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

作者：Sheng Shen, Wendong Liu, Zeng Ge, Hongyan Liang, Xiaoyang Yu, Hu Zhang, Jian Sun, Haitao Guo · 发表于：Journal of Virology · 年份：2023 · DOI：10.1128/jvi.00760-23 · 被引用次数：9 · 研究领域：Hepatitis B Virus Studies、Hepatitis C virus research、RNA Interference and Gene Delivery

ABSTRACT 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 f...