NAT10-dependent N(4)‐acetylcytidine modification mediates PAN RNA stability, KSHV reactivation, and IFI16-related inflammasome activation

N-acetyltransferase 10 (NAT10) is an N(4)‐acetylcytidine (ac(4)C) writer that catalyzes RNA acetylation at cytidine N(4) position on tRNAs, rRNAs and mRNAs. Recently, NAT10 and the associated ac(4)C have been reported to increase the stability of HIV-1 transcripts. Here, we show that NAT10 catalyzes ac(4)C 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 ac(4)C sites in PAN RNA in the context of KSHV infection abolishes PAN ac(4)C modifications, downregulates the expression of viral lytic genes and reduces virion production. NAT10 knockdown or mutagenesis erases ac(4)C modifications of PAN RNA and increases its instability, and prevents KSHV reactivation. Furthermore, PAN ac(4)C modification promotes NAT10 recruitment of IFN-γ-inducible protein-16 (IFI16) mRNA, resulting in its ac(4)C acetylation, mRNA stability and translation, and eventual inflammasome activation. These results reveal a novel mechanism of viral and host ac(4)C modifications and the associated complexes as a critical switch of KSHV replication and antiviral immunity.