Recent discoveries on small non-coding RNAs (sncRNAs) have significantly advanced human genetics and molecular biology, largely due to the identification of a fundamental role of sncRNAs as gene regulators. tRNA- derived RNA Fragments (tRFs) represent a recently discovered sncRNA family that is quickly being recognized as ubiquitously expressed in organisms ranging from prokaryotes to humans; however, tRFs' biological functions and the mechanism(s) underlying them are largely unknown. Respiratory syncytial virus (RSV) is the single most common cause of lower respiratory tract illness in infants. Our recent publication has shown that the sncRNAs most highly induced by RSV belong to the tRF family. Notably, at least the two most abundant RSV-inducible tRFs have a gene trans-silencing function that is mechanistically distinct from that of miRNAs, and one of these two, called tRF5-GluCTC, promotes RSV replication and regulates genes at a post-transcriptional level. The biogenesis of this tRF is also specific, and mediated by a particular endonuclease (angiogenin, ANG), and not by other nucleases. However, the molecular mechanisms underlying the biological functions and induction of tRF5-GluCTC are not known. Our central hypothesis in this project is that the RSV-induced tRF5-GluCTC is not a random by-product of tRNA degradation, but rather a functional molecule important for host-RSV interactions. This project will focus on exploring the molecular mechanisms underlying the regulatory effects of tRF5-GluCTC on RSV replication, and the host responses thereto. Our experiments will identify the targets of tRF5-GluCTC in RSV- infected airway epithelial cells (Aim 1), determine the viral factor(s) controlling the trans-silencing activity of tRF5- GluCTC (Aim 2), and define the viral component(s) contributing to ANG-mediated biogenesis of tRFs (Aim 3). Preliminary data suggest that the RSV proteins N and P will be a focus of Aims 2 and 3, respectively. The overall goal of this project is to use a combination of molecular virology, protein and RNA biochemistry, and cellular and structural biology techniques to elucidate tRF-mediated regulatory mechanisms for both RSV replication and the host responses it elicits. This novel regulation should provide a key new perspective on RSV-host interactions and undoubtedly facilitate the discovery of new gene regulatory mechanisms in response to RSV infection. Our results on antiviral target identification, targeting mechanisms, and tRF biogenesis will also provide new insights important for the design of preventive and therapeutic strategies for RSV infection. Given the early stage of studies on tRFs, the knowledge and techniques obtained in this study on tRF5-GluCTC will be important for exploring the functions of other tRFs in various biological systems, and therefore will broadly benefit the research community.