Elucidating antiviral defenses against diverse immunodeficiency viruses using a novel, high-throughput CRISPR/Cas9 screening method.

项目总结 病毒病原体从动物到人类的人畜共患传播导致了大多数新出现的 传染病，这些疾病对人类健康构成重大和日益严重的威胁。而宿主天生就是 免疫--即I型干扰素的产生和随后干扰素的产生 具有抗病毒活性的刺激基因(ISG)在确定病毒的趋向性和限制性方面发挥着重要作用 病毒的跨物种传播，在识别和表征限制ISGs方面仍有大量工作要做 病毒嗜性。我们的实验室已经确定，虽然人类免疫缺陷病毒(HIV-1)和非人类 灵长类猴免疫缺陷病毒(SIV)至少能够部分逃避细胞内干扰素介导的防御 从它们的同源宿主来看，它们对来自非自然宿主的细胞中的这种防御仍然非常敏感。自.以来 HIV-1和HIV-2都是由人类和灵长类不同SIV物种的跨物种传播引起的 免疫缺陷病毒是研究宿主先天免疫关系的一个丰富的模型系统 和病毒适应。为了确定抑制SIV感染的人类基因，我们最近修改了一种 描述了一种高通量、基于CRISPR的筛选方法，用于识别具有抗病毒活性的人ISGs SIVmac239。我们的初步数据表明，我们的模型系统正在按设计工作，正如已经确定的 抑制人类细胞中SIVmac感染的候选基因的数量。在目标1中，我们将确定 已证实具有抗SIVmac239活性的新型ISGs的作用机制。我们将循序渐进地表演 剖析病毒生命周期中抑制点的机制研究。我们还将联合演出- 免疫沉淀和共定位研究以确定与病毒相关的成分和细胞辅助因子 抑制机制。在目标2中，我们将使用我们建立的渠道来筛选抑制多样性的ISGs SIV毒株。初步数据有力地表明，我们将能够直接使用我们开发的系统 以筛选不同的SIV毒株。我们将评估多种SIV毒株对干扰素的敏感性，并对那些 对抑制性ISG敏感。我们假设既有保守的也有明显的抑制因子 目标是各种SIV物种。这些方法结合在一起，将揭示抑制 人类细胞中不同的SIV，为HIV-1和HIV-2在进化中的适应提供了洞察 才能成功地殖民人类。

PROJECT SUMMARY The zoonotic transmission of viral pathogens from animals to humans contributes to the majority of emerging infectious diseases, which pose a substantial and increasing threat to human health. While host innate immunity—namely the production of Type I Interferon (IFN) and the subsequent production of Interferon Stimulated Genes (ISGS) with antiviral activities—plays an important role in determining viral tropism and limiting the cross-species transmission of viruses, much work remains on identifying and characterizing ISGs limiting viral tropism. Our lab has determined that while the human immunodeficiency virus (HIV-1) and non-human primate simian immunodeficiency viruses (SIV) are able to at least partly evade IFN-mediated defenses in cells from their cognate host, they remain exquisitely sensitive to such defenses in cells from unnatural hosts. Since HIV-1 and HIV-2 both arose from the cross-species transmission of diverse SIV species, the human and primate immunodeficiency viruses are a rich model system for studying the relationship between host innate immunity and viral adaptation. In order to identify human genes inhibiting SIV infection, we have modified a recently described high-throughput, CRISPR-based screening assay to identify human ISGs with activity against SIVmac239. Our preliminary data suggests that our model system is working as designed, as had identified a number of candidate genes that inhibit SIVmac infection in human cells. In Aim 1, we will determine the mechanism of action of novel ISGs with confirmed activity against SIVmac239. We will perform stepwise mechanistic studies to dissect the point of inhibition in the viral lifecycle. We will additionally perform co- immunoprecipitation and co-localization studies to identify viral components and cellular cofactors relevant to mechanism of inhibition. In Aim 2, we will use our established pipeline to screen for ISGs inhibiting diverse SIV strains. Preliminary data strongly suggests that we will be able to directly use the system we have developed to screen diverse strains of SIV. We will assess multiple SIV strains for sensitivity to IFN and screen those that are sensitive for inhibitory ISGs. We hypothesize that there will be both conserved and distinct inhibitory factors targeting the various SIV species. Combined, these approaches will reveal novel restriction factors that inhibit diverse SIVs in human cells, providing insight into the evolutionary adaptations that HIV-1 and HIV-2 made in order to successfully colonize humans.