The Initial Hepatitis B Virus-Hepatocyte Genomic Integrations and Their Role in Hepatocellular Oncogenesis

Abstract

Hepatitis B virus (HBV) remains a dominant cause of hepatocellular carcinoma (HCC). Recently, it was shown that HBV and woodchuck hepatitis virus (WHV) integrate into the hepatocyte genome minutes after invasion. Retrotransposons and transposable sequences were frequent sites of the initial insertions, suggesting a mechanism for spontaneous HBV DNA dispersal throughout the hepatocyte genome. Several somatic genes were also identified as early insertional targets in infected hepatocytes and woodchuck livers. Head-to-tail joints (HTJs) dominated amongst fusions, indicating their creation by non-homologous end-joining (NHEJ). Their formation coincided with the robust oxidative damage of hepatocyte DNA. This was associated with the activation of poly(ADP-ribose) polymerase 1 (PARP1)-mediated dsDNA repair, as reflected by the augmented transcription of PARP1 and XRCC1; the PARP1 binding partner OGG1, a responder to oxidative DNA damage; and increased activity of NAD⁺, a marker of PARP1 activation, and HO1, an indicator of cell oxidative stress. The engagement of the PARP1-mediated NHEJ repair pathway explains the HTJ format of the initial merges. The findings show that HBV and WHV are immediate inducers of oxidative DNA damage and hijack dsDNA repair to integrate into the hepatocyte genome, and through this mechanism, they may initiate pro-oncogenic processes. Tracking initial integrations may uncover early markers of HCC and help to explain HBV-associated oncogenesis.

Keywords: HBV; dsDNA repair; early hepadnavirus–host DNA integration; hepatocellular carcinoma; oncogenesis; retrotransposons; virus-induced oxidative DNA damage; woodchuck hepatitis virus.

Figure 1

Schematic step-by-step outline of methods used for the detection of HBV integration and the identification of the sequences acting as the host’s HBV insertional sites and HBV DNA breakpoints in infected hepatocyte. Total DNA was extracted from infected cells and treated with restriction enzyme NcoI, which cut HBV DNA at the single site and the HBV-merged host’s sequence at unknown sites. The resulting hybrid DNA fragments were circularized with T4 DNA ligase and then linearized with BsiHKAI endonuclease to facilitate PCR amplification and cloning. Nested PCR was performed with forward (F1 and F2) and reverse (R1 and R2) primers. The exception was a situation in which a particular band was well identifiable after direct PCR by agarose gel electrophoresis and clearly displayed the HBV signal with nucleic acid hybridization (NAH). NAH was routinely used to verify the presence of HBV sequences and to augment the detection of the HBV–host DNA merges even when the bands carrying the merges were not apparent on the gels. This was followed by the cloning of HBV-positive amplicons and bidirectional sequencing of the clones. The aim was to sequence 20–30 clones from each band analyzed. For more information, see the text and references [19,31,32]. For identification of WHV–host DNA merges, the approach used was identical to that above, but restriction enzymes and PCR primers were specific for WHV, as reported in [19,31].

Figure 2

Examples of the earliest HBV and WHV DNA fusions with human or woodchuck genomic sequences detected between 15 min and 1 h after exposure to virus in different infection systems investigated in our studies. With the exception of the overlapping homologous junction (OHJ) created by the HBV X gene (HBV X) and human neurotrimin (NTM) gene displayed on the left side of the top panel, all other virus–host genomic fusions shown have the format of the head-to-tail junction (HTJ). In the bottom left panel, the virus–host DNA merge was formed by the preS sequence of WHV S (envelope) gene (WHV PS) and it was detected in a liver biopsy obtained from a woodchuck one hour after injection with WHV, as described in the text. Other abbreviations: SINE, short-interspersed nuclear element (retrotransposon); MAML-2, mastermind-like 2; WHV X, WHV X gene; host, unidentified woodchuck genomic sequence.

Figure 3

Schematic presentation of the HBV X gene break points forming fusions with the human genomic sequences that were detected in hepatocyte-compatible HepRG and HepG2-NRCP-C4 cells within the first 24 h after exposure to HBV. Yellow squares show breakpoints that formed junctions detected at 30 min and 1 h after exposure to virus, red squares show those identified at 3 h post-infection, and blue squares show those detected at 24 h post-infection. Blue squares with stars show breakpoints reported at 24 h after infection in reference [36]. Numbers mark nucleotide positions according to the HBV DNA GenBank X79185 sequence. Abbreviations: URR, upstream regulatory region of the HBV core promoter; BCP, basal core promoter; DR1, direct repeat region; Enh II, HBV enhancer II.

Figure 4

Graphic presentation of the earliest detections and changes over time in the presence of virus genome (DNA), its replication (mRNA and cccDNA) and integration into the hepatocellular genome, indicators of hepatocyte oxidative stress and oxidative DNA damage, as well as markers of the activity of components of the DNA repair pathway during the first 72 h after exposure to HBV or WHV. The profiles represent cumulative data from HBV and WHV infections in human and woodchuck hepatocyte-compatible cells and in woodchucks infected with WHV. White triangles mark the time at which peak expression or activity was found. Whites square in the OGG1 line represent a 5.6-fold increase in the gene expression over uninfected control cells, which did not achieve a statistically significant difference. For more details, see reference [33]. Abbreviations: cccDNA, virus covalently closed circular DNA; ROS, reactive oxygen species; RNS, reactive nitrogen species; HO1, heme oxygenase-1; PARP1, poly(ADP-ribose) polymerase 1; XRCC1, X-ray repair cross-complementing protein 1; NAD⁺, nicotinamide adenine dinucleotide; OGG1, 8-oxyguanidine DNA glucose 1, and nt, not tested beyond this time point.