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.