Conditional replication and secretion of hepatitis B virus genome uncover the truncated 3' terminus of encapsidated viral pregenomic RNA.

Hepatitis B virus (HBV) pregenomic RNA (pgRNA) is packaged into capsid where reverse transcription takes place to synthesize viral DNA genome, and the encapsidated pgRNA is the predominant species of serum HBV RNA in patients as a serological biomarker. In this study, by utilizing various conditional HBV replication and secretion systems, we analyzed the intracellular and extracellular capsid pgRNA and revealed that the 3' terminus of capsid pgRNA is scatteredly distributed between DR2 and poly(A) tail, except that the viral polymerase priming-defective mutant Y63D retained the sequence upstream of 3' DR1. Mechanistically, the heterogeneity of capsid RNA 3' terminus is due to the endogenous viral RNaseH activity during reverse transcription and exogenous MNase digestion during capsid RNA isolation; cellular ribonucleases may also participate in this process as the Y63D pgRNA 3' terminus in the immunoprecipitated capsid without prior MNase treatment remains truncated into 3' DR1. The major pgRNA splicing variant 1 of 2.1 kb and the artificial 3' DR1 and ε deletion mutants also possess a truncated 3' end of capsid RNA, indicating that the underrepresentation of the 3' end of encapsidated pgRNA is independent of pgRNA length or 3' terminal sequences. Altogether, our study suggests that the 3' region of HBV capsid pgRNA downstream of 3' DR1 is likely positioned outside of the capsid or loosely encapsulated, and thus is ribonuclease accessible. Furthermore, the detailed features of capsid pgRNA 3' terminus will shed light on HBV pgRNA encapsidation and reverse transcription and aid the development of better diagnostics of serum HBV RNA. IMPORTANCE The biogenesis and clinical application of serum HBV pgRNA have been a research hotspot in recent years. This study further characterized the heterogeneity of the 3' terminus of capsid RNA by utilizing a variety of experimental systems conditionally supporting HBV genome replication and secretion, and reveal that the 3' truncation of capsid pgRNA is catalyzed by cellular ribonuclease(s) and viral RNaseH at positions after and before 3' DR1, respectively, indicating the 3' DR1 as a boundary between the encapsidated portion of pgRNA for reverse transcription and the 3' unprotected terminus, which is independent of pgRNA length and the 3' terminal sequence. Thus, our study provides new insights into the mechanism of pgRNA encapsidation and reverse transcription, as well as the optimization of serum HBV RNA diagnostics.