Resolution of viral R-loop by DHX9 promotes hepatitis B virus replication.

Hepatitis B virus (HBV) is a hepatotropic DNA virus with a compact 3.2 kb genome that replicates via reverse transcription. HBV covalently closed circular DNA (cccDNA) transcription shares molecular features with R-loop. DExH-box helicase 9 (DHX9) has been shown to be associated with R-loop accumulation, genome instability, and HBV replication. However, the role of DHX9-mediated R-loop homeostasis in HBV transcription has not yet been elucidated. In this study, we identified a critical role of DHX9 in resolving R-loops at transcription termination during HBV transcription. We demonstrate that HBV replication promotes viral R-loop accumulation. DHX9 promotes HBV R-loop resolution, HBV cccDNA transcription termination, and subsequent nuclear export of the 3.5 kb HBV RNA, facilitating HBV DNA replication. These findings reveal DHX9-mediated R-loop resolution as a potential therapeutic target for antiviral intervention.IMPORTANCEThe persistence of HBV cccDNA poses a major challenge to achieving a cure for chronic hepatitis B. While R-loop structures have been implicated in the life cycles of viruses, their formation and functional significance in HBV replication have remained largely unexplored. This study provides the first evidence that the cellular helicase DHX9 resolves transcription-replication conflicts by dissolving R-loops formed during HBV replication. Our findings also identify DHX9-mediated R-loop resolution as a critical mechanism supporting the HBV life cycle. Consequently, targeting DHX9 presents a promising therapeutic strategy to silence the persistent HBV cccDNA reservoir.