Investigating virus-host interactions with prime editing and genetic code expansion

项目总结 宿主蛋白在所有病毒的复制中起着至关重要的作用。这代表了一个漏洞，可能 有可能被利用来开发针对目前在人类中传播的病毒的干预措施，以及新的 以及新出现的病毒威胁。然而，实现这一潜力需要机械性地理解有多具体 宿主因素在病毒感染过程中起作用。这项提案将开发一种通用的筛查方法来 研究宿主蛋白促进病毒复制的机制。这将通过以下方式实现 基因组工程和化学生物学的最新进展，使宿主能够进行定点修饰 含有非天然氨基酸的因子。通过基因重新编码与复制有关的&gt；100宿主蛋白 甲型流感病毒(IAV)，我们将研究(1)耗尽特定宿主因子对病毒复制的影响 以及病毒蛋白的亚细胞定位；(2)病毒感染对其表达和亚细胞的影响 宿主蛋白的定位；(3)宿主因子耗竭对细胞的组装和形态发生的影响。 新的病毒颗粒。如果成功，该项目将提供对IAV宿主依赖性的洞察，并建立 研究宿主蛋白在额外的、进化分化的复制中的功能的资源 呼吸道病毒。

PROJECT SUMMARY Host proteins play essential roles in the replication of all viruses. This represents a vulnerability that could potentially be exploited to develop interventions against viruses currently circulating in humans as well as new and emerging viral threats. However, realizing this potential requires mechanistic understanding of how specific host factors function during viral infection. This proposal will develop a versatile screening methodology to investigate the mechanisms through which host proteins enhance virus replication. This will be achieved using recent developments in genome engineering and chemical biology that enable site-specific modification of host factors with unnatural amino acids. By genetically re-coding >100 host proteins implicated in the replication of influenza A virus (IAV), we will investigate (1) the effects of depleting specific host factors on virus replication and the subcellular localization of viral proteins; (2) the effects of viral infection on the expression and subcellular localization of host proteins; and (3) the effects of host factor depletion on the assembly and morphogenesis of new virus particles. If successful, this project will provide insight into the host dependency of IAV and establish a resource for investigating host protein function in the replication of additional, evolutionarily-divergent respiratory viruses.