Quantitative analysis of the splice variants expressed by the major hepatitis B virus genotypes

Abstract

Hepatitis B virus (HBV) is a major human pathogen that causes liver diseases. The main HBV RNAs are unspliced transcripts that encode the key viral proteins. Recent studies show that some of the HBV spliced transcript isoforms are predictive of liver cancer, yet the roles of these spliced transcripts remain elusive. Furthermore, a total of 9 major HBV genotypes were isolated from discrete geographical regions of the world, it is likely that these genotypes may express a broad variety of spliced transcript isoforms. To systematically study the HBV splice variants, we transfected the human hepatoma cells Huh7 with 4 HBV genotypes (A2, B2, C2, and D3), followed by deep RNA-sequencing. We found that 12-25% of HBV RNAs were splice variants, which were reproducibly detected across independent biological replicates. This accounted for a total of 6 novel and 6 previously identified splice variants. In particular, two highly abundant novel splice variants, in which we called the putative splice variants 1 and 5 (pSP1 and pSP5), were specifically expressed at high levels in genotypes D3 and B2, respectively. In general, the HBV splicing profiles varied across the genotypes except for the known spliced pgRNAs SP1 and SP9, which were present in all 4 major genotypes. Counterintuitively, these singly spliced SP1 and SP9 had a suboptimal 5′ splice site, suggesting that splicing of HBV RNAs is tightly controlled by the viral post-transcriptional regulatory RNA element.

Surrogate Markers for Hepatitis B Virus Covalently Closed Circular DNA

“…116 118 Additionally, over 20 splice variants of the pgRNA 111 115 116 119 120 and preS2/S mRNA 112 have been described to be present in the supernatant of transfected cell lines 115 and in patient sera. 115 121 122 123 The quasispecies of serum HBV RNA have been shown to reflect the genomic sequence of cccDNA, 111 adding to the argument that serum HBV RNA could be an accurate surrogate marker for cccDNA.…”