Introduction:HBV integration occurs at double-stranded DNA breaks in the host genome in ~1/10000 cells. Most hepatocellular carcinomas(HCC) contain integrations, suggesting they are linked but the mechanism remains unknown. HBV X protein(HBx) can induce genomic instability by degrading SMC5/6 complex, but it is unclear if HBx derived from integrations is functional. We aimed to characterise the functionality of HBx from integrated HBV DNA. We hypothesise that HBx expressed from integrations in infected cells induces more DNA damage, leading to more integrations, fostering a feed-forward loop and HCC development.
Methods:We generated 118 HepG2-NTCP-derived clones with de novo integrations using a recombinant reporter HBV expressing a Zeocin-resistance gene(in place of HBs). Integrations were quantified using digital droplet PCR. Integration junctions were sequenced using either inverse PCR or targeted sequencing. Expression of HBx transcripts was measured using quantitative 5’ rapid amplification of cDNA ends(5’RACE). Functional HBx was determined using a trans-complementation assay by infecting cells with HBx-null HBV and measuring HBeAg by ELISA.
Results:The HepG2-NTCP clones each contained 1-2 copies of integrated HBV DNA. Sequence analysis of integration junctions found that HBx regions required for SMC5/6 degradation(amino acids 45-140)were present in >70% of integrations, consistent with observations in human liver. Quantitative 5’RACE revealed that HBx expression in the clones ranged from 3-518 copies/1000ng RNA, averaging ~4-fold lower than HBV-infected cells that express HBx from cccDNA. In a screen of 9 clones, we identified 2 clones that could trans-complement HBx-null HBV and restore HBeAg expression. Cells with existing integrations were 2-12 fold more susceptible to integrations after a new HBV infection.
Discussion:Cells with HBV integrations can express functional HBx and may be more susceptible to additional integrations. Ongoing work will determine the role of HBx in this process.
Conclusions:Integration-derived HBx could be key to driving HCC progression and may be a target for HCC prevention.