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

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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.