Cell Type Diversity in Hepatitis B Virus RNA Splicing and Its Regulation

Abstract

Although RNA splicing of hepatitis B virus (HBV) is a commonly observed in livers of hepatitis B patients as well as in the cultured cells replicating the viral genome, its biological significance in the HBV life cycle and the detailed regulatory mechanisms are still largely unclear. In this study, we found cell-type dependency of HBV splicing of the 3.5 kb pregenomic RNA, which is efficiently spliced in human hepatoma cells but not in cells derived from human hepatic stellate, mouse hepatoma and human non-hepatic cells. It may be likely that RNA splicing is one of the determinants of host range restriction of HBV. Given the finding indicating the difference in cell-type dependency of the splicing efficiency between HBV and simian virus 40, we carried out intron-swapping experiments. The results suggest the presence of putative exonic splicing enhancer that possibly works in the cell-type dependent fashion. Together with further mutational analyses, a novel 50-nt intronic splicing silencer, whose secondary structure is well conserved among the HBV strains, was identified. It appears that this intronic silencer functions effectively independent of cell backgrounds.

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

“…We also identified previously reported splice variants SP1, 5, 6, 7, 9 and 13** across the HBV genotypes (11,14,24,38) (Fig 1B and 2). Notably, SP1 and SP9 were consistently detected in all the genotypes.…”

“…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. We identified a number of novel splice variants across HBV genotypes as well as previously identified variants of yet unknown functions.…”

“…In addition, different HBV genotypes may produce distinct spliced transcript isoforms whose precise roles are largely unknown (11)(12)(13)(14) . At least 17 spliced transcripts of pgRNA (14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26) and 4 spliced transcripts of preS2/S (27,28) were identified in various sources including liver, serum, and transfected cells. Interestingly, a recent study showed that HBV RNA splicing is more efficient in human hepatoma cells than other tested cell-types (14) .…”

“…At least 17 spliced transcripts of pgRNA (14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26) and 4 spliced transcripts of preS2/S (27,28) were identified in various sources including liver, serum, and transfected cells. Interestingly, a recent study showed that HBV RNA splicing is more efficient in human hepatoma cells than other tested cell-types (14) . Furthermore, spliced pgRNA SP1 is the most commonly detected (16,17,19,21,(29)(30)(31)(32)(33) , although SP3 and SP9 have also been commonly observed in some studies (13,14,26) .…”

“…Interestingly, a recent study showed that HBV RNA splicing is more efficient in human hepatoma cells than other tested cell-types (14) . Furthermore, spliced pgRNA SP1 is the most commonly detected (16,17,19,21,(29)(30)(31)(32)(33) , although SP3 and SP9 have also been commonly observed in some studies (13,14,26) .…”