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 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.

Targeted long-read sequencing reveals clonally expanded HBV-associated chromosomal translocations in patients with chronic hepatitis B

“…Finally, we performed a splice isoform analysis using the known HBV splice isoforms, SP1 to SP17 [30,31]. As previously reported, SP1 was the most common splice isoform identified in our patient collection (Figure 4D) [32]. The ratio of spliced to non-spliced HBV RNAs ranged from 2% to over 20% and was highest in HBeAg-positive patients.…”

“…4 D). 32 The ratio of spliced to non-spliced HBV RNAs ranged from 2% to over 20% and was highest in patients who were HBeAg-positive. Using a 3ʹ poly(A) analysis, the majority of spliced HBV RNAs originated from cccDNA, and accordingly, splicing was more prevalent in HBeAg-positive patients with higher cccDNA transcriptional burden.…”

Inflammatory Gene Expression Associates with Hepatitis B Virus cccDNA- but Not Integrant-Derived Transcripts in HBeAg Negative Disease

“…Primers targeting the 3′ end of all transcripts (3′T) will amplify both cccDNA- and iDNA-derived RNAs, whereas those targeting the repeat region (RR), which is absent in the majority of id-RNAs, will only amplify cccDNA-derived RNAs [ 14 , 29 ] ( Figure 3 A). The 3′T primers will amplify pC/pg splice variants [ 11 ] and two of the three known HBx truncated transcripts [ 30 ]. Both 3′T and RR primer pairs target regions conserved among diverse genotypes and showed a linear amplification of HBV genotype D over a 5-log range ( Supplementary Figure S4A,B ).…”

“…HBV transcribes six major viral RNAs of decreasing length with a common 3′ polyadenylation signal together with a number of spliced RNAs of unknown function [ 11 ]. The major RNAs include: precore (pC), which encodes e antigen (HBeAg); pregenomic (pg RNA), which is translated to yield core protein or antigen (HBcAg) and polymerase; and preS1, which encodes large surface envelope (L-HBsAg) and S RNA, and encodes medium (M-HBsAg; Pres1 and S), and small surface envelope (S-HBsAg, S) glycoproteins (HBsAg) and the X transcript codes for the multi-functional x protein (HBx).…”

Surrogate Markers for Hepatitis B Virus Covalently Closed Circular DNA

“…HBV RNA molecules transcribed from cccDNA also include the 5' truncated RNA with 3' poly-A tail 128,134 and the HBx RNA 133,135 . Additionally, over 20 splice variants of the pgRNA 128,132,133,136,137 and preS2/S mRNA 129 have been described to be present in the supernatant of transfected cell lines 132 and in patient sera 132,[138][139][140] . The quasispecies of serum HBV RNA have been shown to reflect the genome of cccDNA 128 , adding to the argument that serum HBV RNA could be an accurate surrogate marker for cccDNA.…”