GEOapplication/xmlftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE330nnn/GSE330506/primaryOK200Other Mus musculusHomo sapiensOtherhttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE330506GEOGSEfalseac4C stabilizes a G-quadruplex to enforce HBV pgRNA clearanceHepatitis B virus (HBV) pregenomic RNA (pgRNA) is essential for viral replication, but the host factors that degrade it remain poorly understood. 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. The ac4C-G4-GRSF1 axis represents a potential therapeutic target for HBV cure.2026/05/13GSE330506GSM9727870GSM9727850GSM9727872GSM9727871GSM9727874GSM9727852GSM9727873GSM9727851GSM9727876GSM9727854GSM9727853GSM9727875GSM9727878GSM9727856GSM9727855GSM9727877GSM9727858GSM9727879GSM9727857GSM9727838GSM9727859GSM9727837GSM9727839GSM9727880GSM9727861GSM9727860GSM9727841GSM9727863GSM9727862GSM9727840GSM9727865GSM9727843GSM9727864GSM9727842GSM9727867GSM9727845GSM9727844GSM9727866GSM9727847GSM9727869GSM9727868GSM9727846GSM9727849GSM972784820301155201641729480330506 Mus musculusHomo sapiens