Quantitative analysis of the splice variants expressed by the major hepatitis B virus genotypes
Chun Shen Lim 1 , Vitina Sozzi 2 , Margaret Littlejohn 2 , Lilly K W Yuen 2 , Nadia Warner 2 , Brigid Betz-Stablein 3 4 , Fabio Luciani 4 , Peter A Revill 2 5 , Chris M Brown 1
Affiliations
Affiliations
1
Department of Biochemistry, School of Biomedical Sciences, University of Otago, Dunedin, New Zealand.
2
Victorian Infectious Diseases Reference Laboratory, Royal Melbourne Hospital at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia.
3
Present address: Dermatology Research Centre, Diamantina Institute, University of Queensland, Brisbane, Queensland, Australia.
4
Systems Medicine, School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, New South Wales, Australia.
5
Department of Microbiology and Immunology, University of Melbourne, Melbourne, Victoria, Australia.
PMID: 33439114 DOI: 10.1099/mgen.0.000492
Free article
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.