的生物体，从人类到线虫，利用一个独特的C。秀丽线虫病毒 感染系统C.线虫在生物学的许多基础发现中发挥了关键作用，但它在 由于缺乏能够自然感染C. 优雅的奥赛病毒（Orsay Virus，ORV）是近年来发现的第一个也是迄今为止唯一一个已知的C.优雅， 为病毒感染所必需的宿主因子的遗传解剖提供了新的途径。初步数据显示， 化学诱变筛选C. elegans的研究，鉴定了ORV所必需的两个宿主基因 感染第一个基因，sid-3，编码一种非受体酪氨酸激酶，它与人的酪氨酸激酶是同源的。 “激活的Cdc 42相关激酶”（ACK 1/TNK 2）。第二个基因，viro-2，编码人类的一个直系同源物， Wiskott Aldrich综合征蛋白（N-WASP）。ORV是一种无包膜的正义RNA病毒; ORV与人类病原体家族中的小核糖核酸病毒、杯状病毒和星状病毒最密切相关。删除 TNK 2或N-WASP在人A549细胞中的表达导致脑心肌炎病毒滴度降低约1000倍 （EMCV）。在TNK 2缺失的细胞中也观察到科萨基病毒B3（CVB 3）减少约100倍。引人注目的是， N-WASP是TNK 2的激酶活性的已知底物，表明这些基因可能包含一个或多个N-WASP基因。 病毒利用的关键途径。为了支持这种可能性，C. nck-1，也就是 与TNK 2和N-WASP结合的人NCK蛋白的直系同源物，也被发现对于 ORV感染。TNK 2以前没有涉及病毒感染，代表了一种新的哺乳动物 前病毒宿主因子是通过在C.优雅的目的是：1）利用C.优雅作为一个 发现引擎，以全面识别和表征对ORV感染至关重要的宿主因素。2)获得 对TNK 2和N-WASP的机制洞察，这些基因影响EMCV和CVB 3感染的阶段 并定义它们的前病毒功能所需的必需蛋白质结构域和底物。在 此外，C.在Aim 1中鉴定的具有人类直系同源物的线虫候选基因， 拟表型SID-3和viro-2是最有可能在同一途径中起作用的候选者，将被优先考虑 类似的分析。3)最后，将研究小鼠中TNK 2缺失对EMCV和CVB 3感染的影响。 定义了这一建议将产生新的见解基本宿主基因和途径所必需的病毒 感染，这可能是抗病毒药物开发的新靶点。

项目成果

Huntingtin-interacting protein family members have a conserved pro-viral function from Caenorhabditis elegans to humans.

亨廷顿蛋白相互作用蛋白家族成员具有从秀丽隐杆线虫到人类的保守的促病毒功能。

• DOI: 10.1073/pnas.2006914117
• 发表时间: 2020
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Jiang,Hongbing;SandovalDelPrado,LuisEnrique;Leung,Christian;Wang,David
• 通讯作者: Wang,David

Orsay Virus Infection of Caenorhabditis elegans Is Modulated by Zinc and Dependent on Lipids.

• DOI: 10.1128/jvi.01211-22
• 发表时间: 2022-11-23
• 期刊: Journal of virology
• 影响因子: 5.4