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 have shown that some of the HBV spliced transcript isoforms are predictive of liver cancer, yet the roles of these spliced transcripts remain elusive. Furthermore, there are nine major HBV genotypes common in different regions of the world, these genotypes may express different spliced transcript isoforms. To systematically study the HBV splice variants, we transfected human hepatoma cells, Huh7, with four HBV genotypes (A2, B2, C2 and D3), followed by deep RNA-sequencing. We found that 13–28 % of HBV RNAs were splice variants, which were reproducibly detected across independent biological replicates. These comprised 6 novel and 10 previously identified splice variants. In particular, a novel, singly spliced transcript was detected in genotypes A2 and D3 at high levels. The biological relevance of these splice variants was supported by their identification in HBV-positive liver biopsy and serum samples, and in HBV-infected primary human hepatocytes. Interestingly the levels of HBV splice variants varied across the genotypes, but the spliced pregenomic RNA SP1 and SP9 were the two most abundant splice variants. Counterintuitively, these singly spliced SP1 and SP9 variants had a suboptimal 5′ splice site, supporting the idea that splicing of HBV RNAs is tightly controlled by the viral post-transcriptional regulatory RNA element.
Highlights
Hepatitis B virus is a common human pathogen that is a major cause of liver cirrhosis and liver cancer
Hepatitis B virus (HBV) genotypes A to D expressed a large proportion of spliced transcript isoforms, representing
Our study has used RNA-seq to identify the type and proportion of splice variants produced for HBV genotypes A2, B2, C2 and D3, with a higher degree of sensitivity than 454 sequencing and conventional reverse-transcription PCR [13, 14, 21, 24], all of which require an intervening step to generate cDNA, followed by PCR
Summary
Hepatitis B virus is a common human pathogen that is a major cause of liver cirrhosis and liver cancer. The genomic DNA of HBV is approximately 3.2kb, but can be transcribed into the greater than genome length pregenomic RNA (pgRNA) and preC RNA (pcRNA) [1]. The pgRNA encodes the core (C) and polymerase (P) proteins, whereas the pcRNA encodes the pre-core (PC) protein that is subsequently processed into the Hepatitis B e Antigen (HBeAg). The HBV genome is transcribed into several subgenomic transcripts, namely the preS1, preS2, S, and X mRNAs. The preS1, S2, and S mRNAs encode the 3 surface (S) structural proteins of the HBV particles and subviral particles (HBsAg). The X mRNA encodes the HBx protein
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