Haploid genetic screen to identify host genes required for RNA virruses

RNA病毒以极高的突变率复制，使它们能够迅速对直接针对病毒蛋白质的常规药物产生耐药性。病毒的复制和传播主要依赖于细胞基因。因此，使用这些宿主基因作为药物靶点可以为耐药性的发展创造更高的屏障。为了鉴定宿主基因对各种不同的新出现或再出现的RNA病毒的感染至关重要，我们将使用一种创新的方法在人类细胞中进行遗传筛选。这种方法是基于独特的单倍体或近单倍体人类细胞系，其中可以大量创建敲除等位基因，从而创建一个基本上所有基因都发生突变的细胞文库。再加上深度测序，这使我们能够筛选人类基因组中对病毒复制至关重要的基因。我们已将该技术应用于鉴定埃博拉病毒所需的进入因子，发现NPC1是不同丝状病毒的细胞内受体。在这里，我们建议进行一系列的比较遗传筛选宿主基因所需的RNA病毒纲要，包括不同株甲型流感病毒，委内瑞拉马脑炎病毒。拉克罗斯脑炎病毒、黄热病病毒、西尼罗河病毒、甲型肝炎病毒和丙型肝炎病毒。这些筛选将产生候选基因，这些候选基因需要在相关的组织培养和动物模型中进行严格验证。为此，我们将开发aav - shrna - RNAi工具。这些将用于：1)验证宿主细胞候选基因鉴定和认为重要的病毒病原体的复制；2)比较宿主靶点敲除与宿主候选基因直接靶向病毒mrna的效果；3)从小分子抑制剂的多效性毒性中识别与候选蛋白直接敲低直接相关的小分子抑制剂的毒性。

RNA viruses replicate with extremely high mutation rates allowing them to quickly develop resistance to conventional drugs that directly target viral proteins. Viruses critically rely on cellular genes for their replication and spread. Therefore, using these host genes as drug targets could create a much higher barrier to the development of drug resistance. To identify host genes critical to infection of a wide range of different emerging or reemerging RNA viruses, we will use an innovative method for genetic screens in human cells. This approach is based on unique haploid or near-haploid human cell lines in which it is possible to create knockout alleles en masse creating a library of cells with mutations in essentially all genes. Coupled with deep sequencing this allows us to screen the human genome for genes that are critical for viral replication. We have applied this technique to identify entry factors required for Ebola virus and found that NPC1 is the intracellular receptor for different filoviruses. Here we propose to perform a series of comparative genetic screens for host genes required for a compendium of RNA viruses including different strains of influenza A virus, Venezuelan equine encephalitis virus. La Crosse encephalitis virus, yellow fever virus, West Nile virus, hepatitis A virus and hepatitis C virus. These screens will generate candidate genes that need to be rigorously validated in relevant tissue culture and animal models. For this we will develop AAV-shRNAs- RNAi tools. These will be used to: 1) validate host cell candidate genes identified and deemed important for the replication of viral pathogens; 2) compare efficacy of host target knockdown with direct targeting of viral mRNAs with host candidate genes; 3) identify the toxicity of small molecule inhibitors directly related to direct knockdown of the candidate protein from toxicity related to pleiotropic effects of the small molecule inhibitors.