From the University Hospital Freiburg, Department of Internal Medicine II/Molecular Biology, Hugstetter Str. 55, D-79106 Freiburg, Germany
Received for publication, July 17, 2000, and in revised form, November 20, 2000
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ABSTRACT |
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Hepatitis B virus (HBV), a small DNA containing
virus that replicates via reverse transcription, causes acute and
chronic B-type hepatitis in humans. The limited success of current
therapies for chronic infection has prompted exploration of alternative strategies. Capsid-targeted viral inactivation is a conceptually powerful approach that exploits virion structural proteins to target a
degradative enzyme specifically into viral particles. Its principal
feasibility has been demonstrated in retroviral model systems but not
yet for a medically relevant virus outside the retrovirus family.
Recently, we found that C proximal fusion to the HBV capsid protein of
the Ca2+-dependent nuclease (SN) from
Staphylococcus aureus yields a chimeric protein, coreSN,
that in Escherichia coli coassembles with the wild-type
capsid protein into particles with internal SN domains. Here we show
that, in HBV co-transfected human hepatoma cells, less than 1 coreSN
protein per 10 wild-type core protein subunits reduced titers of
enveloped DNA containing virions by more than 95%. The antiviral
effect depends on both an enzymatically active SN and on the core
domain. CoreSN does not block assembly of RNA containing nucleocapsids
but interferes with proper synthesis of viral DNA inside the capsid, or
leads to rapid DNA degradation. Our data suggest an intracellular
nuclease activation that, owing to the characteristics of HBV
morphogenesis, is nonetheless highly virus specific. HBV may therefore
be particularly vulnerable to the capsid-targeted viral inactivation approach.
Hepatitis B virus
(HBV),1 an enveloped DNA
containing virus that replicates via reverse transcription (1), is the
causative agent of B type hepatitis in humans. Chronic infections
affect more than 350 million people worldwide, they have potentially severe consequences such a liver cirrhosis and hepatocellular carcinoma
(2), and current treatments are of limited efficacy (3, 4). The
sustained response rate of the approved high-dose interferon- Whereas only primary human, or chimpanzee, hepatocytes are susceptible
to HBV infection in cell culture, a few human hepatoma cell lines like
Huh7 and HepG2 support virus production upon transfection with cloned
viral DNA. The late steps of the infectious cycle (Fig. 1A)
are hence understood in some detail (7, 8), and they present novel
targets for intervention. After infection, the nucleocapsid transports
the partially double-stranded circular 3.2-kb DNA genome to the nucleus
(9) for conversion into covalently closed circular DNA; this molecule
is the transcriptional template for several subgenomic and genomic RNAs
that all act as mRNAs. Of these, the pregenomic RNA (pgRNA) is
first used to translate both the capsid, or core, protein and the
reverse transcriptase, P protein. Then P protein, together with
cellular chaperones (10), binds to a stem-loop structure, The restriction of HBV genome replication to the nucleocapsid makes
this nucleoprotein particle an attractive target for intervention. Apart from nucleic acid-based strategies (19, 20) dominant negative
core protein variants have been described that passively interfere with
nucleocapsid assembly (21-24). A conceptually more powerful approach
is capsid-targeted viral inactivation (CTVI) or, generally,
virion-targeted viral inactivation, which exploits a viral capsid
protein or other virion-associated protein as carrier to target a
degradative enzyme specifically into virus particles (25, 26);
alternatively, nucleic acid-based effectors such as ribozymes may be
fused to viral packaging signals and thus be used against viruses that,
like retroviruses but unlike HBV, encapsidate more than one genome, or
genome segment (27, 28). For the protein based approach, the nuclease
from Staphylococcus aureus (SN) is considered particularly
useful because it requires Ca2+ for activity (29).
Intracellular Ca2+ levels are usually below 1 µM, providing a safeguard against attacks on cellular
nucleic acids. Serum levels of Ca2+, by contrast, are in
the millimolar range; hence SN incorporated into a virus particle is
thought to be activated upon release from the cell.
The principal feasibility of the approach, pioneered using the yeast
retrotransposon Ty1 (25), has been well documented for the model of
Moloney murine leukemia virus, a simple C-type retrovirus (30-34).
Adapting the approach to human immunodeficiency virus has been hampered
by the poor expression and inefficient incorporation into particles of
human immunodeficiency virus Gag fusion proteins (35). Effectors fused
to the accessory Vpr and Vpx proteins, although efficiently
incorporated, can be subject to inactivation by the retroviral protease
(36), and the Vpr carrier may induce cell cycle arrest and apoptosis
(35). Using expression in E. coli we have recently shown
that a chimeric protein consisting of the N-terminal 155 aa of the HBV
core protein followed by the complete SN protein, coreSN, coassembles
with wild-type core protein to particles with internal SN domains (37).
Here we investigated if, and how, this fusion protein is able to
interfere with HBV replication. We show that low levels of coreSN
drastically reduce the titers of replication-competent HB virons in
supernatants from transfected Huh7 cells, and we present evidence for
an intracellular but virus-specific nuclease activation that may make
HBV particularly vulnerable to the CTVI approach.
