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: 9918848
• 负责人: DAVID WANG
• 金额: $ 39.35万
• 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 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9918848
• 关键词: A549; ACK1 Gene; Actins; Affect; Animal Model; Antiviral Agents; Astroviridae; Binding Proteins; Biology; Caenorhabditis elegans; Candidate Disease Gene; Caspase; Cell Culture Techniques; Cell Death; Cells; Chemicals; Coxsackie Viruses; Coxsackievirus Infections; Data; Development; 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; 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.

概括 该项目的目的是识别和表征进化保存的寄主基因 通过利用独特的秀丽隐杆线虫病毒，对从人到线虫的生物的病毒感染很重要 感染系统。秀丽隐杆线虫在生物学的许多基本发现中都发挥了关键作用，但它在 病毒发病机理的研究受到缺乏能够自然感染的已知病毒的限制。 秀丽隐杆线。最近发现的奥赛病毒（ORV），第一个，迄今为止仅是秀丽隐杆线虫的已知病毒， 为病毒感染必不可少的宿主因子提供了新的途径。在初步数据中 开发了秀丽隐杆线虫中的化学诱变筛选，鉴定了两个对ORV必不可少的宿主基因 感染。第一个基因SID-3编码与人类直系同源的非受体酪氨酸激酶 “活化的CDC42相关激酶”（ACK1/TNK2）。第二个基因Viro-2编码人类的直系同源 Wiskott Aldrich综合征蛋白（N-WASP）。 ORV是一种非发达的阳性RNA病毒。在病毒中 ORV患有人类病原体的家庭与picorna-，calici-和Astroviridae最密切相关。删除 人类A549细胞中的TNK2或N-WASP的含量约为1000倍降低了脑膜炎病毒的滴度 （EMCV）。在TNK2删除的细胞中，还观察到Coxsackie病毒B3（CVB3）的降低〜100倍。令人惊讶的是， N-WASP是TNK2激酶活性的已知底物，表明这些基因可能构成A 病毒利用的关键途径。为了支持这种可能性，秀丽隐杆线虫中的第三个基因，NCK-1，这是 还发现与TNK2和N-WASP结合的人NCK蛋白的直系同源物对于 ORV感染。 TNK2以前尚未与病毒感染有关，代表了一种新型的哺乳动物 秀丽隐杆线虫中的基因筛查发现的促病毒宿主因子。目的是：1）利用秀丽隐杆线虫作为 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TNK2和N-WASP的机械洞察力，EMCV和CVB3感染的阶段受这些基因影响 将定义其促病毒功能所需的必需蛋白质结构域和底物。在 此外，在具有人类直系同源物的AIM 1中鉴定出的秀丽隐杆线虫候选基因中，该子集 将在同一途径中起作用的最可能候选者SID-3和Viro-2优先考虑 用于类似的分析。 3）最后，TNK2缺失在小鼠中对EMCV和CVB3感染的影响 定义。该提案将产生对病毒必要的基本宿主基因和途径的新见解。 感染可能是抗病毒发育的新靶标。