Depleting HBV rcDNA by lamivudine or HBV-specific siRNA enhances cccDNA deamination by APOBEC/AID.
Currently, HBV rcDNA levels can be effectively reduced by a number of approaches, including the use of reverse transcriptase inhibitors such as lamivudine (LAM), which has a long history of clinical use for treating CHB patients51, and by siRNA therapeutics, which exhibited potent antiviral activity in clinical trials52. Metabolite of LAM is incorporated into newly synthesized HBV DNA on the template of pgRNA, causing chain termination, and competitively inhibits HBV reverse transcriptase activity53; siRNA suppresses viral RNA, including pgRNA, and reduces HBV DNA and protein levels54.
To elucidate the role of HBV rcDNA in APOBEC/AID-mediated deamination of cccDNA, we CRISPR-activated A3A, A3B, or AID in cells treated with either LAM or DMSO as vehicle (Figure 2), or co-transfected with HBV-targeting siRNA (targeting the X region of HBV) or mock control (Figure 3). Four days post transfection, HBV replication (assessed by measuring HBsAg, intracellular HBV DNA, and cccDNA levels) and cccDNA deamination were analyzed. HBsAg levels were not affected by LAM, CRISPRa, or combined treatment (Figure 2A), while siRNA potently reduced HBsAg expression (Figure 3A). CRISPRa reduced intracellular HBV DNA levels, but LAM or siRNA alone were as efficient as combined treatment (Figure 2B, 3B). Expectedly, cccDNA was not affected by LAM treatment, whereas CRISPRa of A3A, A3B, or AID diminished cccDNA by >2-fold, similar to our previous results31 (Figure 2C). The combination of CRISPRa and LAM treatment exhibited comparable efficacy to CRISPRa monotherapy, while selectively activating A3A with LAM demonstrated significantly greater efficiency in reducing cccDNA than CRISPRa alone (Figure 2C). At the same time, the reduction in cccDNA levels in the siRNA + CRISPRa groups was significantly more prominent than in individual treatment groups. The findings suggest that both LAM and siRNA affect anti-cccDNA activity of A3A, A3B, and AID, with LAM showing limited improvement and siRNA demonstrating more pronounced effects.
Further 3D-PCR analysis revealed stark differences in the rates of APOBEC/AID-induced deamination of cccDNA and of secreted and intracellular HBV DNA between control and LAM/siRNA groups (Figure 2D-I, Figure 3D-I). It appears that, as a biochemically primary target for APOBEC/AID, the less abundant HBV rcDNA becomes much more actively deaminated. Upon LAM treatment, secreted HBV DNA was much more actively deaminated by A3A and A3B (Figure 2G), and moderately improved deamination of cccDNA (AID) (Figure 2E) and intracellular HBV DNA (A3B) (Figure 2I). Using siRNA, the results were very pronounced, as increased deamination of intracellular and secreted HBV DNA was observed (Figure 3G, I), as well as of cccDNA by A3A and AID (Figure 3E).
Finally, NGS analysis of cccDNA and secreted rcDNA demonstrated stark differences in deamination between mock control and siRNA groups (Figure 4) consistent with 3D-PCR results (Figure 3D-G). Suppressing HBV replication with siRNA induced massive deamination of cccDNA by A3A and AID (Figure 4A). A3B-induced deamination was already prolific in the mock group, but was at the control level in siRNA group (Figure 4A). Potential explanation could be in degradation of heavily deaminated cccDNA as shown previously30. Nucleotide context deamination analysis demonstrated typical APOBEC/AID-induced mutations with preference of GA/GC context in secreted HBV DNA over GC/GG in cccDNA (Figure S1).
These results indicate that reduced levels of HBV rcDNA are more prone to deamination, while reduced HBV rcDNA levels also increase availability of cccDNA to APOBEC/AID enzymes. The lack of complete uniformity in deamination results between APOBEC/AID groups and LAM/siRNA groups may be explained by the complex dynamics of HBV DNA/cccDNA decline and deamination.