Initial sites of hepadnavirus integration into host genome in human hepatocytes and in the woodchuck model of hepatitis B-associated hepatocellular carcinoma

Abstract

Hepatitis B virus (HBV) and the closely related woodchuck hepatitis virus (WHV) are potent carcinogens that trigger development of primary hepatocellular carcinoma (HCC). The initial sites of hepadnavirus-host genome integration, their diversity and kinetics of formation can be central to virus persistence and the initiation and progression of HCC. To recognize the nature of the very early virus-host interactions, we explored de novo infection of human hepatocyte-like HepaRG cells with authentic HBV and naive woodchucks with WHV. HepaRG were analyzed from several minutes post exposure to HBV onwards, whereas woodchuck liver biopsies at 1 or 3 h and 6 weeks post infection with WHV. Inverse PCR and clonal sequencing of the amplicons were applied to identify virus-host genomic junctions. HBV and WHV DNA and their replication intermediates became detectable in one hour after virus exposure. Concomitantly, HBV DNA integration into various host genes was detected. Notably, junctions of HBV X gene with retrotransposon sequences, such as LINE1 and LINE2, became prominent shortly after infection. In woodchucks, insertion of WHV X and preS sequences into host genome was evident at 1 and 3 h post infection (h.p.i.), confirming that hepadnavirus under natural conditions integrates into hepatocyte DNA soon after invasion. The HBV and WHV X gene enhancer II/core promotor sequence most often formed initial junctions with host DNA. Moreover, multiple virus-virus DNA fusions appeared from 1 h.p.i. onwards in both infected hepatocytes and woodchuck livers. In summary, HBV DNA integrates almost immediately after infection with a variety of host's sequences, among which tandemly repeating non-coding DNAs are common. This study revealed that HBV can engage mobile genetic elements from the beginning of infection to induce pro-oncogenic perturbations throughout the host genome. Such swift virus insertion was also evident in natural hepadnaviral infection in woodchucks.

Figure 1

Study design and detection of HBV DNA- or WHV DNA-specific signals by invPCR/NAH after infection of HepaRG cells or healthy woodchucks. (a) HepaRG cells infected with authentic HBV from patients NL01.A, NL02.C and NL03.E with chronic hepatitis B. (b) Woodchucks Cw1 to Cw4 intravenously infected with WHVtm/3 inoculum. Liver biopsy 1 (Bx-1) obtained prior to experiment, LBx-2 at 1 or 3 h after injection with WHV, and LBx-3 acquired 6 weeks thereafter. Open circles show the acquisition time of cells or liver biopsies and closed circles represent samples in which virus DNA hybridization signals suggesting virus–host integration were detected. A, C and E, HBV genotypes; F, female; M, male.

Figure 2

HBV integration with the neurotrimin (NTM) gene in HepaRG cells after 1-h exposure to NL02.C inoculum. (a) Schematic presentation of integration of HBx sequence (continuous lines) with NTM (shaded boxes) detected in eight separate clones (1–8). All clones displayed the same four-bp overlapping homolgous virus–host junction. Locations of the junction in Ch11q25 and in relation to the HBx sequence are shown. (b) Sequencing electropherograms depict in detail the nucleotides forming the OHJ. HBV and NTM sequences are marked by continuous and dashed lines, respectively. Enh-II, HBV enhancer II; DR1, direct repeat 1.

Figure 3

HBV DNA integration with LINE1 (L1) after 24-h exposure to HBV NL02.C. (a) Schemes showing HBx (continuous lines) integrated with LINE1 (shaded boxes) found in six independent clones (1–6). All clones demonstrated the same sequence of the head- to-tail junction. Location of the junction in Ch15 at q11.2 and in relation to HBx sequence are shown. (b) Electropherograms detailing the breaking point between HBV (continuous line) and LINE1 (dashed line). See the legend to Figure 2 for more details.

Figure 4

HBV integration with transposon LINE2 (L2) at 3 and 7 days post infection of HepaRG cells with NL01.A. (a) Integration of HBx (continuous lines) with LINE2 (shaded boxes) detected in four clones (1–4) obtained after 3 d.p.i. and in five clones (1–5) acquired at 7 d.p.i. (1–5). Different lengths of the LINE2 sequences identified, details on the HBV and LINE2 nucleotides forming the 11-bp homologous junction, except a singular nucleotide mismatch (T versus G), and location of the junction in relation to the HBx sequence and Ch11q13.4 are presented. (b) Electropherograms showing details on the contributions of HBV (continuous line) and LINE2 (dashed line) nucleotides to formation of the OHJ.

Figure 5

HBV integration with myosin III B (Myo3B) encoding gene in HepaRG cells at 2 and 4 weeks post infection with NL01.A. (a) Schematic presentation of integration of HBx sequence (continuous lines) with Myo3B (shaded boxes) detected in five (1–5) and four (1–4) clones obtained at 2 and 4 w.p.i., respectively. Different lengths of the Myo3 sequences, HBV and Myo3B nucleotides forming the eight-bp OHJ, and location of the integration in Ch11q13.1 and within the HBx sequence are shown. (b) Electropherograms detailing nucleotides forming the junction in relation to sequences of HBV (continuous line) and Myo3B (dashed line).

Figure 6

HBV junction with zinc finger protein 782 (ZNF782) encoding gene in HepaRG cells at 2 and 4 weeks post infection with NL01.A. (a) Integration of HBx sequence (continuous lines) with ZNF782 gene (shaded boxes) identified in two clones (1–2) obtained at 2 w.p.i. and in six clones (1–6) obtained at four w.p.i. Lengths of the ZNF782 sequences identified, nucleotides of the HBV and ZNF782 HTJ, and location of the junction in the HBx sequence and Ch11q25 are shown. (b) Electropherograms detailing the junction nucleotides in relation to the HBV (continuous line) and the ZNF782 (dashed line) sequences.

Figure 7

HBV DNA integration with retrotransposon human satellite DNA II (HSAT-II) DNA at 2 weeks after infection with NL03.E. (a) Schematic presentation of HBx (continuous lines) integrated with HSAT-II (shaded boxes) identified in 12 independent clones (1–12). All clones demonstrated the same HTJ sequence and locations within Ch16p11.2. (b) Electropherograms show in detail nucleotides forming the breaking point between HBV (continuous line) and HSAT-II (dashed line).

Figure 8

Schematic representation of the HBV X gene breaking points forming junctions with the human genomic sequences. Slim arrows identify breaking points which formed junctions detected in single clones, whereas bold arrows represent those identified in multiple clones. Four black circles depict HBV TATA elements. BCP, basal core promoter; DR, direct repeat region; Enh-II, HBV enhancer II region; Pg RNA, pre-genomic RNA; URR, upstream regulatory region. Numbers mark nucleotide positions according to HBV DNA GenBank X70185 sequence.