Plasmid Constructs--
Effector plasmids pCS1-coreSN and
pCS1-coreSNmut were generated by transferring DNA fragments encoding
the chimeric proteins from the prokaryotic vectors pPLC-coreSNwt and
pPLC-coreSNmut43/87 (37) into plasmid pCS1-C1 (38). This plasmid
contains the cytomegalovirus immediate early (CMV-IE) promoter and a
polyadenylation signal from SV40. Control constructs encoding an SN
fusion to a mutant core protein with aa 80 changed from Ala to
Lys (pCS1-coreA80K-SN), and SN without core protein domain were
obtained by conventional polymerase chain reaction-mediated mutagenesis
and cloning into vectors pCS1-coreSN and pcDNA6/Myc-His
(Invitrogen), respectively. pCS1-SN and pCS1-SNmut code for aa 1-149
of mature SN and its inactivated double mutant, preceded by the
dipeptide MD and followed by a C-terminal His-tag; pcDNA6-SN and
pcDNA6-SNmut specify active and inactive SN with the same 6-aa
propeptide sequence as present in coreSN (see Fig. 1) plus a methionine
at the N terminus and a C-terminal His-tag. The HBV expression plasmid
pCHT-9/3091 carries a slightly overlength HBV genome under control of
the CMV-IE promoter (38). Transfection efficiencies were monitored by
co-transfection of plasmid pTR-UF5 (39) encoding a fluorescence
enhanced green fluorescent protein (GFP).
Immunological Techniques--
For Western blot detection of
wild-type core and coreSN proteins (37, 40), either a polyclonal rabbit
antiserum raised against denatured recombinant coreSN protein, or the
monoclonal antibody 10E11, recognizing an epitope between amino acids 8 and 20 on denatured core protein (41), served as primary antibodies; for HBsAg monoclonal antibody 4/7B (42) was used. Detection was
performed using appropriate peroxidase-coupled secondary antibodies and
a chemiluminescent substrate (ECL-Plus, Amersham Pharmacia Biotech).
Blots were exposed to x-ray film or, for quantitation, to a Diana
charge-coupled device camera; band intensities were evaluated using
AIDA software (both Raytest). For immunoprecipitations (38), usually
polyclonal rabbit antisera raised against native recombinant core
protein aa 1-149 (serum H800) or coreSN particles were employed. In
the experiments aimed at directly demonstrating coincorporation of
coreSN in mixed particles with wild-type core protein, monoclonal
antibody mc312 was used. This antibody recognizes a linear epitope
within aa 76-84 of core protein (41, 43) which largely coincides with
a loop exposed on the spikes of core particles. Therefore, the core
variant A80K with a lysine instead of alanine at position 80 reacts
much less efficiently with mc312.
Transfections--
The human hepatoma cell line HuH7 was used
throughout (38). Transfections were performed with FuGENE6 reagent
(Roche Molecular Biochemicals) as recommended by the manufacturer. For
typical co-transfections 50 µl of FuGENE6 and a total of 21 µg of
plasmid DNA/10-cm dish were used. If required, constant amounts of DNA and CMV-IE promoter copies were maintained by adding an appropriate quantity of plasmid pTR-UF5.
Isolation of Secreted and Intracellular HBV
Particles--
Particles contained in culture supernatants collected
from day 3 to 4 post-transfection were enriched by polyethylene glycol precipitation (44), and loaded on a CsCl step gradient (0.9 ml each of
1.5, 1.4, 1.3, 1.2, and 1.1 g/ml CsCl in 10 mM
Tris/Cl Characterization of Viral Proteins and Nucleic Acids--
For
dot blot analysis, equal aliquots from the CsCl gradient fractions were
applied to a positively charged Nylon membrane (Roche Molecular
Biochemicals), using a dot blot apparatus (Bio-Rad). DNAs were detected
by a random primed HBV DNA probe (High Prime DNA Labeling Kit, Roche
Molecular Biochemicals) and quantitated using a PhosphorImager (Fuji
BAS 1500). Core proteins and HBsAg were detected by Western blotting as
described above. For Southern blots, DNAs contained in viral particles
from CsCl gradients, or in immunoprecipitated intracellular cores, were
isolated by proteinase K digestion in the presence of 0.5% SDS, and
purified using the QiaAmp tissue kit (Qiagen). If desired, aliquots
were further treated with avian myoblastosis virus reverse
transcriptase (AMV-RT) as previously described (38). DNAs were
separated on 1% agarose gels and transferred to nylon membranes using
0.4 M NaOH. Detection was performed with either a
32P radiolabeled, or a digoxygenin-labeled probe (Dig High
Prime DNA Labeling Kit; Roche Molecular Biochemicals) as indicated. For
Northern blotting of total RNA about one-third of the cells on a 10-cm
dish were lysed in RLT buffer and the RNA was purified using the RNeasy
Mini kit (both Qiagen). Encapsidated RNA was obtained accordingly from
core specific immunopellets. RNAs were separated on 1.2%
agarose-formaldehyde gels, transferred to nylon membrane in 10 × SSC buffer, and detected using the 32P radiolabeled HBV
probe. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA served
as control. Native agarose gel electrophoresis of cytoplasmic cores was
performed as described (40), using about 1 to 2% of the cytoplasmic
lysate from a 10-cm dish pretreated with DNase I for 30 min at 37 °C
to remove nonencapsidated DNA. After denaturation (0.5 M
NaOH, 1 M NaCl) and neutralization (0.5 M Tris,
pH 7.5, 3 M NaCl) core proteins were detected using H800 serum, and DNA by hybridization with the 32P-labeled probe.
