Discussion
In this work, for the first time, we assessed how HBV rcDNA affects
antiviral and, specifically, cccDNA-targeting activity of major
APOBEC/AID enzymes. Experiments with transcriptionally silenced cccDNA
revealed markedly increased deamination of cccDNA by A3A, A3B, and AID
(Figure 1). This prompted us to perform experiments with modern
antiviral therapeutics that block or terminate HBV rcDNA production,
namely lamivudine and siRNA targeting X gene of HBV. These experiments
revealed increased deamination of the remaining rcDNA and, more
importantly, increased cccDNA deamination (Figure 2, 3) with massive
deamination demonstrated by NGS analysis (Figure 4). These observations
suggest a new, background defense strategy utilized by the virus to
counter innate immunity and to preserve cccDNA integrity by saturating
antiviral enzymes with its genome intermediate, HBV rcDNA. This unique
mechanism of immune evasion is somewhat similar to what HBV does to
dampen adaptive immunity in CHB patients, when HBV produces excessive
amounts of HBsAg compared to virions to establish antigen overload that
exhausts antigen-presenting cells and leads to immune
tolerance57,58. In this particular “hide-abide”
strategy, HBV produces abundant HBV rcDNA, the single-stranded regions
of which are the primary targets for APOBEC/AID cytidine deaminases.
Overloading and saturating APOBEC/AID with ssDNA targets minimizes the
effect of these enzymes on cccDNA, contributing to viral persistence. To
the best of our knowledge, this is a newly characterized mechanism of
HBV immune evasion that has not been reported previously for HBV or
other viruses, adding to the already complex interaction of viruses with
the host immune system.
Our results also discourage from further use of APOBEC/AID as potential
anti-HBV therapeutics. APOBEC/AID enzymes have long been known to be
important drivers of human cancers. Widespread APOBEC/AID-mediated
mutations in genomic DNA of cancer patients have been
detected56. More recent evidence adds knowledge about
RNA-targeting properties of some APOBEC/AID enzymes dysregulating the
host cell functioning59 and inducing death in animals
with, specifically, A3B overexpression20. The promise
of APOBEC/AID as safe molecular tools came from the pioneering study by
Lucifora et al .42 who demonstrated that A3A and
A3B do not deaminate the host genome due to binding HBc protein of HBV.
However, we recently observed that A3A, A3B, and AID exhibit no off-site
deamination only in human cells with very active HBV replication,
whereas in cells with low viral loads, deamination of the host genome
occurs31. In this study, we imitated the real-life
therapeutic situation of suppressing HBV replication by antivirals,
thereby reducing HBV viral loads, and then activated APOBEC/AID
expression. In this scenario, A3A and A3B per se were not mutagenic, but
the use of siRNA or LAM provoked host mutagenesis of the cancer-relatedTP53 gene by A3A and A3B. Thus, using CRISPR deaminases able to
directly interact with cccDNA60 together with improved
versions of cytidine deaminases lacking RNA-targeting properties may
represent a more rational therapeutic approach61.
At the same time, CRISPR activation and CRISPR interference approaches
demonstrate high efficacy in highly specific and transient activation of
antiviral genes for suppressing viral
replication19,31,62,63. The search for the critical
antiviral factors for CRISPR activation is necessary to further its use
as an antiviral therapeutic18.
To conclude, this study provides evidence of a new mechanism of immune
evasion unique to HBV that works by saturating APOBEC/AID immunity with
the rcDNA genomic form to preserve cccDNA pool integrity. Analysis of
host genome deamination indicates severely hazardous activity of
APOBEC/AID on host DNA when HBV viral loads are reduced, compromising
its use as a potential therapy.