tRNA-derived fragments restrict KSHV lytic replication through miRNA-like repression of host pro-viral signalling networks

Transfer RNA derived fragments (tRFs) are emerging regulators of gene expression. Despite mounting evidence of their functional relevance in cellular and pathological processes, their roles in virus infection remain poorly understood. Here, we show that host tRFs are dynamically reprogrammed during Kaposi sarcoma-associated herpesvirus (KSHV) lytic replication and function as potent antiviral effectors. Transcriptome-wide profiling revealed extensive remodelling of the tRF landscape following viral reactivation, identifying a subset of tRFs; tRFs (tRF-His-GTG, tRF-Gly-CCC, and tRF-Lys-CTT) that are robustly induced independently of parental tRNA abundance. Functional analyses demonstrate that these tRFs suppress viral gene expression and infectious virion production. Mechanistically, we show that tRF-His-GTG and tRF-Lys-CTT engage canonical RNA silencing pathways by associating with AGO2 complexes and mediating sequence-specific repression of host target genes. Among these targets, we identify SHF as a critical pro-viral factor that promotes PI3K/Akt signalling, cellular survival and efficient viral replication. Collectively, our findings define a previously uncharacterised host defence pathway in which tRFs restrict KSHV replication by targeting host factors required for efficient viral propagation, thus expanding the functional repertoire of non-coding RNAs in antiviral immunity.