Project Summary/Abstract Host transfer RNAs are cleaved into fragments during infection by diverse bacteria and viruses, yet what triggers this molecular event and how tRNA fragments (tRFs) impact infection remain poorly understood. This proposal leverages powerful molecular, genetic, and biochemical approaches during gammaherpesvirus infection to profile differential tRNA cleavage and to assess how tRNA cleavage and/or the resulting tRFs regulate the antiviral response. The overall objective in this proposal is to profile tRFs in infected primary cells and define whether tRNA cleavage or tRFs modulate gammaherpesvirus infection. This work explores the central hypothesis that tRNAs are cleaved in response to viral sensing and that gene regulatory functions of tRFs further establish an antiviral state. To test this hypothesis, Aim 1 employs a highly innovative sequencing strategy to accurately sequence tRNAs and their derivatives induced by gammaherpesvirus infection in primary, immune-competent cells. This aim will identify abundant, and likely functionally relevant, infection- induced tRFs, as well as introduce a sequencing tool with broad applicability to the tRF field. Aim 2 tests the hypothesis that tRF biogenesis is part of the host antiviral response by assessing gammaherpesvirus replication in response to decreased or increased expression of tRFs or tRNA endonucleases. These experiments will result in fundamental knowledge explaining why tRFs are generated in response to diverse viruses and reveal broad potential for tRNA-based antiviral therapeutic strategies. The work proposed here is significant, as it will pioneer the concept of antiviral tRFs, introduce a widely applicable sequencing strategy, and will ultimately reveal novel tRNA-centric nodes of gene expression control that can be manipulated to prevent and treat infectious disease.