Experimental Design--
For lack of a feasible infection system
we used co-transfection of HuH7 cells with an HBV target plasmid,
pCHT-9/3091, and a coreSN effector plasmid, pCS1-coreSN, as an assay
system. The target plasmid produces, under control of the CMV-IE
promoter, substantial amounts of pgRNA and the subgenomic RNAs
generating all gene products required to form complete virions (38). It should be re-emphasized that, in this setting, there is neither reinfection of the cells nor a significant accumulation of intranuclear HBV covalently closed circular DNA, hence virtually all virions produced derive from the transfected plasmid. The coreSN
gene (Fig. 1B) in pCS1-coreSN,
although also controlled by the CMV-IE promoter, is only moderately
expressed, possibly for lack of introns and/or other elements promoting
nuclear export in the mRNA. Here this low expression appeared
advantageous since even one fusion protein per particle should be
sufficient to exert an antiviral effect (Fig. 1A). To
distinguish nuclease-dependent effects from passive steric
hindrance we analyzed in parallel a homologous fusion protein,
coreSNmut, with two point mutations in the SN part that drastically
reduce enzymatic activity (45).
To test whether coreSN affected expression and/or stability of the
wild-type core protein, Huh7 cells were co-transfected with a constant
amount of plasmid pCHT-9/3091, and various concentrations of
pCS1-coreSN. Core proteins were immunoprecipitated from cytoplasmic lysates using a polyclonal antiserum against denatured recombinant core
protein, H800, that cross-reacts with the chimeric protein (37). For
sensitive Western blot detection a polyclonal antiserum against
denatured recombinant coreSN protein was used which reacts much better
with coreSN than with wild-type core protein. Indeed, a new band at the
expected 36-kDa position was dose-dependently detected
(Fig. 2A). Even at the highest
coreSN concentration the intensity of the 21-kDa wild-type core protein
band was not significantly reduced; the slightly weakened 21-kDa band
in the HBV-only transfected sample is due to some loss of the immune
matrix as evidenced by the weaker band for the immunoglobulin heavy
chain.
To evaluate the antiviral potency of coreSN it was important to know
the relative ratios between both proteins in the transfected cells.
Using recombinant coreSN and wild-type core protein of known
concentration for calibration we found that the monoclonal antibody
10E11, recognizing a linear epitope close to the N terminus of
denatured core protein (41), reacted equally well with both proteins on
Western blots (Fig. 2B). This was true when the proteins were directly loaded at equimolar concentration, and also after immunoprecipitation using H800 serum. For cytoplasmic samples from
cells co-transfected with the HBV construct and equal amounts of the
coreSN or coreSNmut plasmid this assay, after densitometric scanning,
showed that both chimeras were present between 5% and, maximally, 10%
of the wild-type protein.
A potential concern was that, at the relatively low concentrations in
this eukaryotic setting, the coreSN protein would not be cointegrated
into wild-type core particles. Obtaining formal proof for coassembly,
e.g. by coimmunoprecipitation, was not trivial for two
reasons: (i) using an anti-SN antibody to demonstrate co-precipitation
of wild-type core protein would require that the SN domains be exposed
on the particle surface, in contrast to our previous Escherichia
coli data which strongly suggested an interior location of SN
(37); (ii) by necessity of the approach, the core protein part in
coreSN must mimic the structure of wild-type core protein as closely as
possible, otherwise efficient coassembly would be compromised. To
resolve this problem, we resorted to the monclonal anti-core antibody
mc312 which recognizes a linear epitope between aa positions 76 and 84 (41, 43). This sequence overlaps with a surface exposed loop located on
the spikes of the core particles (Fig.
3A). Because an entire protein
such as GFP can be inserted into this loop without a major impact on
protein folding (40), a single aa exchange within the loop sequence would conceivably not affect the assembly capability of a
correspondingly altered core protein but inhibit binding of mc312. We
therefore generated a mutant core protein, coreA80K, in which the
authentic alanine at position 80 is replaced by lysine. This protein
formed particles in E. coli, and it reacted very poorly with
mc312 on Western blots as well as in immunoprecipitation; a
corresponding coreA80K-SN fusion protein (Fig. 3A)
was also assembly-competent.2
This fusion protein should therefore not be precipitated by mc312 unless it is associated with wild-type core protein. This assumption was first confirmed using mixtures of recombinant wild-type core and
coreA80K-SN protein (data not shown). Next, a eukaryotic expression vector for the mutant fusion protein, pCS1-coreA80K-SN, was generated which, except for the single codon exchange, is identical to the coreSN
vector. This plasmid was transfected in Huh7 cells, either alone or
together with the HBV expression plasmid. Subsequently, equal aliquots
of cytoplasmic lysates from both transfections were subjected to
immunoprecipitation with mc312 and, for control, with the polyclonal
antiserum against native coreSN. The precipitates were then analyzed by
Western blotting using the polyclonal antiserum against denatured
coreSN. As shown in Fig. 3B, no antigen-specific signal was
seen in the single transfection when mc312 was used for
immunoprecipitation while the protein was well detectable in the
precipitate obtained with the polyclonal antiserum. By contrast,
coreA80K-SN was precipitated by mc312 when coexpressed with wild-type
core protein. This result indicated that wild-type and fusion protein
did indeed interact. To further prove that this interaction occurred in
the context of complete capsids, aliquots of the same two lysates were
subjected to sedimentation in sucrose gradients. Under the conditions
chosen, wild-type core particles are typically found in the center of
the gradient (37). The corresponding fractions were again used for
immunoprecipitation with mc312. As before, mc312 did not precipitate
coreA80K-SN alone but did so when it was coexpressed with wild-type
protein (Fig. 3B, right panel). Together these data
indicated that the mutant coreSN fusion protein was able to form mixed
particles with the wild-type core protein in eukaryotic cells. The
suitability of coreA80K-SN as a model compound for coreSN was confirmed
by its similar activity against particle-borne HBV DNA (see below).
