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

摘要 该项目的目标是识别和表征进化上保守的宿主基因 利用一种独特的线虫病毒，对从人类到线虫的生物中的病毒感染具有重要意义 感染系统。线虫在生物学的许多基本发现中发挥了关键作用，但它在 病毒发病机制的研究一直受到缺乏能够自然感染C. 优雅女装。奥赛病毒(Orsay Virus，ORV)是目前已知的第一种也是唯一一种线虫病毒， 为病毒感染所必需的宿主因子的基因解剖提供了一条新的途径。在初步数据中， 在线虫中建立了化学诱变筛选，鉴定了两个ORV必需的宿主基因 感染。第一个基因sid-3编码一种与人类同源的非受体酪氨酸激酶。 “激活的CDC42相关激酶”(AcK1/TNK2)。第二个基因是病毒2，编码人类的同源基因。 Wiskott Aldrich综合征蛋白(N-WASP)。ORV是一种无包膜的正义RNA病毒；在病毒中 有人类病原体的家族中，ORV与短小病毒科、杯状病毒科和星状病毒科关系最密切。删除 在人A549细胞中应用TNK2或N-WASP可使脑心肌炎病毒滴度降低约1000倍 (EMCV)。在TNK2缺失的细胞中，柯萨奇病毒B3(CVB3)的感染也减少了约100倍。令人惊讶的是， N-WASP是TNK2激酶活性的已知底物，表明这些基因可能包括一个 病毒利用的关键途径。为了支持这种可能性，线虫中的第三个基因nck-1，即 与TNK2和N-WASP结合的人NCK蛋白的同源蛋白也被发现对 ORV感染。TNK2以前没有被认为与病毒感染有关，它代表了一种新的哺乳动物 在线虫遗传筛选中发现的亲病毒宿主因子。其目的是：1)利用线虫作为一种 发现引擎，全面识别和表征对ORV感染至关重要的宿主因素。2)获取 TNK2和N-WASP基因对EMCV和CVB3感染阶段影响的机制研究 并将定义其亲病毒功能所需的基本蛋白结构域和底物。在……里面 此外，在Aim 1中确定的具有人类同源基因的线虫候选基因中，有一个子集 表观复制sid-3和病毒2是最有可能在同一途径中起作用的候选病毒，将被优先考虑 进行类似的分析。3)小鼠TnK2缺失对EMCV和CVB3感染的影响 已定义。这一提议将对病毒所必需的基本宿主基因和途径产生新的见解 感染，这可能是抗病毒开发的新靶点。

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.