ac4C stabilizes a G-quadruplex to enforce HBV pgRNA clearance

Using ac4C-RIP-seq, CLIP-seq, and RedaC:T-amplicon sequencing, we identify an N4-acetylcytidine (ac4C) RNA modification-dependent pathway that targets HBV pgRNA for decay. We find that ac4C modifications, deposited by NAT10, stabilize a G-quadruplex (G4) structure within pgRNA.

This ac4C-stabilized G4 structure is recognized and bound by two RNA-binding proteins, hnRNPH1 and GRSF1. hnRNPH1 binding destabilizes the G4 and accelerates pgRNA decay, while GRSF1 directs the RNA to mitochondria for clearance by the RNA degradosome (PNPase/hSUV3). The viral HBx protein upregulates NAT10, forming a negative feedback loop that restricts HBV replication. Together, our findings reveal a multilayered host surveillance mechanism that eliminates viral pgRNA via RNA modification and structural elements.