CoreSN Protein Drastically Reduces HBV DNA in Extracellular
Virions--
The foremost aim of any antiviral strategy is to reduce
the number of infectious virus particles. Therefore we first compared the amounts of genome-containing enveloped virions in supernatants from
cells transfected with only HBV, or co-transfected with HBV and equal
amounts of the coreSN and core SNmut plasmids. A peculiarity of
efficient HBV constructs such as pCHT-9/3091 is the release, by an
unknown mechanism, of nonenveloped cores (38). We therefore used CsCl
density gradients to separate enveloped and naked particles. Aliquots
from the gradient fractions were analyzed by dot blot (Fig.
4A) for HBV-specific nucleic
acids, core protein, and HBsAg which is produced in large excess over
virions. HBV-only transfected cells showed two peaks of HBV nucleic
acid: a weaker one in fractions 10 to 13 at a density around 1.25 g/ml
(expected for enveloped virions), and a stronger one in fractions 16 to
19 at around 1.35 g/ml (expected for naked cores). The presence of core
protein in these fractions was shown by anti-core immunoblotting. HBsAg appeared in fractions 8 to 12 around the expected density of 1.20 g/ml.
The presence of active coreSN protein, but not of the inactive variant
coreSNmut, dramatically changed the DNA pattern: the virion peak was
virtually absent, and the DNA peak from naked cores was substantially
reduced. This reduction was not due to global effects on viral gene
expression because the signal intensities and distributions of core
protein and HBsAg were similar in all three samples.
Next we analyzed the DNAs present in the corresponding fractions by
Southern blotting. For better comparison, all samples were run on one
gel and transferred to one membrane for hybridization and detection
(Fig. 4B). For the HBV-only transfected cells, the virion
peak contained mostly mature DNAs at about 3.2 kb, and a weak band at
the position of single-stranded DNA (fractions 10 to 13). Naked cores
(fractions 16 to 19) contained ssDNA plus additional immature DNA
species extending up to the 3.2-kb position. The samples from
co-transfection with coreSN, on the same exposure, gave no visible
signals in the virion fractions and only weak signals in the naked core
fractions; these consisted of ssDNA and a smear of slower, and of
faster migrating material that was more pronounced than in the absence
of coreSN (panel marked 4x). Further overexposure (panel
marked 10x) revealed small amounts of DNA in the virion
fractions, broadly distributed with no accumulation at the position of
full-length genomes. By contrast, co-transfection with coreSNmut gave,
in general, signals similar to those from HBV-only transfected cells.
Virion DNA was somewhat reduced and contained a higher proportion of
ssDNA. In the naked core fractions, signals were similarly strong but
the smear above the ssDNA did not extend as far up.
Quantitation of the overall signal intensities using a PhosphorImager
revealed that coreSN diminished the DNA in enveloped particles to
2.5%, or less, of that observed in its absence. Coexpressed coreSNmut
reduced the virion signal only to about 70%. For the naked capsids,
the signals were lowered to about 10% by coreSN, while coreSNmut had
little effect (reduction to 94%). These data confirmed that coreSN
efficiently interferes with production of complete virions, and that
most of this interference depends on an active nuclease domain. Whether
the number of physical virus particles was reduced could not be
definitely answered because the large excess of HBsAg precludes a
complete separation from virions; similarly, the core blot signals were
not sharply separated between the virion and core particle fractions,
possibly due to the presence of empty capsids.
Antiviral Activity of CoreSN Depends on the Presence of the Core
Protein Domain--
That coreSN dramatically reduced DNA containing
virions but had no significant effect on HBsAg production (and on core
protein, packaged viral pgRNA, or cellular RNA; see below) strongly
suggested a virus-specific action. However, to further prove this
specificity we also tested the consequences of expressing active SN,
and its enzymatically disabled counterpart SNmut, without the core
protein domain. Two possible outcomes were envisaged: either the
nuclease would exert a generally toxic effect and nonspecifically
inhibit gene expression in, or lead to the death of, the subset of
successfully transfected cells (roughly 10% of the cells with our
procedure). Alternatively, the low intracellular Ca2+
concentration may be insufficient for activation of the enzyme, and no
discernable difference between cells expressing active, inactive, or no
SN would be expected. Especially the latter case would imply a major
difference between free SN, and coreSN incorporated into nucleocapsids.
Two types of expression plasmids for SN and its enzymatically disabled
double mutant SNmut were constructed. The first is derived from plasmid
pCS1 and encodes the mature nuclease, i.e. aa 1-149,
preceded by the dipeptide MD and followed by a His-tag, resulting in a
product with a nominal molecular mass of 17.8 kDa. The second is
derived from the commercial vector pcDNA6/Myc-His and contains, in
addition, the same 6-aa propeptide sequence as present in coreSN; its
nominal molecular mass is 18.4 kDa. The rationale was that the slightly
different products should be discernable by SDS-polyacrylamide gel
electrophoresis and thus aid in identifying the corresponding proteins
by Western blotting using the anti-coreSNdenat antiserum, as was indeed
the case (see Fig. 5).
