NAT10-dependent N⁴-acetylcytidine modification mediates PAN RNA stability, KSHV reactivation, and IFI16-related...

N-acetyltransferase 10 (NAT10) is an N⁴-acetylcytidine (ac⁴C) writer that catalyzes RNA acetylation at cytidine N⁴ position on tRNAs, rRNAs and mRNAs. Recently, NAT10 and the associated ac⁴C have been reported to increase the stability of HIV-1 transcripts. Here, we show that NAT10 catalyzes ac⁴C addition to the polyadenylated nuclear RNA (PAN), a long non-coding RNA encoded by the oncogenic DNA virus Kaposi's sarcoma-associated herpesvirus (KSHV), triggering viral lytic reactivation from latency. Mutagenesis of ac⁴C sites in PAN RNA in the context of KSHV infection abolishes PAN ac⁴C modifications, downregulates the expression of viral lytic genes and reduces virion production. NAT10 knockdown or mutagenesis erases ac⁴C modifications of PAN RNA and increases its instability, and prevents KSHV reactivation. Furthermore, PAN ac⁴C modification promotes NAT10 recruitment of IFN-γ-inducible protein-16 (IFI16) mRNA, resulting in its ac⁴C acetylation, mRNA stability and translation, and eventual inflammasome activation. These results reveal a novel mechanism of viral and host ac⁴C modifications and the associated complexes as a critical switch of KSHV replication and antiviral immunity.