Transcriptionally silenced HBV cccDNA exhibits increased susceptibility to deamination by A3A, A3B, and AID.
Currently, there is still disagreement on whether HBV cccDNA is directly mutated by APOBEC/AID. Lucifora et al . provided extensive evidence of cccDNA deamination by A3A and A3B42, but this effect was later questioned by Seeger and Sohn43and Nair and Zlotnik44, who observed very low cccDNA mutation rates and hypothesized preferential deamination of rcDNA. Using primer extension analysis and clone sequencing, Chen et al . also did not detect cccDNA deamination by any APOBEC/AID enzymes overexpressed from DNA vectors28. Contrastingly, Faure-Dupuy et al . observed prolific deamination of HBV cccDNA by A3B, and first attempted to assess anti-HBV activity of A3B at transcriptionally inactive HBV ΔX genomes, which have a deleted X gene that is required for HBV transcription and rcDNA formation. The group showed robust mutation by A3B at both transcriptionally active and inactive cccDNA30. Our own group also showed that even very transient upregulation of A3A, A3B, or AID profoundly reduced and hyper-edited cccDNA31. cccDNA degradation by A3B was confirmed in another study that focused on the role of HIF-1α in suppressing A3B in CHB patients45.
In this study, we used a well-established model of transcriptionally inactivated, hypermethylated recombinant cccDNA (meth-rcccDNA) that does not form pgRNA or rcDNA7,14,46 to assess direct cccDNA deamination by APOBEC/AID factors with the most potent anti-HBV activity. CRISPRa of A3A, A3B, or AID similarly reduced both recombinant cccDNA (rcccDNA) and meth-rcccDNA by >50–75% (Figure 1A). Analysis of cccDNA deamination by 3D-PCR and semi-quantitative 3D-PCR, techniques enabling preferential amplification of deaminated nucleotides17, revealed extensive deamination of cccDNA in the order A3A > A3B > AID (Figure 1B). Notably, this order of anti-cccDNA activity may be related to different target gene overexpression levels and duration by CRISPRa, as A3B is known to be the most potent DNA mutator of the APOBEC/AID family members. Contrastingly to similar reduction of HBV DNA levels, meth-cccDNA harbored >100-fold more mutations than cccDNA (Figure 1B).