HBV DNA Integration: Molecular Mechanisms and Clinical Implications

Abstract

Chronic infection with the Hepatitis B Virus (HBV) is a major cause of liver-related morbidity and mortality. One peculiar observation in cells infected with HBV (or with closely‑related animal hepadnaviruses) is the presence of viral DNA integration in the host cell genome, despite this form being a replicative dead-end for the virus. The frequent finding of somatic integration of viral DNA suggests an evolutionary benefit for the virus; however, the mechanism of integration, its functions, and the clinical implications remain unknown. Here we review the current body of knowledge of HBV DNA integration, with particular focus on the molecular mechanisms and its clinical implications (including the possible consequences of replication-independent antigen expression and its possible role in hepatocellular carcinoma). HBV DNA integration is likely to influence HBV replication, persistence, and pathogenesis, and so deserves greater attention in future studies.

Keywords: hepatitis B virus; hepatocellular carcinoma; integration; non-homologous end joining; viral persistence.

Figure 1

Open reading frames (ORFs) of hepatitis B virus (HBV) circular DNA form. Coloured blocks represent each of the four main open reading frames, and the associated promoters (Pro) and enhancers (EN1 and EN2, light blue ovals) are shown. The direct repeat regions (DR1 and DR2, grey boxes) play important roles in hepatitis B virus (HBV) replication (seen in Figure 2). The large, medium and small forms of HBsAg are translated from the preS1 + preS2 + S, preS2 + S and S alone ORF, respectively. Pol is encoded by the polymerase ORF. HBV core antigen (HBcAg) is translated from the core ORF alone. HBV e antigen (HBeAg) is formed by the cleavage of the translated product from PreC + core ORF. HBV X protein (HBx) is translated from the X ORF.

Figure 2

The replication cycle of HBV (a) with a focus on the steps of intra-capsid reverse transcription (b); (a) After the virus undergoes receptor-mediated entry via heparan sulphate proteoglycans and sodium taurocholate cotransporting polypeptide (NTCP), the nucleocapsid is transported to the nucleus, where the relaxed circular DNA (rcDNA) genome is converted into covalently closed circular (ccc)DNA by the host proteins. Covalently closed circular DNA (cccDNA) acts as the viral template for messenger RNAs (mRNA) and pregenomic RNA (pgRNA; red line), which is encapsidated together with the viral polymerase (red circle) that mediates reverse transcription (purple area); (b) During reverse transcription, Pol has three distinct functions: primer synthesis, RNA/DNA-dependent DNA polymerisation, and RNaseH-mediated RNA degradation. pgRNA is greater than genome length and thus contains redundant regions (light blue shading). After binding to the 5’-epsilon (ε) region of the pgRNA (left blue box), Pol synthesises a three nucleotide (nt) oligonucleotide (GAA), using ε as a template. The trinucleotide primer is covalently attached to the Pol, and this complex then translocates to the direct repeat 1* region (DR1*), located in the 3’region. Pol reverse transcribes a negative-sense DNA strand (black) using the 3 nt oligonucleotide as a primer and the pgRNA as a template. Simultaneously with reverse transcription, Pol hydrolyses the RNA template with its ribonuclease H (RNaseH) activity lagging 18 nt behind the site of reverse transcription. Since Pol is covalently attached to the 5’ end of the synthesised negative-sense DNA strand, a loop structure forms. The pgRNA is hydrolysed up to 18 nt from the 5’ end. The remaining 18 nt RNA oligonucleotide acts as a primer for the synthesis of the positive-sense DNA strand. In ~90% of nucleocapsids, the 18 nt RNA primer translocates to the DR2 sequence, leading to the synthesis of rcDNA. In the remaining 10%, the RNA primer remains bound to the DR1 region, priming double stranded linear DNA (dslDNA) synthesis. After reverse transcription, the mature nucleocapsids can either be secreted as virions or cycle to the nucleus to add to the cccDNA pool. dslDNA can also integrate into the host cell genome.

Figure 3

ORFs of HBV dslDNA form. As with Figure 1, coloured blocks represent each of the four main open reading frames. Associated promoters (Pro), enhancers (EN1 and EN2, light blue ovals), and direct repeat regions (DR1 and DR2) are also depicted. The approximate nts of the 5’ and 3’ ends are shown (numbering based on HBV DNA sequence from Genbank Accession #AB241115). As a result of the dslDNA forms generated by in situ priming (Figure 2b), the X ORF is truncated at its C‑terminus (by at least three amino acids) and the pre-core/core promoter is separated from its ORF.

Figure 4

A model of potential molecular factors in HBV DNA integration. Various secreted HBV forms (top row) may be involved in bringing the potential molecular substrates for HBV DNA integration (black) into the cell. Both reported (solid arrows) and as-yet purely hypothetical pathways (dashed arrows) for HBV DNA integration into the host cell genome (light blue double lines) via potential cellular double-stranded DNA (dsDNA) repair mechanisms (dark blue) are shown, depicted with the key host enzymes involved. The substrates for integration include full-length HBV dslDNA, though the HBV single-stranded DNA (ssDNA) and spliced variant dsDNA forms may also contribute. Further, it is currently unknown whether viral proteins (red) play either an inhibitory or inducing role in HBV DNA integration. Integration occurs at dsDNA breaks in the host cell genome, and relevant host factors that induce these breaks are shown in purple. MMEJ, microhomology‑mediated end-joining; NHEJ, non-homologous end-joining; SSA, single-stranded annealing; ROS, reactive oxygen species; S/MARs, scaffold/matrix attachment regions.