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

项目摘要 许多新出现的人畜共患病病毒（传播给人类的动物病毒）具有高致病性， 可能导致致命的流行病甚至全球性的流行病。强调了人畜共患病毒构成的风险 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， 这些病毒在引起大量人类疾病之前是未知的。鉴于风险动物 病毒对人类的危害，许多研究人员已经转向病毒的发现-使用基因组测序工具， 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之前识别它们。而 这些努力将证明对我们理解动物病毒生态学是非常宝贵的，我试图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。这项建议的要点如下。 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病风险至关重要。然而，在这方面， 在人类细胞中复制受阻的动物病毒将具有与动物病毒相同的表型 面对20块：在人体细胞上产生低滴度或不产生滴度。但这两者之间有一个关键的区别 两个障碍--一种动物病毒面对一个障碍会造成更大的人畜共患病风险，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， 将区分动物病毒在人类细胞中复制时遇到的障碍很少和有很多。 宿主遗传学在病毒的种属特异性中起着关键作用。所用宿主蛋白的差异 对于病毒进入（细胞受体）、复制（细胞辅因子）和抗病毒免疫（限制因子）， 在新的宿主物种中作为病毒感染的有效屏障。使用假设驱动的研究和高- 通过基因组方法，我将系统地描述这种宿主-病毒相互作用的景观， 未充分研究的灵长类病毒家族（simarteriviruses）。在目标1中，我将评估各种 simarteriviruses与人类版本的细胞受体，CD 163。在目标2中，我将使用CRISPR 筛选以鉴定simarterivirus复制所需的宿主辅因子。然后，我将使用进化的签名， 积极的自然选择，以确定那些宿主蛋白可能从事simarteriviruses在一个物种特异性 方式在目标3中，我将鉴定阻断simarterivirus复制的抗病毒蛋白。利用一系列的基因 敲除和互补，我将评估人类中simarterivirus复制的遗传障碍 细胞（目标1-3）。综合起来，这些目标将建立一个实验框架，以评估人畜共患病 他说：“这是一种未被充分研究的动物病毒的风险，并将为我提供新的训练，在表演和写作方面， 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的筛选和分析。

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.