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。

基本信息

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
项目类别：
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

项目摘要

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.

概括该项目的目标是识别和表征进化上保守的宿主基因利用独特的秀丽隐杆线虫病毒，对从人类到线虫等生物体的病毒感染非常重要线虫感染系统在生物学的许多基本发现中发挥了关键作用，但它的用途由于缺乏能够自然感染念珠菌的已知病毒，病毒发病机制的研究受到限制。最近发现的奥赛病毒（ORV）是第一个也是迄今为止唯一已知的线虫病毒，为病毒感染所必需的宿主因子的基因解析提供了一种新途径。开发了秀丽隐杆线虫化学诱变筛选，鉴定了 ORV 必需的两个宿主基因第一个基因 sid-3 编码与人类同源的非受体酪氨酸激酶。 “激活的 Cdc42 相关激酶”(ACK1/TNK2) 第二个基因，viro-2，编码人类的直系同源物。 Wiskott Aldrich 蛋白综合征 (N-WASP) 是一种无包膜正义 RNA 病毒； ORV 与人类病原体的家族中，与小病毒科、杯状病毒科和星状病毒科关系最密切。人 A549 细胞中 TNK2 或 N-WASP 导致脑心肌炎病毒滴度降低约 1000 倍 (EMCV)。令人惊讶的是，在 TNK2 缺失的细胞中也观察到柯萨奇病毒 B3 (CVB3) 减少了约 100 倍。 N-WASP 是 TNK2 激酶活性的已知底物，表明这些基因可能包含为了支持这种可能性，线虫中的第三个基因 nck-1 是病毒利用的关键途径。与 TNK2 和 N-WASP 结合的人类 NCK 蛋白的直系同源物也被发现对于 ORV 感染之前并未涉及病毒感染，并且代表了一种新型哺乳动物。通过线虫基因筛选发现的前病毒宿主因子目的是：1) 利用线虫作为宿主因子。发现引擎全面识别和表征 ORV 感染的关键宿主因素 2) 获得。深入了解 TNK2 和 N-WASP、受这些基因影响的 EMCV 和 CVB3 感染阶段以及它们的前病毒功能所需的基本蛋白结构域和底物将被定义。此外，在目标 1 中确定的具有人类直系同源物的秀丽隐杆线虫候选基因中，有一个子集表型 sid-3 和 viro-2 是最有可能在同一途径中发挥作用的候选者，将被优先考虑 3）最后，小鼠中 TNK2 缺失对 EMCV 和 CVB3 感染的影响。该提案将对病毒所需的基本宿主基因和途径产生新的见解。感染可能成为抗病毒开发的新靶点。

项目成果

期刊论文数量（6）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Vertical transmission in Caenorhabditis nematodes of RNA molecules encoding a viral RNA-dependent RNA polymerase.

编码病毒 RNA 依赖性 RNA 聚合酶的 RNA 分子在秀丽隐杆线虫中垂直传播。

DOI：
发表时间：
2019
期刊：
影响因子：
11.1
作者：
Richaud, Aurélien;Frézal, Lise;Tahan, Stephen;Jiang, Hongbing;Blatter, Joshua A;Zhao, Guoyan;Kaur, Taniya;Wang, David;Félix, Marie
通讯作者：
Félix, Marie

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。