Viral integrations in chronic hepatitis B infection: Purposeless passenger or problematic promoter of persistence?

“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 HBVDNA 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 replicationdefective form, despite the existence of simple mutations that can increase replicationcompetent virion secretion. [2] This suggests that HBVDNA 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 AluPCR assays to quantify either integrated HBVDNA 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 standardofcare 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 HBVDNA 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). In 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 cccDNAspecific 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 HepG22.2. 15 cells, a hepatoma cell line containing two headtotail 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 HBVDNA 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 falsepositive amplification and amplification of PCRmediated 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 endjoining. [7] Other approaches to solidify the assay could include other types of control reactions (e.g., spiking HBVnegative 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 Received: 28 January 2022 | Accepted: 28 January 2022