Figure 1. Effects of A3A, A3B, and AID on transcriptionally active and inactivated cccDNA. (A) Real-time PCR analysis of recombinant cccDNA (rcccDNA) or hypermethylated recombinant cccDNA (meth-rcccDNA) levels upon CRISPR activation of A3A, A3B, and AID. (B) 3D-PCR analysis of deamination in rcccDNA and methylated rcccDNA (top); semi-quantitative 3D-PCR analysis (bottom). (C) Frequency of G→A and C→T deaminated nucleotides in rcccDNA and meth-rcccDNA per read. (D) Analysis of rcccDNA and meth-rcccDNA dinucleotide G→A and C→T mutation context.

We deep-sequenced the X region of rcccDNA and meth-rcccDNA and analyzed the mutation profile caused by APOBEC/AID. Frequency of C→T/G→A mutations was markedly higher in meth-rcccDNA in all studied groups, indicating both higher frequency of C→T/G→A mutations per read and emergence of extensively edited reads (Figure 1C). Nucleotide context analysis of A3A, A3B, and AID showed similar nucleotide preference, characteristic for these factors (Figure 1D). Thus, A3A, A3B, and AID directly deaminate cccDNA and are more efficient in deaminating transcriptionally inactivated meth-cccDNA.

CG content and CpG methylation are significant factors that confer resistance to or lower activity of some APOBEC/AID enzymes⁴⁷. Reports suggest weaker DNA deamination of methylated CpG by A3B⁴⁸ and AID⁴⁹, whereas A3A is generally not affected⁵⁰. Thus, it was particularly surprising to observe more pronounced deamination of meth-rcccDNA than rcccDNA. The major difference between rcccDNA and meth-rcccDNA is that cccDNA launches viral replication and formation of other viral intermediates, mostly HBV rcDNA. Therefore, we endeavored to further assess the role of the most prevalent HBV intermediate – HBV rcDNA – in restricting APOBEC/AID activity.