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

使用新型高通量 CRISPR/Cas9 筛选方法阐明针对多种免疫缺陷病毒的抗病毒防御。

• 批准号: 10324563
• 负责人: Daniel Poston
• 金额: $ 5.18万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10324563
• 关键词: Address; Animals; Anti-Retroviral Agents; Antiviral Agents; Biological Assay; Biological Models; CRISPR screen; CRISPR/Cas technology; Candidate Disease Gene; Cells; Co-Immunoprecipitations; Data; Emerging Communicable Diseases; Epidemic; Future; Genes; Goals; HIV; HIV-2; Health; Host Defense; Human; Human Cell Line; Immunologic Deficiency Syndromes; Individual; Infection; Infectious Agent; Innate Immune System; Interferon Type I; Interferons; Macaca; Mediating; Methods; Monitor; Natural Immunity; Play; Primate Lentiviruses; Primate Retrovirus; Production; Proteins; Publishing; Resistance; Role; SIV; Screening procedure; System; TRIM5 gene; Tropism; Viral; Virus; Virus Diseases; Virus Replication; Work; Zoonoses; base; cofactor; cross-species transmission; design; experimental study; global health; insight; nonhuman primate; novel; pathogen; pathogenic virus; screening; simian human immunodeficiency virus; tool; transmission process; viral transmission

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.

项目摘要 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，对人类健康构成重大和日益严重的威胁。虽然宿主先天 免疫-即产生I型干扰素（IFN）和随后产生干扰素 具有抗病毒活性的刺激基因（ISGS）在决定病毒的嗜性和限制性方面起着重要作用 由于病毒的跨物种传播，在识别和表征ISG限制方面仍有大量工作要做， 病毒向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）能够至少部分逃避细胞中IFN介导的防御 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。以来 HIV-1和HIV-2都是由不同SIV物种（人类和灵长类动物）的跨物种传播引起的 免疫缺陷病毒是研究宿主先天免疫与免疫缺陷之间关系的丰富模型系统。 和病毒的适应。为了鉴定抑制SIV感染的人类基因，我们最近修改了一个 描述了高通量的基于CRISPR的筛选测定，以鉴定具有抗肿瘤活性的人ISG。 SIVmac239.我们的初步数据表明，我们的模型系统正在按设计工作，正如已经确定的那样， 在人类细胞中抑制SIVmac感染的候选基因的数量。在目标1中，我们将确定 具有确认的抗SIVmac 239活性的新型ISG的作用机制。我们将逐步执行 机制研究，以剖析病毒生命周期中的抑制点。我们还将共同努力， 免疫沉淀和共定位研究，以鉴定病毒组分和与 抑制机制。在目标2中，我们将使用已建立的管道筛选抑制多种 SIV毒株。初步数据强烈表明，我们将能够直接使用我们开发的系统 来筛选不同的SIV毒株我们将评估多种SIV毒株对IFN的敏感性， 对抑制性ISG敏感。我们假设，将有保守和独特的抑制因子， 针对不同种类的SIV结合起来，这些方法将揭示新的限制因素，抑制 人类细胞中的多种SIV，提供了对HIV-1和HIV-2在进化过程中所产生的进化适应的深入了解。 为了成功地殖民人类。