induce a persistent antiviral effect. In contrast to
LAM treatment, no rebound of HBV replication
was observed when BS1 treatment stopped (Fig.
2E). Hence, LTbR activation not only suppressed
HBV replication but also caused nuclear cccDNA
degradation, which is needed to achieve virus
LTbR Activation and IFN-a Treatment Induce
Deamination and Apurinic/Apyrimidinic (AP)
Site Formation in cccDNA
To investigate whether cccDNA degradation
upon LTbR activation or IFN-a treatment was a
result of DNA damage, we examined cccDNA
deamination by differential DNA denaturation
PCR (3D-PCR) (21). Lower denaturing temper-
atures were sufficient for cccDNA amplification
from HBV-infected dHepaRG cells and for PHHs
treated with IFN-a or BS1, compared with de-
naturing temperatures needed to amplify cccDNA
from untreated, LAM-treated, or ETV-treated cells
(Fig. 3A and fig. S7, C and D). Using a cocktail
of recombinant proteins containing all enzymes
necessary for DNA repair, we could reverse the
denaturation of cccDNA (Fig. 3A, lower panels).
The fact that the denaturation temperatures of
mock-, LAM-, and ETV-treated cells also shifted
indicated that this modification of HBV cccDNA
existed even without exposure to exogenous drugs.
Deamination of cccDNA (Fig. 3A, right panel)
and a drop in cccDNA levels after treatment with
CBE11 (table S1) were confirmed in vivo in human
liver chimeric uPA-SCID mice infected with HBV.
Sequencing analyses showed that G→A transi-
tions occurred under treatment (Fig. 3B and fig.
S7, A and B), indicating deamination of cytidines
to uridine in the HBV cccDNA minus strand. At
lower denaturation temperatures, G→A transi-
tions became more obvious (Fig. 3C and fig. S7A).
These data showed that both LTbR activation and
IFN-a treatment led to cccDNA deamination in
vitro and in vivo, and help to explain the G→A
hypermutation observed in patient samples (21).
Neither deamination nor mutations of genomic
DNA were observed by 3D-PCR (fig. S8A) or by
deep sequencing of selected housekeeping genes
or of IFN and LTbR target genes (fig. S8B). This
finding indicated that DNA modifications were
specifically targeted to viral cccDNA.
After cytidine deamination, DNA glycosylases
recognize the damaged DNA and cleave N-
glycosidic bonds to release the base and create an
accessible apurinic/apyrimidinic site (AP site)
that can then be cleaved by endonucleases (22).
These AP sites can be repaired, can lead to mu-
tations upon DNA replication, or can induce DNA
degradation (23). We quantified AP sites created
by LTbR activation or IFN-a treatment. However,
no increase of AP sites in total DNA extracts from
dHepaRG cells or PHHs treated with IFN-a or
LTbR agonists (fig. S8C) was found, indicating
again that our treatments did not lead to detect-
able damage in genomic DNA. Because AP sites
in the small (3.2 kb) cccDNA are very likely to be
missed by this analysis, we digested total DNA
extracts with an AP endonuclease (APE1) and then
amplified cccDNA by qPCR. APE digestion fur-
ther decreased cccDNA extracted from dHepaRG
cells and PHHs treated with IFN-a or LTbR ago-
nists but not with LAM (Fig. 3D). Taken together,
our data indicate that both LTbR activation and
IFN-a treatment induced deamination and AP site
formation in HBV cccDNA, leading to its degrada-
tion, but did not affect genomic DNA.
LTbR Activation and IFN-a Treatment
Up-Regulate Expression of Nuclear
IFN-a is known to induce several cytidine deaminases (23, 24). We performed genome-wide
expression profiling of HBV-infected dHepaRG
Fig. 2. LTbR activation inhibits HBV infection and leads to
cccDNA degradation in HepaRG
cells and PHHs. (A and B) HBV-infected dHepaRG cells were
treated with BS1, CBE11, human
immunoglobulin (hu-IgG) control,
or lamivudine (LAM). Treatment
started 24 hours before infection
for 12 days (A) or at 18 dpi for
10 days (B). Levels of the indicated HBV markers as well as cell
viability are given relative to untreated controls (mock). (C) cccDNA
levels were analyzed after 14 days
of BS1 treatment by Southern blot
in HBV-infected dHepaRG and
HBV-replicating HepG2-H1.3 cells.
Supercoiled cccDNA bands were
identified by their expected size
and linearization upon Eco RI digestion (3.2 kb). (D) PHHs were
infected with HBV and treated with
BS1 at 7 dpi for 10 days. Levels
of the indicated HBV markers were
compared to untreated PHHs of
the same donor (donor 3) (mock).
(E) HBV-infected dHepaRG cells
were treated with BS1, hu-IgG
control, or LAM. Intracellular HBV-DNA was analyzed 8 and 14 days
after treatment cessation. Data are
means T SD of replicates from
independent experiments and
were analyzed by t test. *P < 0.05,
**P < 0.01, ***P < 0.001.