Identifying genetic barriers to animal virus replication in human cells: Insights into zoonosis

识别人类细胞中动物病毒复制的遗传障碍：深入了解人畜共患病

• 批准号: 10461714
• 负责人: Cody Jay Warren
• 金额: $ 1.84万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-04 至 2022-08-02
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10461714
• 关键词: Address; Animals; Arterivirus; Biological; Blood; Blood Circulation; CRISPR screen; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Complement; Data; Data Analyses; Disease; Dominant Genes; Ebola virus; Ecology; Epidemic; Face; Family; Genes; Genetic; Genomic approach; Geographic Distribution; Goals; HIV-1; Health; High-Throughput Nucleotide Sequencing; Human; Infection; Integration Host Factors; Interferons; Investments; Knock-out; Membrane Glycoproteins; Metagenomics; Middle East Respiratory Syndrome Coronavirus; Molecular Evolution; Monkeys; Mutation; Natural Immunity; Natural Selections; Natural experiment; Nature; Orthologous Gene; Pathogenicity; Phenotype; Play; Primates; Process; Proteins; Public Health; Relative Risks; Research Personnel; Risk; Role; SARS coronavirus; SIV; Series; Severe Acute Respiratory Syndrome; Species Specificity; Testing; Training; Viral; Viral Load result; Virus; Virus Diseases; Virus Replication; Work; Writing; Zoonoses; antiviral immunity; cDNA Library; cofactor; cross-species transmission; functional genomics; genome analysis; genome sequencing; genome-wide; insight; interest; knock-down; macrophage; novel; pandemic disease; prevent; prospective; receptor; receptor binding; success; tool; transcription factor; transmission process; virus host interaction

Project Summary Many emerging zoonotic viruses (animal viruses that transmit to humans) are highly pathogenic, having the potential to cause deadly epidemics or even global pandemics. The risks zoonotic viruses pose are highlighted by the emergence of the SARS/MERS coronaviruses, Ebola virus, and HIV-1, all of which are related to animal viruses that were unknown before they caused substantial cases of disease in humans. Given the risk animal viruses pose to humans, many researchers have turned to viral discovery—using genome sequencing tools and metagenomic analyses, researchers hope to identify novel animal viruses before they emerge in humans. While such efforts will prove invaluable to our understanding animal virus ecology, I seek to expand this work by experimentally evaluating the zoonotic risk an animal virus poses. The crux of this proposal is as follows. Experimental assessment of animal virus replication in human cells is crucial to evaluate zoonotic risk. However, an animal virus facing one block to replication in a human cell will have the same phenotype as an animal virus facing twenty blocks: producing low or no titers on human cells. But there is a critical difference between these two scenarios—an animal virus facing one block poses a greater zoonotic risk because it requires fewer adaptive mutations to replicate in human cells. Here, I propose to develop and demonstrate an experimental pipeline that will distinguish between animal viruses facing few blocks to replication in human cells from those with many. Host genetics plays a critical role in the species specificity of viruses. Divergence in host proteins used for virus entry (cellular receptors), replication (cellular cofactors), and antiviral immunity (restriction factors) can serve as potent barriers to virus infection in a new host species. Using hypothesis-driven studies and high- throughput genomic approaches, I will systematically characterize this host-virus interaction landscape for an understudied family of primate viruses (simarteriviruses). In Aim 1, I will assess the compatibility of diverse simarteriviruses with the human version of their cellular receptor, CD163. In Aim 2, I will employ a CRISPR screen to identify host cofactors required for simarterivirus replication. Then, I will use evolutionary signatures of positive natural selection to identify those host proteins likely to engage simarteriviruses in a species-specific manner. In Aim 3, I will identify antiviral proteins that block simarterivirus replication. Using a series of gene knockdowns and complementations, I will evaluate the genetic barriers to simarterivirus replication in human cells (Aims 1-3). Taken together, these aims will establish an experimental framework to evaluate the zoonotic risk of an understudied animal virus, and will provide me with new training in 1) performing and writing about molecular evolution analyses, 2) developing high-throughput sequencing projects and data analysis, and 3) functional genomics, defined as the execution and analysis of genome-scale screens for phenotypes of interest.

项目摘要 许多新兴的人畜共患病毒（传播到人类的动物病毒）具有高度致病性，具有 引起致命流行病甚至全球流行病的潜力。人畜共患病毒姿势的风险突出显示 SARS/MERS冠状病毒，埃博拉病毒和HIV-1的出现都与动物有关 在引起人类疾病的大量病例之前未知的病毒。鉴于风险动物 病毒构成人类，许多研究人员转向病毒发现 - 使用基因组测序工具和 宏基因组分析，研究人员希望在人类出现之前鉴定出新型动物病毒。尽管 这样的努力对我们的理解动物病毒生态学将是无价的，我试图扩大这项工作 实验评估动物病毒位置的人畜共患风险。该提议的症结如下。 人类细胞中动物病毒复制的实验评估对于评估人畜共患病风险至关重要。然而， 在人类细胞中面临一个块复制的动物病毒将与动物病毒具有相同的表型 面对二十个块：在人类细胞上产生低或没有滴度。但是这些有关键的区别 两种情况 - 面向一个块的动物病毒带来更大的人畜共患风险，因为它需要更少的适应性 在人类细胞中复制的突变。在这里，我建议开发和演示一个实验管道， 将区分人类细胞中几乎没有块的动物病毒与许多人的复制。 宿主遗传学在病毒的规范中起关键作用。使用的宿主蛋白的发散 对于病毒进入（细胞接收器），复制（细胞辅助因子）和抗病毒免疫学（限制因子）可以 作为新宿主物种中病毒感染的潜在障碍。使用假设驱动的研究和高 吞吐量基因组方法，我将系统地表征此宿主病毒相互作用景观 研究的灵长类病毒家族（Simarterivirues）。在AIM 1中，我将评估潜水员的兼容性 simarteriviruses具有其细胞接收器的人类版本CD163。在AIM 2中，我将使用CRISPR 屏幕以识别Simarterivirus复制所需的主机辅助因子。然后，我将使用进化签名 积极的自然选择，以识别可能在规格特定的特定规格中参与Simarterivirus的宿主蛋白 方式。在AIM 3中，我将确定阻止Simarterivirus复制的抗病毒蛋白。使用一系列基因 敲低和完成，我将评估人类simarterivirus复制的遗传障碍 细胞（目标1-3）。综上所述，这些目标将建立一个实验框架来评估人畜共努 患动物病毒的风险，将为我提供1）表演和写作的新培训 分子进化分析，2）开发高通量测序项目和数据分析，以及3） 功能基因组学，定义为对感兴趣表型的基因组规模筛选的执行和分析。

项目成果

APOBEC3: Friend or Foe in Human Papillomavirus Infection and Oncogenesis?

• DOI: 10.1146/annurev-virology-092920-030354
• 发表时间: 2022-09-29
• 期刊: Annual review of virology
• 影响因子: 11.3

Quantification of virus-infected cells using RNA FISH-Flow.

• DOI: 10.1016/j.xpro.2023.102291
• 发表时间: 2023-05-18
• 期刊: STAR PROTOCOLS
• 作者: Warren, Cody J.;Barbachano-Guerrero, Arturo;Huey, Devra;Yang, Qing;Worden-Sapper, Emma R.;Kuhn, Jens H.;Sawyer, Sara L.
• 通讯作者: Sawyer, Sara L.