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.

N ⁴ -乙酰胞苷 (ac ⁴ C) 是一种保守但最近重新发现的 tRNA、rRNA 和 mRNA 上的 RNA 修饰，由N -乙酰转移酶 10 (NAT10) 催化。赖氨酸酰化是一种普遍存在的控制蛋白质功能的蛋白质修饰。我们的最新研究表明，NAT10 依赖性 ac ⁴ C 修饰发生在致癌 DNA 病毒卡波西肉瘤相关疱疹病毒 (KSHV) 编码的聚腺苷酸化核 RNA (PAN) 上，可以诱导 KSHV 从潜伏期重新激活并激活炎症小体。然而，目前尚不清楚 KSHV 重新激活期间 NAT10 中是否发生新的赖氨酸酰化，以及 NAT10 的这种酰化如何调节 tRNA ac ⁴ C 修饰。在这里，我们发现 NAT10 作为关键域的写入者被 α-微管蛋白乙酰转移酶 1 (ATAT1) 乳酰化，发挥 RNA 乙酰转移酶功能，从而提高 tRNA ^Ser-CGA-1-1的 ac ⁴ C 水平。 tRNA ^Ser-CGA-1-1中 ac ⁴ C 位点的诱变抑制了其 ac ⁴ C 修饰、病毒裂解基因的翻译效率和病毒颗粒的产生。从机制上讲，KSHV PAN 协调 NAT10 和 ATAT1 以增强 NAT10 乳酰化，导致 tRNA ^Ser-CGA-1-1 ac ⁴ C 修饰，最终促进 KSHV 重新激活。我们的研究结果揭示了 NAT10 中的一种新的翻译后修饰，并扩大了对 KSHV 复制过程中 tRNA 相关的 ac ⁴ C 修饰的理解，这可用于设计 KSHV 相关疾病的治疗策略。