Viral integrations in chronic hepatitis B infection: Purposeless passenger or problematic promoter of persistence?
Authors: Thomas Tu, Henrik Zhang
Journal: Hepatology (2022)

SEE ARTICLE ON PAGE 196 "Why does HBV integrate into the host genome?" This is a problem that has challenged the viral hepatitis field for decades. As opposed to the episomal covalently closed circular DNA (cccDNA) form on which hepatitis B replication is dependent, integrated HBV‐DNA genomes do not produce new virus (previously reviewed in Tu et al.).[1] Instead, this rare phenomenon (occurring in 1 per ~1000 cells) can express subgenomic transcripts encoding some viral antigens, notably HBsAg, which has been associated with viral persistence (possibly through immune modulation, though this is still controversial). HBV devotes ~10% of its virions to precursors of this replication‐defective form, despite the existence of simple mutations that can increase replication‐competent virion secretion.[2] This suggests that HBV‐DNA integration is important for viral fitness, but to date no studies have shown this. Within this context, a study by Erken et al. in this issue of Hepatology investigated the role of integrations in chronic HBV infection.[3] The researchers developed nested Alu‐PCR assays to quantify either integrated HBV‐DNA copies or the subgenomic RNA transcripts derived from integrants. They used these assays to analyze liver tissues of participants of a clinical trial given a combination of standard‐of‐care nucleos(t)ide analogue (NA) therapy and pegylated interferon (IFN) alpha. Chronic HBV infections are usually lifelong, even with these therapies, but a minority of patients cleared virus infection (indicated by loss of HBsAg). As their key result, the researchers report that the level of HBV‐DNA integrations at baseline was lower in those who achieved infection clearance, even after controlling for multiple variables. Thus, this study suggests a potential link between integration and viral persistence (Figure 1).FIGURE 1: A hypothesized mechanism by which integration contributes to viral persistence. Integration of HBV DNA into the host genome may be important for viral fitness and play a role in viral persistence. Greater viral integration is associated with higher HBsAg levels, which may suppress immune clearance of HBV cccDNA by yet‐unknown mechanisms. Conversely, low viral integration may act as a predictor for favorable outcomes of combination NA and IFN therapies. Determining levels of HBV viral integration and, subsequently, preventing new integrations may assist in increasing the efficacy of future functional cures. Created with BioRender.comIn general, their results are consistent with previous studies showing that transcription from integrated HBV DNA is common in patients and is associated with higher HBsAg levels[4,5] and viral replication.[6] However, it is difficult to know the contribution of cccDNA to these factors given that the cccDNA‐specific transcripts were not measured. Although there is hope that the measurement of integrations could become a novel predictive tool for patients who might overcome HBV infection with treatment, several issues should be addressed first. Importantly, any new assay needs a robust foundation. The presented data for assay establishment have been performed on HepG2‐2.2.15 cells, a hepatoma cell line containing two head‐to‐tail dimers of the HBV genome. The characteristics of this transgene (with termini at 3182 nucleotides [nt] of the viral genome inserted into a plasmid backbone, producing HBV transcripts ending at the viral poly A signal) differ markedly from the integrants resultant from HBV infection (less than genome length, viral termini at ~1832 nt, and producing transcripts terminating hundreds of nucleotides before the viral poly A signal). These structural differences likely alter the detection of both integrated HBV DNA and their transcripts. Other structural considerations of integrants in patient tissues would also affect their detection (e.g., the closer an integration is to a host Alu sequence, the easier it will be amplified). A general concern about quantifying HBV‐DNA integrations is the specificity of detection, given that these forms are usually the least prevalent forms of HBV DNA in a DNA extract of infected tissue. Particularly with a nested PCR design as used here, great care needs to be taken to minimize (or at least define) the level of false‐positive amplification and amplification of PCR‐mediated recombination events. Deep sequencing of the amplicons could shed light into the extent that this is occurring: Limited homology between the viral and host sequences is observed, given that most integration occurs by nonhomologous end‐joining.[7] Other approaches to solidify the assay could include other types of control reactions (e.g., spiking HBV‐negative liver DNA extracts with HBV virion DNA or replicative intermediates). Nonspecificity in detection could explain why these results contradict some previous studies showing that integration rates either stay constant or increase during the course of HBV infection.[4,8,9] Given that the assay is semiquantitative, it may be difficult to extend for clinical use. Given that it is based on a nested PCR, a linear increase of integrations in the sample are not matched by a linear increase in signal. Moreover, these levels will be highly sensitive to variability in amplicon transfer from the first to second PCR, particularly without an appropriate control gene. Finally, absolute cutoffs would need to be developed; the classification of patients in this study was based on the level being higher or lower than average. If this novel assay is shown to be robust in future studies, then these findings represent evidence that HBV‐DNA integrations play a role in viral persistence and could act as a predictor for patients who respond favorably to combination IFN and NA treatment. Moreover, recent studies have shown that inhibiting HBV replication may prevent new integrations and/or limit the expansion of established integrations.[6] Combined with this work, an argument could be made that earlier treatment of patients (though not curative) would reduce HBV‐DNA integration burden and facilitate functional cures driven by upcoming investigational therapies. Thus, this work potentially improves the clinical care of persons with chronic hepatitis B and provides them with hope that a cure is that much closer to a reality. CONFLICT OF INTEREST Nothing to report. AUTHOR CONTRIBUTIONS TT led the conception and design of the review. HZ prepared the figure and legend. Both authors contributed to drafting and revising the paper, and had final approval of the version to be published.