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.

项目概要 许多新出现的人畜共患病毒（传播给人类的动物病毒）具有高致病性，具有 人们强调了人畜共患病毒造成致命流行病甚至全球大流行的可能性。 SARS/MERS 冠状病毒、埃博拉病毒和 HIV-1 的出现，所有这些病毒都与动物有关 考虑到动物的风险，这些病毒在引起人类大量疾病之前是未知的。 由于病毒对人类构成威胁，许多研究人员已转向病毒发现——使用基因组测序工具和 通过宏基因组分析，研究人员希望在新的动物病毒出现在人类身上之前识别出它们。 这对于我们了解动物病毒生态学的努力将证明是无价的，我寻求通过以下方式扩展这项工作 通过实验评估动物病毒造成的人畜共患风险。该提案的关键如下。 动物病毒在人类细胞中复制的实验评估对于评估人畜共患风险至关重要。 在人类细胞中复制受到阻碍的动物病毒将具有与动物病毒相同的表型 面对二十个区块：对人体细胞产生低滴度或不产生滴度，但这些之间存在关键区别。 两种情况——一种动物病毒面对一个区块会带来更大的人畜共患风险，因为它需要更少的适应性 在这里，我建议开发并展示一个实验管道，该管道可以在人类细胞中复制。 将区分在人类细胞中复制受到很少阻碍的动物病毒和受到许多阻碍的动物病毒。 宿主遗传学在病毒的物种特异性中发挥着至关重要的作用。 对于病毒进入（细胞受体）、复制（细胞辅助因子）和抗病毒免疫（限制因子）可以 利用假设驱动的研究和高水平的研究，可以作为新宿主物种中病毒感染的有效屏障。 通过吞吐量基因组方法，我将系统地描述这种宿主-病毒相互作用景观 在目标 1 中，我将评估不同灵长类病毒家族的兼容性。 simarteriviruses 及其细胞受体 CD163 的人类版本 在目标 2 中，我将使用 CRISPR。 筛选以确定 simarterivirus 复制所需的宿主辅因子 然后，我将使用 的进化特征。 积极的自然选择来识别那些可能以物种特异性方式与 simarterivirus 结合的宿主蛋白 在目标 3 中，我将使用一系列基因来识别阻止 simarterivirus 复制的抗病毒蛋白。 敲低和互补，我将评估 simarterivirus 在人类中复制的遗传障碍 总而言之，这些目标将建立一个评估人畜共患病的实验框架。 未充分研究的动物病毒的风险，并将为我提供新的培训：1）表演和写作 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，定义为对感兴趣的表型进行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.