As an initial test for general toxicity we monitored the effect of SNwt
and SNmut on co-transfected GFP. Over 5 days we did not observe
significant differences in the number of GFP-positive cells, or the
intensity of GFP-fluorescence transfected with active, inactive, or no
SN. This was confirmed biochemically using Western blotting against GFP
which also did not reveal significant differences in the amounts of GFP
protein (see below). This strongly suggested that even expression of
enzymatically active SN was not generally deleterious to the
transfected cells.
Next we performed triple transfections, using combinations of plasmids
encoding HBV (pCHT-9/3091), GFP (pTR-UF5), and one each of the
different SN expression plasmids. Cells transfected with expression
plasmids for coreA80K-SN plus either GFP or HBV were analyzed in
parallel. Western blots of equal aliquots from the individual
transfections, developed using the polyclonal anti-coreSNdenat serum,
showed the expected results (Fig. 5A). The antiserum
detected the coreSN fusions with either wild-type or coreA80K core part (Fig. 5A, upper panel, lanes 1, 2, and 8), and
the 21-kDa wild-type core protein, in similar amounts, in all
transfections containing the HBV expression plasmid (lanes
2-8). In addition, slightly faster migrating bands with small but
distinct mobility differences were seen in samples transfected with the
nonfused SN expression constructs pCS1-SN (Fig. 5A, upper panel,
lanes 3 and 4) and pcDNA6-SN (lanes 5 and 6), strongly suggesting they represented SN and SNmut. Equally sized aliquots of the same lysates were analyzed in parallel (Fig. 5B, lower panel) with monoclonal antibodies to core
protein and GFP. The intensities of the wild-type core protein signals paralleled those seen with the polyclonal anti-coreSN serum, and the
GFP-specific signals were of comparable intensity in all corresponding samples. The slight signal reduction for wild-type core protein and GFP
in the cells transfected with plasmid pcDNA6-SN (lane 5)
was due to a slightly lower transfection efficiency because the band
corresponding to SN itself as well as a nonspecific background band
were also somewhat decreased. Importantly, no difference was seen for
SN versus SNmut from the pCS1 vectors although the detectable SN amounts were, if anything, higher than from the pcDNA6 derivatives (Fig. 5A, upper panel, lanes 3 and
5). These data suggested that free SN lacking a core protein
domain neither inhibited wild-type core protein nor GFP expression, and
they confirmed the absence of a general inhibitory effect on host cell expression by the SN proteins.
Next we compared the DNA signals from enveloped virions, and from naked
cores obtained without additive, or with the active and inactive
nonfused SN proteins encoded by pCS1-SN and pCS1-SNmut by subjecting
the cell culture supernatants from selected transfections to CsCl
density gradient centrifugation. As controls, supernatants from cells
co-transfected with the original coreSN fusion protein and its variant
coreA80K-SN were analyzed in parallel. Fractions covering the density
range of enveloped virions were pooled, and the DNA was analyzed by
Southern blotting (Fig. 5B, left panel). As before, coreSN
led to a drastic signal reduction, and a similarly pronounced decrease
was seen with the coreA80K-SN variant (lane cA80K-SN),
corroborating its suitability as a model compound in the above
described coimmunoprecipitation experiments. By contrast, the nonfused
nuclease (lane SN) had no detectable effect when compared
with the HBV only (lane 0) or inactivated free SN
(lane SNmut) samples. Very similar results were obtained
when the DNAs from released naked cores were analyzed (Fig. 5B,
right panel). Together, these data further corroborated the
specific targeting to viral particles of coreSN, but not free SN, by
means of its core protein domain.
coreSN Protein Moderately Reduces the Nucleic Acid Content but Not
the Amount of Cytoplasmic Cores--
To analyze at what stage of
virion morphogenesis coreSN was acting we next compared the
intracellular nucleocapsids produced in the absence or presence of
coreSN and coreSNmut. Native agarose gel electrophoresis allows to
analyze whether the core protein is present as intact particles (46).
After transfer to a membrane, both core protein and the nucleic acid
contained in the particles can be detected. Cytoplasmic lysates from
singly and doubly transfected cells all contained similar amounts of
particulate core protein (Fig.
6A). By contrast, the
HBV-specific nucleic acid signal from co-transfection with coreSN was
selectively decreased. Semiquantitation by densitometric scanning
showed a reduction to about 30% of that in the other samples (Fig.
6B) when normalized to the scanned protein signals; similar
values (between 15 and 30%) were obtained in several independent
experiments. In contrast to encapsidated DNA, RNA may not be stable
during the procedure and hence cannot reliably be
determined.3 Cores with
coreSN could therefore have contained a higher proportion of pgRNA that
was not properly reverse transcribed to DNA, or have a defect in pgRNA
encapsidation.
To test for a packaging defect we performed Northern blots comparing
total cytoplasmic and encapsidated HBV RNAs (Fig. 6C); for
normalization and proof of absence of nonencapsidated RNA in the
core-derived samples the blot was also probed for GAPDH. All total RNA
preparations gave similar signals, suggesting no significant
intracellular activity of coreSN against free viral or cellular RNAs.
For the encapsidated RNAs, full-length pgRNA was slightly weakened in
the presence of coreSN; however, the smear of smaller products was
proportionally stronger. This indicated that coreSN did not influence
RNA packaging but possibly led to moderate RNA degradation.
