Specific and Nonhepatotoxic
Degradation of Nuclear Hepatitis B
Julie Lucifora,1,2 Yuchen Xia,1 Florian Reisinger,1 Ke Zhang,1 Daniela Stadler,1
Xiaoming Cheng,1 Martin F. Sprinzl,1,3 Herwig Koppensteiner,1 Zuzanna Makowska,4
Tassilo Volz,5 Caroline Remouchamps,6 Wen-Min Chou,1 Wolfgang E. Thasler,7
Norbert Hüser,8 David Durantel,9 T. Jake Liang,10 Carsten Münk,11 Markus H. Heim,4
Jeffrey L. Browning,12 Emmanuel Dejardin,6 Maura Dandri,2,5 Michael Schindler,1
Mathias Heikenwalder,1†‡ Ulrike Protzer1,2†‡
Current antiviral agents can control but not eliminate hepatitis B virus (HBV), because HBV establishes
a stable nuclear covalently closed circular DNA (cccDNA). Interferon-a treatment can clear HBV but
is limited by systemic side effects. We describe how interferon-a can induce specific degradation of
the nuclear viral DNA without hepatotoxicity and propose lymphotoxin-b receptor activation as a
therapeutic alternative. Interferon-a and lymphotoxin-b receptor activation up-regulated APOBEC3A
and APOBEC3B cytidine deaminases, respectively, in HBV-infected cells, primary hepatocytes, and
human liver needle biopsies. HBV core protein mediated the interaction with nuclear cccDNA, resulting
in cytidine deamination, apurinic/apyrimidinic site formation, and finally cccDNA degradation that
prevented HBV reactivation. Genomic DNA was not affected. Thus, inducing nuclear deaminases—for
example, by lymphotoxin-b receptor activation—allows the development of new therapeutics that,
in combination with existing antivirals, may cure hepatitis B.
Hepatitis B virus (HBV) infection remains a major public health threat, with more than 350 million humans chronically infected worldwide at risk of developing end-stage
liver disease and hepatocellular carcinoma. Each
year, more than 600,000 people die from the consequences of chronic HBV infection. A prophylactic vaccine has been available for hepatitis B
for almost 30 years, but the overall number of
chronic infections remains high.
HBV is a small, enveloped DNA virus repli-
cating via an RNA intermediate. The encapsidated
viral genome consists of a 3.2-kb partially double-
stranded relaxed circular DNA (rcDNA) mol-
ecule. The virus has optimized its life cycle for
long-term persistence in the liver (1). Upon trans-
location to the nucleus, the rcDNA genome is
converted into a covalently closed circular DNA
(cccDNA), which serves as the template for
viral transcription and secures HBV persistence.
Nucleoside or nucleotide analogs are efficient an-
tivirals but only control and do not cure HBV in-
fection owing to the persistence of HBV cccDNA.
Therefore, long-term treatment is required, which
is expensive and may lead to concomitant resist-
ance (2). Interferon (IFN)–a is licensed for hepa-
titis B therapy, and treatment with this cytokine
can result in virus clearance in a proportion of
patients; however, its efficacy is limited and
high doses are not tolerated (3). Thus, efficient
and nontoxic elimination of cccDNA in hepato-
cytes is a major goal of HBV research.
Using animal models, it has been shown that
HBV replication—in particular, the cccDNA content of the liver—can be affected by noncytopathic
mechanisms involving cytokines such as interferons and tumor necrosis factor (TNF), which influence RNA and capsid stability (4–7). Here, we
describe an antiviral mechanism that interferes with
cccDNA stability and is distinct from influences
of antiviral cytokines on cccDNA activity (8).
High-Dose IFN-a Leads to cccDNA
Degradation in HBV-Infected Hepatocytes
IFN-a is known to exert transcriptional, post-transcriptional, and epigenetic antiviral effects on
HBV (8–12). To study the effect of IFN-a on
HBV cccDNA, we used HBV-infected, differentiated HepaRG (dHepaRG) cells and primary
human hepatocytes (PHHs). These are human
cell types susceptible to HBV infection (13, 14)
and responsive to IFN-a treatment in vitro (fig.
S1A). IFN-a treatment did not lead to detectable
hepatotoxicity, even at very high doses (fig. S1B).
Treating dHepaRG cells with IFN-a (500 or
1000 IU/ml) controlled HBV-DNA synthesis as
efficiently as the nucleoside analog lamivudine
(LAM) at 0.5 mM (5 times the median effective
concentration, EC50). IFN-a, however, unlike
LAM, also significantly reduced expression of
HBV-RNA and hepatitis B surface (HBsAg) and
e (HBeAg) antigens (Fig. 1A and fig. S1C).
