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Respiratory Syncytial Virus (RSV) is a significant cause of lower respiratory tract infections (LRTI) across all demographics, with increasing mortality and morbidity among high-risk groups such as infants under two years old, the elderly, and immunocompromised individuals. Although newly approved vaccines and treatments have substantially reduced RSV hospitalizations, accessibility remains limited, and response to treatment varies. This underscores the importance of comprehensive studies on host–RSV interactions. tRNA-derived RNA fragments (tRFs) are recently discovered non-coding RNAs, notable for their regulatory roles in diseases, including viral infections. Our prior work demonstrated that RSV infection induces tRFs, primarily derived from the 5′-end of a limited subset of tRNAs (tRF5), to promote RSV replication by partially targeting the mRNA of antiviral genes. This study found that tRFs could also use their bound proteins to regulate replication. Our proteomics data identified that PABPC1 (poly(A)-binding protein cytoplasmic 1) is associated with tRF5-GluCTC, an RSV-induced tRF. Western blot experimentally confirmed the presence of PABPC1 in the tRF5-GluCTC complex. In addition, tRF5-GluCTC is in the anti-PABPC1-precipitated immune complex. This study also discovered that suppressing PABPC1 with its specific siRNA increased RSV (-) genome copies without impacting viral gene transcription, but led to less infectious progeny viruses, suggesting the importance of PABPC1 in virus assembly, which was supported by its interaction with the RSV matrix protein. Additionally, PABPC1 knockdown decreased the production of the cytokines MIP-1α, MIP-1β, MCP-1, and TNF-α. This is the first observation suggesting that tRFs may regulate viral infection via their bound proteins.

tRNA-derived RNA fragments (tRFs) are a recently discovered family of small noncoding RNAs (sncRNAs). We previously reported that respiratory syncytial virus (RSV) infection induces functional tRFs, which are derived from a limited subset of parent tRNAs, in airway epithelial cells. Such induction is also observed in nasopharyngeal wash samples from RSV patients and correlates to RSV genome copies, suggesting a clinical significance of tRFs in RSV infection. This work also investigates whether the modification of parent tRNAs is changed by RSV to induce tRFs, using one of the most inducible tRFs as a model. We discovered that RSV infection changed the methylation modification of adenine at position 57 in tRNA glutamic acid, with a codon of CTC (tRNA-GluCTC), and the change is essential for its cleavage. AlkB homolog 1, a previously reported tRNA demethylase, appears to remove methyladenine from tRNA-GluCTC, prompting the subsequent production of tRFs from the 5′-end of tRNA-GluCTC, a regulator of RSV replication. This study demonstrates for the first time the importance of post-transcriptional modification of tRNAs in tRF biogenesis following RSV infection, providing critical insights for antiviral strategy development.