Next we analyzed the DNAs contained in the core particles by Southern
blotting. DNA from pure wild-type cores, expectedly, produced a smear
of replicative intermediates extending up to the position of 3.2 kb
linear HBV DNA, with a prominent single-stranded DNA band (Fig.
7A). A similar pattern was
obtained with coreSNmut. In the samples containing coreSN, however, all
signals were reduced, with the strongest remaining band at the position
of ssDNA (Fig. 7B). In several experiments, quantitation by
PhosphorImaging showed a reduction to 15 to 30% compared with the
other samples. This reduction in DNA but not RNA content by coreSN was
compatible with either a failure in reverse transcription, or a
preferential degradation of the reverse transcribed DNA. Evidence
favoring the first possiblity was obtained by analyzing the same
samples after incubation with AMV-RT and dNTPs (Fig. 5, lanes + AMV). Authentic replicative intermediates isolated from cores can
be extended into full-length products using an exogenously added polymerase (47). However, in contrast to the HBV-only, and HBV plus
coreSNmut samples, only a small proportion of the material from
coexpressed coreSN was extended to the 3.2-kb position (Fig. 7B). A semiquantitative evaluation by densitometric scanning
revealed this full-length fraction to account for about 50% of the
total signal intensity for the HBV-only and coreSNmut samples but at most 10% for the coreSN sample. This suggests the existence of breaks
in the RNA and/or DNA templates that impede elongation.
In this report we show the successful application of CTVI to an
important nonretroviral human pathogen, HBV. A chimera of the HBV
capsid protein with the S. aureus nuclease, coreSN,
drastically reduced the titers of DNA-containing enveloped HB virions
in supernatants from cells co-transfected with an efficient HBV
expression plasmid. The antiviral mechanism depends on nuclease
activity because only minor effects were observed with the
enzymatically inactive chimera coreSNmut; likewise, the presence of the
core protein domain is essential as no significant effects were
observed with nonfused SN. CoreSN prevents proper reverse transcription
of the encapsidated RNA, or leads to rapid degradation of the genomic
DNA. This "destruction from within" (26) is the hallmark of CTVI;
unexpectedly, it appears to proceed, to a significant extent, intracellularly.
Antiviral Mechanism of CoreSN--
CoreSN protein, when
recombinantly expressed in E. coli, fulfilled two
fundamental criteria for CTVI: it cointegrates into wild-type capsids,
and the nuclease domains are internally localized (37); all data in the
present study fully confirm this notion for a eukaryotic setting.
However, it was not a priori clear whether this would
translate into a detectable antiviral effect against HBV. In
particular, the Ca2+ dependence of SN should require the
particles to reach a Ca2+-rich milieu. If this was
exclusively the extracellular space it would, in addition, require that
the HBV envelope be permeable to Ca2+ ions, a factor for
which no information exists.
Despite these concerns, coreSN, at 5% to at most 10% the
concentration of wild-type core protein, led to an at least 20-fold, and probably higher, reduction in the titers of extracellular DNA
containing enveloped particles. By contrast, coreSNmut exerted only
minor effects, proving the dependence on nuclease activity of the
antiviral mechanism. The apparent lack of full-length DNA forms as well
as the failure of nonfused SN to show an antiviral effect corroborated
a direct action of coreSN on the packaged genomes. Therefore, the
particles had been in contact with sufficient concentrations of
Ca2+ for nuclease activation, either after release from the
cells, or during export, or inside the cells. Attempts to obtain
evidence for extracellular activation by keeping the cells in medium
with fetal calf serum as the only Ca2+ source (final
concentration about 0.3 mM), or by deliberate addition of
Ca2+ to the supernatants gave no conclusive answer because
the results were essentially identical in all cases (data not shown).
This suggested the nuclease had acted before we analyzed the particles. We therefore also investigated intracellular cores. Whereas their concentration was neither influenced by coreSN nor by coreSNmut protein, selectively the cores produced in the presence of coreSN contained about 70 to 90% less DNA. This reduction was not due to
interference with encapsidation of the RNA template, hence coreSN
either inhibited reverse transcription, or it led to a preferential
degradation of viral DNA. To further minimize degradation after cell
lysis, we performed controls with lysis buffers containing 10 mM, or even 100 mM EDTA to chelate the
Ca2+ ions released from intracellular Ca2+
stores during work-up (data not shown), again with no significant impact. While post-lysis degradation remains formally possible, the
data are fully consistent with an intracellular activation of the
nuclease. Direct evidence against a post-lysis artifact is our ability
to isolate RNA containing cores in similar amounts in the absence and
presence of coreSN although SN degrades both RNA and DNA. Second, even
a very low level activation, resulting in a single cut anywhere within
the pgRNA, or the (
A 70-90% reduction in the DNA content of intracellular cores, and a
greater than 95% reduction in extracellular enveloped particles was
reproduced in several independent experiments with coreSN, and also
with the assembly-competent variant coreA80K-SN. We therefore propose
the following stepwise interference model (compare Fig. 1A):
in the cytoplasm, coreSN is cointegrated into core particles; the
nuclease is activated to a low but sufficient extent to damage the
encapsidated viral nucleic acid, resulting in fewer mature
nucleocapsids. A second important component is the
hepadnavirus-specific coupling of genome maturation and envelopment: of
the fewer DNA containing nucleocapsids in the cytoplasm, even fewer
harbor genomes mature enough for export. Passive steric hindrance
probably contributes to both factors because the most pronounced of the
generally modest effects of coreSNmut were seen with enveloped virions.
