Lactylation of NAT10 promotes N⁴‐acetylcytidine modification on tRNA^Ser-CGA-1-1 to boost oncogenic DNA virus KSHV reactivation

N⁴-acetylcytidine (ac⁴C), a conserved but recently rediscovered RNA modification on tRNAs, rRNAs and mRNAs, is catalyzed by N-acetyltransferase 10 (NAT10). Lysine acylation is a ubiquitous protein modification that controls protein functions. Our latest study demonstrates a NAT10-dependent ac⁴C modification, which occurs on the polyadenylated nuclear RNA (PAN) encoded by oncogenic DNA virus Kaposi’s sarcoma-associated herpesvirus (KSHV), can induce KSHV reactivation from latency and activate inflammasome. However, it remains unclear whether a novel lysine acylation occurs in NAT10 during KSHV reactivation and how this acylation of NAT10 regulates tRNAs ac⁴C modification. Here, we showed that NAT10 was lactylated by α-tubulin acetyltransferase 1 (ATAT1), as a writer at the critical domain, to exert RNA acetyltransferase function and thus increase the ac⁴C level of tRNA^Ser-CGA-1-1. Mutagenesis at the ac⁴C site in tRNA^Ser-CGA-1-1 inhibited its ac⁴C modifications, translation efficiency of viral lytic genes, and virion production. Mechanistically, KSHV PAN orchestrated NAT10 and ATAT1 to enhance NAT10 lactylation, resulting in tRNA^Ser-CGA-1-1 ac⁴C modification, eventually boosting KSHV reactivation. Our findings reveal a novel post-translational modification in NAT10, as well as expand the understanding about tRNA-related ac⁴C modification during KSHV replication, which may be exploited to design therapeutic strategies for KSHV-related diseases.