Introduction
Hepatitis B virus (HBV) is a virus that infects human hepatocytes and
causes acute or chronic infection of the liver (chronic hepatitis B
virus infection, or CHB). Continuous replication of HBV in CHB results
in progressing liver damage, leading to liver cirrhosis or
hepatocellular carcinoma1. HBV has a complicated viral
life cycle, starting from infection of human hepatocytes by HBV virions
containing the partially double-stranded, relaxed circular DNA (rcDNA)
viral genome. This is followed by nuclear import of rcDNA with the
assistance of HBV core protein2 and its conversion
into covalently closed circular DNA (cccDNA), a highly persistent
genomic form that transcribes all viral RNA, including pregenomic RNA
(pgRNA)3. HBV RNA is translated into viral proteins
(X, core, surface proteins, and E-antigen), whereas pgRNA is
reverse-transcribed by viral polymerase to produce
rcDNA3. HBV cccDNA is not only very stable (persisting
through the lifespan of resting hepatocytes; estimated half-life
< 40 days to >9–26 months in patients undergoing
nucleot(s)ide analog therapy)4–6, but is also
replenished via intracellular amplification (re-import of generated
rcDNA back to the nucleus)7–9 and
re-infection10. Destroying HBV cccDNA or promoting its
decay in hepatocytes can lead to a cure of HBV
infection7,11. However, no medications are approved to
target HBV cccDNA. While nucleot(s)ide analogs, interferons, and
Bulevirtide (inhibitor of HBV entry) suppress HBV replication and reduce
the risks of CHB outcomes, they do not cure the disease. Recent findings
demonstrate that combining Bulevirtide with anti-HBV antisense
oligonucleotides may result in sustained silencing of HBV cccDNA
transcriptional activity12. HBV cccDNA can also be
directly cleaved and destroyed by site-specific CRISPR/Cas9
nucleases7,13–16. Notably, some factors of innate
immunity can limit HBV replication, while others, such as APOBEC/AID
cytidine deaminases, can induce HBV cccDNA mutational inactivation
and/or degradation17.
In humans, the APOBEC/AID includes 10 members: APOBEC1, APOBEC2,
APOBEC3A (A3A), APOBEC3B (A3B), APOBEC3C (A3C), APOBEC3D (A3D), APOBEC3F
(A3F), APOBEC3G (A3G), APOBEC3H (A3H), and AID18. A3A,
A3B, A3C, A3D, A3F, A3G, A3H, and AID can directly deaminate cytidines
in single-stranded DNA, introducing C to T and G to A mutations on the
complementary strand; while APOBEC1, A3A, and A3G deaminate RNA, forming
C to U mutations19. Recent findings indicate that
elevated (but not endogenous) levels of A3B can also edit RNA at
specific hotspots, causing lethality in a model of inducible A3B
expression in mice20. APOBEC2 is unable to edit
nucleic acids.
APOBEC/AID are famous mutators, implicated in the development of
numerous cancers21, and important factors that
restrict foreign RNA and DNA, such as viral nucleic acids or
intracellular DNA leaks, e.g., from nucleus or
mitochondria19,22. APOBEC/AID suppress replication of
many viruses infecting humans, like human immunodeficiency virus, herpes
simplex virus 1, Epstein–Barr virus, hepatitis C virus, human papilloma
virus, and HBV19,23. Pioneering studies demonstrated
HBV-editing activity of A3G, A3C, A3H, and A3F24–26.
A3G was also shown to inhibit HBV without cytidine deamination by
suppressing pgRNA packaging into viral capsids and inhibiting reverse
transcription27. More recently, Chen et al .
extensively evaluated APOBEC/AID cytidine deamination activity of HBV
rcDNA, demonstrating that deaminating activity decreases in the order
A3B >> A3G > A3H or
A3C28, while the activity of A3A, A3D, A3H, and A3F
was very low or undetectable. Several studies provided evidence that
A3A, A3B, and AID can hyper-edit and destroy HBV cccDNA without off-site
mutagenesis17,29,30. However, we recently described
that transiently overexpressed A3A, A3B, and AID enzymes induce frequent
mutations in cancer-related genes in the human genome in cells with low
levels of HBV replication even upon transient activation, while A3G
inflicts DNA double-stranded breaks 31.
Due to their potent anti-HBV activity, induction of APOBEC/AID is
considered a promising antiviral strategy for potentially curing CHB
patients. Intracellular expression of cytidine deaminases in the liver
can be induced by different agonists, including interferon alpha
(IFN-α)17, IFN-γ32,
IFN-λ33, lymphotoxin-β receptor
agonist17, and others. Otherwise, APOBEC/AID can be
transcriptionally controlled by dCas-based molecular activators (CRISPR
activation systems, CRISPRa)19,31,34. CRISPRa enables
precise activation and tunable control of target gene expression and
monogenic or simultaneous activation of several antiviral
factors35. Typically, CRISPRa consists of a
nucleolytically dead Cas9 protein (dCas) recruited to the regulatory
regions of genes via single-guide RNA (sgRNA)36,37.
Either dCas9 or sgRNA could also harbor units that mediate activation of
transcription34.
HBV’s interactions with innate immunity are complex18;
HBV is frequently regarded as a stealthy virus that evades immune
recognition38, and a resistant one as it is only
moderately sensitive to the IFN-α antiviral
response39. Some evidence indicates active countering
of innate immunity by the virus (via STAT1
signaling40, TLR2 recognition41,
etc.). In this study, we demonstrate the existence of another type of
mechanism: saturation of APOBEC/AID factors with their primary target,
HBV rcDNA. This “background defense” is provided by the vast
overabundance of HBV rcDNA compared to 0–50 intracellular copies of
cccDNA and by the preference of APOBEC/AID for single-stranded DNA. We
demonstrate that transcriptionally inactivated cccDNA is more
efficiently deaminated by APOBEC/AID than the transcriptionally active
form. Reducing HBV rcDNA with a reverse transcriptase inhibitor or
HBV-specific siRNA markedly enhances cccDNA deamination by APOBEC/AID.
This positions HBV rcDNA as an important player in viral suppression
mechanisms. However, our results indicate that A3A and A3B, which were
not shown to deaminate the host genome in some previous studies,
deaminate the host genome when rcDNA is depleted. A3C, A3D and A3H
demonstrated weak rcDNA deamination and did not exhibit
cccDNA-deaminating properties, but A3C and A3H increased antiviral
activity of anti-HBV siRNA. At the same time, we did not detect off-site
deamination by A3C, A3D, or A3H in a limited set of cancer-related
genes.