Finally, the genomes present in secreted virions may be subject to
further nuclease attack such that, in sum, genome damage plus
secretion inhibition amount to the observed greater than 20-fold
overall inhibition.
While an intracellular nuclease activation seemingly contradicts
previous reports (25, 34), including safety aspects, the two viewpoints
may be reconciled considering the intracellular assembly mechanism of
HBV, and the very low level activation required, namely a single hit in
any of the strands used as template. Like for Moloney murine leukemia
virus Gag-SN fusions (32) we found no evidence that coreSN is
cytotoxic; similarly, nonfused SN had neither a significant negative
influence on cell viability, nor on GFP or HBV expression, suggesting
SN itself had no, or only a marginal intracellular activity. A low
level activity cannot be excluded because it would not be directed
against one specific but against many different targets, and therefore
remain undetectable. By contrast, coreSN integrated into the
intracellularly assembled HBV core particles is kept in close spatial
proximity to its specific nucleic acid substrate for extended time
periods; this situation differs from Moloney murine leukemia virus for
which, as a typical C-type retrovirus, nucleocapsid assembly and
budding occur simultaneously at the plasma membrane. In addition, many
signaling pathways involve the intermittent release of Ca2+
into the cytoplasm and, given the inner volume of the HBV capsid (48),
about 6 Ca2+ ions per particle suffice to make up for a
local 1 mM concentration. Hence all of the data are
consistent with the specific action of coreSN on the encapsidated
genome but not on cellular, and total viral RNAs and/or protein
products. It should be borne in mind that in the transfection system,
in contrast to the commonly used retroviral model infection systems
(see below), HBV expression relies solely on the transfected plasmid.
Because the cells cannot be reinfected, this synthesis route would not
measurably be affected even by the complete interruption of infectious
HBV progeny formation.
Antiviral Efficacy of HBV coreSN Protein--
In our
co-transfection system, we measured a nominal reduction to about 2.5%
in the DNA content of virions by coreSN. Because the residual signals
were weak and broadly distributed, this number may be slightly higher
but could be substantially lower. In addition, the lack of a feasible
infection system precluded determination of infectious HBV particles;
given the incompleteness of most of the remaining DNA genomes, their
number could lag far behind that of physical particles. Assuming a
20-fold reduction caused by coreSN is therefore a conservative
estimate. In the retroviral model systems infectious particle titers
can be directly measured. In prophylactic assays, cells are
intracellularly pre-immunized (49) with an effector gene, and then are
challenged with replication-competent virus at a low multiplicity of
infection. In this setting, from 30 to up to a few thousand-fold
reductions compared with untreated cells have been reported (30, 32,
34). However, because the challenge virus can replicate, the difference
between treated and untreated cells is amplified during each growth
cycle (32). In therapeutic assays, the effector gene is transduced into
persistently infected cells. Except for a recently reported (31)
1000-fold inhibition, mostly reductions in the range of 10-60-fold
have been observed in this format (30, 32, 33), comparable to our
results. In one case, the Gag-nuclease protein was determined to
account for about 25% of the total particulate Gag protein (32). That
coreSN exerted a similar inhibition at a severalfold lower ratio to the
wild-type protein indicates that its antiviral potency against HBV is
as high as that of the Gag-nuclease proteins against Moloney murine
leukemia virus.
CoreSN also appears to be at least as effective as other gene-based
anti-HBV inhibitors. Most nucleic acid-based approaches led to a lesser
reduction of HBV production (19). Relatively high inhibition rates were
described for dominant-negative core protein derivatives (21, 23); in
particular, a core protein fusion to part of the envelope protein was
reported to reduce viral replication by more than 95% at an effector
to target ratio of 1:15 (22). The major antiviral mechanisms appear to
be inhibition of capsid shell formation (22) and passive interference
with RNA packaging and/or reverse transcription (24). Considering the
active mechanism of coreSN it should be even more detrimental to HBV infectivity.
Application of CTVI to HBV may be a valuable new tool to combat this
important viral infection. However, several important problems, in
addition to delivery efficiency, need to be solved before any
therapeutic application can be thought of. An essential intermediate
step is to prove the concept in an in vivo setting. Toward
this end, we are currently generating adenoviruses carrying the
coreSN gene for tests in HBV-transgenic mice and
other surrogate systems (8). Even more important will be experiments in
naturally hepatitis B virus-infected woodchucks. They should also
clarify whether induction of cytotoxic T cells against the transduced coreSN protein would soon abolish its antiviral efficacy or, by contrast, would further contribute to virus elimination by
concomitantly inducing a response against wild-type core protein in the
infected cells.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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therapy is about 30%. Nucleoside analogues such as lamivudine markedly
reduce viral load but suffer, inter alia, from the emergence
of drug-resistant virus variants (5). This situation has spurred
interest in alternative approaches to interfere with HBV replication
(6).
, close to
the 5'-end of the pgRNA (11, 12). Complex formation mediates assembly
of immature RNA-containing nucleocapsids (13), and initiation of
reverse transcription (14-17). DNA synthesis occurs inside the
nucleocapsid and involves several template switches that lead to the
characteristic relaxed circular (RC) DNA genome containing a complete
(
)-strand and variously extended (+)-strands. Mature DNA-containing
nucleocapsids can re-escort the genome to nucleus, or be exported as
enveloped virions by budding into a post-endoplasmic
reticulum/pre-Golgi compartment. Both events apparently require that at
least the (
)-DNA strand be completed (1, 18).
