Helicases DDX5 and DDX17 promote Hepatitis B Virus transcription termination heterogeneity in infected human hepatocytes

Background & Aims: Transcription termination fine tunes gene expression and contributes to specify the function of RNAs in eukaryotic cells. Transcription termination of hepatitis B virus (HBV) is subjected to the recognition of the canonical polyadenylation signal (cPAS) common to all viral transcripts. The regulation of the usage of this cPAS and its impact on viral gene expression and replication is currently unknown. Approach & Results: To unravel the regulation of HBV transcript termination, we used a 3’ RACE-PCR assay together with single molecule sequencing both in in vitro infected hepatocytes and in chronically infected patients. The detection of a previously unidentified transcriptional readthrough indicated that the cPAS was not systematically recognized during HBV replication in vitro and in vivo. After gene expression downregulation, followed by RNA and chromatin immunoprecipitation experiments, we showed the role of the RNA helicases DDX5 and DDX17 in promoting viral transcriptional readthrough, which was, furthermore, associated to HBV RNA destabilization. Moreover, downregulation of DDX5 and DDX17 allowed the precise termination of HBV transcripts at cPAS, which was associated with increased viral replication. Conclusions: Our findings identify DDX5 and DDX17 as crucial determinants for HBV transcriptional fidelity and as host restriction factors for HBV replication.