In patients, interruption of LAM treatment
results in a rebound of HBV replication (2). Using
IFN-a, we observed only a partial rebound, or
none at all, in HBV-infected dHepaRG cells after
treatment cessation (Fig. 1A). Because dHepaRG
cells do not allow virus spread, reduction of
HBeAg and the lack of rebound indicated an ef-
fect of IFN-a on the established HBV cccDNA
transcription template separate from the known
antiviral effects on viral replication (14). By
cccDNA-specific quantitative polymerase chain
reaction (qPCR), we determined an 80% re-
duction of cccDNA after 10 days of treatment
(Fig. 1B). Reduction of cccDNA was confirmed
by Southern blot analysis (fig. S1D) and was
dose-dependent (fig. S1E). cccDNA reduction
could be induced at any time point (Fig. 1C) and
persisted over time (Fig. 1, A and C). The effect
was corroborated in HBV-infected PHHs (Fig. 1D).
In contrast to IFN-a, LAM and the even more
potent nucleoside analog entecavir (ETV) at very
high doses (0.5 mM; 1000 times EC50) only in-
hibited reverse transcription, and thus HBV
replication, but not viral persistence (Fig. 1E).
Pretreatment with ETV did not enhance the effect
of IFN-a (Fig. 1F), indicating that IFN-a induces
the decay of established HBV cccDNA. Because
the doses of IFN-a used to achieve this effect were
high, we screened for other cytokines showing sim-
ilar antiviral effects at moderate doses.
LTbR Activation Controls HBV and Leads to
cccDNA Degradation in HBV-Infected Cells
IFN-g and TNF-a are known to control HBV in a
noncytopathic fashion (4, 7) but cannot be used
as therapeutics because they cause severe side
effects. We tested the effect of lymphotoxin (LT)
b receptor (LTbR) activation as an alternative
therapeutic option. The TNF superfamily members LTa, LTb, and CD258 are the physiological
ligands for LTbR and activate either inflammatory
or anti-inflammatory pathways or induce apoptosis
(15). Like hepatocytes (16), dHepaRG (14) and
HepG2-H1.3 cells permit HBV replication (17)
and express LTbR (fig. S2, A and B). To activate
LTbR, we used a superagonistic tetravalent bispecific antibody (BS1) and a bivalent anti-LTbR
monoclonal antibody (CBE11) (18, 19). As expected, LTbR agonists activated canonical (20) and
noncanonical nuclear factor kB (NF-kB) pathways to trigger p100 cleavage (fig. S2C), RelA
phosphorylation (fig. S2D), nuclear RelB and
RelA translocation (fig. S2, E and F), and up-regulation of known target genes (fig. S2G) without causing any detectable hepatocytotoxicity
1Institute of Virology, Technische Universität München–
Helmholtz Zentrum München, 81675 Munich, Germany.
2German Center for Infection Research (DZIF), Munich and
Hamburg sites, Germany. 31st Medical Department, University
Hospital Mainz, 55131 Mainz, Germany. 4Department of Biomedicine, University Hospital Basel, 4031 Basel, Switzerland.
5Department of Internal Medicine, University Medical Center
Hamburg-Eppendorf, 20246 Hamburg, Germany. 6GIGA-Research
Laboratory of Molecular Immunology and Signal Transduction,
University of Liège, 4000 Liège, Belgium. 7Department of General, Visceral, Transplantation, Vascular and Thoracic Surgery,
Grosshadern Hospital, Ludwig Maximilians University, 81377
Munich, Germany. 8Department of Surgery, University Hospital
Rechts der Isar, Technische Universität München, 85748 Munich,
Germany. 9INSERM U1052, CNRS UMR 5286, Cancer Research
Center of Lyon, University of Lyon, LabEx DEVweCAN, 69007
Lyon, France. 10Liver Diseases Branch, National Institute of
Diabetes and Digestive and Kidney Diseases, Bethesda, MD
20892, USA. 11Clinic for Gastroenterology, Hepatology and In-fectiology, Medical Faculty, Heinrich-Heine University, 40225
Düsseldorf, Germany. 12Department of Immunobiology, Biogen
Idec, Cambridge, MA 02142, USA.
*These authors contributed equally to this work.
†Corresponding author. E-mail: firstname.lastname@example.org (U.P.);
‡These authors contributed equally to this work.