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

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, 2 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. IMPORTANCEHBV infection affects over 257 million people worldwide. HBV is a major cause of liver diseases including cancer and there is no cure. Some HBV RNAs are spliced variants and their roles are largely unclear, although some splice variants have been previously found to be associated with liver cancer. HBV exists as 9 genotypes worldwide with marked differences in replicative capacity and disease sequelae. Whether HBV splice variants vary for the different genotypes is yet to be investigated in depth. Here we sequenced RNAs from 4 major HBV genotypes using a cell culture system. We found 6 new and 6 previously known splice variants across these genotypes. Some novel splice variants were present at high levels, suggesting they could be functionally important. Interestingly, although HBV has adapted to human hosts for over 50,000 years, the most frequently spliced location shared little flanking sequence similarity with that of humans.