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靶向病毒蛋白的常规药物产生耐药性。病毒严重依赖细胞基因进行复制和传播。因此，使用这些宿主基因作为药物靶标可以为耐药性的发展创造更高的屏障。为了鉴定对感染各种不同的新出现或重新出现的RNA病毒至关重要的宿主基因，我们将使用一种创新的方法在人类细胞中进行遗传筛选。该方法基于独特的单倍体或近单倍体人类细胞系，其中可以创建敲除等位基因，从而创建基本上所有基因都具有突变的细胞文库。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。我们已经应用这种技术来识别埃博拉病毒所需的进入因子，并发现NPC 1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所需的宿主基因的RNA病毒，包括不同株的流感病毒A，委内瑞拉马脑炎病毒的纲要。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，需要在相关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）验证所鉴定的并被认为对病毒病原体的复制重要的宿主细胞候选基因; 2）比较宿主靶标敲低与用宿主候选基因直接靶向病毒mRNA的功效; 3）从与小分子抑制剂的多效性效应相关的毒性中鉴定与候选蛋白质的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.