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DISCUSSION
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, pH 7.5, 100 mM NaCl). After 17 h at 35,000 rpm at 20 °C in a SW 50.1 rotor (Beckman), 25 200-µl
fractions were collected from the top. Densities were determined by
refractive index. Intracellular cores were obtained 4 days
post-transfection from cytoplasmic Nonidet P-40 lysates and subsequent
immunoprecipitation with antiserum H800 (38). In some experiments, core
particles were separated from nonassembled core and/or coreSN protein
by sedimentation in 10 to 60% sucrose gradients as previously
described (37).
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ABSTRACT
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DISCUSSION
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View larger version (41K):
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Fig. 1.
Essential intracellular steps of HBV
replication and potential intervention points for coreSN protein.
A, simplified view of progeny virion formation. Nuclear
covalently closed circular DNA is the template for viral RNA synthesis;
besides other transcripts (not shown) pgRNA (wavy line) is
translated into core (C) and P protein (P);
TP denotes the terminal protein domain of P which becomes
covalently fixed to the ( )-strand DNA. P protein binds to the
stem-loop, initiating replication and association of core protein
subunits. Inside the nucleocapsid, P protein reverse transcribes the
RNA via replicative intermediates (RI) into relaxed circular
(RC) DNA. Mature, DNA containing nucleocapsids bud, via
interaction with the envelope proteins (not shown), into a
post-endoplasmic reticulum (ER)/pre-Golgi compartment and are secreted
as enveloped virions. Alternatively, they can reescort the DNA genome
to nucleus. Apart from passively interfering with capsid shell
assembly, pgRNA encapsidation, or reverse transcription, coreSN could
actively degrade any of the packaged nucleic acids; even a single hit
in one of the intermediates would prevent formation of mature capsids
and, possibly, their export as enveloped virion. Note that in the
transfection system used here, there is neither reinfection nor
accumulation of nuclear covalently closed circular DNA; hence
essentially all progeny virions are plasmid-derived. B,
structure of coreSN protein. Bars on the left
indicate the primary sequences of the core and SN protein;
numbers represent amino acid positions. The junction is
shown in detail. E43S and R87G denote aa
exchanges present in coreSNmut that inhibit nuclease activity. The
three-dimensional model on the right is derived from EM and
x-ray studies. Only the fold in front of aa Pro144 is known
in detail. SN has similar dimensions as a core protein subunit but is
only symbolized by a box.
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Fig. 2.
Coexpression of coreSN and wild-type HBV core
protein in HuH7 cells. A, dose-dependence. HuH7 cells
were transfected with HBV and coreSN encoding plasmids at the indicated
ratios. Core proteins in cytoplasmic lysates were immunoprecipitated
and detected by Western blotting using an anti-coreSN antiserum that
preferentially recognizes the fusion protein. Immunoglobulin heavy
chains of the precipitating antibodies are denoted by hc.
B, semiquantitative determination of the relative ratio of
coreSN to wild-type core protein. For calibration, E. coli-derived coreSN and wild-type core protein at the same molar
concentration, alone, or in combination (lanes mix), were
either directly loaded, or first immunoprecipitated, and then detected
using monoclonal antibody 10E11. Lysates from HuH7 cells transfected
with HBV alone (lane 0), or co-transfected with equal
amounts of the coreSN plasmids as indicated were analyzed on the same
blot.
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Fig. 3.
Coassembly of coreSN with wild-type core
protein. A, outline of the coprecipitation assay.
Monoclonal antibody mc312 recognizes an epitope between aa 76 and 84 of
wild-type core protein. This sequence (left) overlaps with a
surface exposed loop at the tips of core particle spikes, schematically
outlined for core protein dimers on the right. In variant
coreA80K-SN the central alanine residue at position 80 was mutated to
lysine (symbolized by the gray crosses). The variant should
not be precipitated by mc312 unless it is physically associated with
wild-type core protein as in mixed particles. The Y-shaped
objects symbolize antibody mc312 (not drawn to scale), the
zigzag lines on the wild-type core dimer denote the basic
C-terminal region not required for assembly but for nucleic acid
binding. B, immunoprecipitation. Plasmid pCS1-coreA80K-SN
was transfected into Huh7 cells, either without or with HBV plasmid
pCHT-9/3091. Equal aliquots from cytoplasmic lysates were used for
direct immunoprecipitation with mc312, or with a polyclonal serum
directed against native recombinant coreSN particles (left
panel). In addition, an aliquot from the co-transfection lysate
was subjected to sucrose gradient sedimentation, and
immunoprecipitation was performed from the pooled fractions that
typically contain core particles (right panel). Precipitates
were resolved by SDS-polyacrylamide gel electrophoresis and detected
with a polyclonal rabbit serum against denatured coreSN protein,
anti-rabbit antibody-peroxidase conjugate, and a chemiluminescent
substrate. The bands marked with an asterisk are derived
from the polyclonal rabbit immunoprecipitation antiserum;
hc, heavy chain.
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Fig. 4.
Inhibition of HB virion production by coreSN
protein. A, dot blot of density fractionated viral
particles from supernatants of transfected HuH7 cells. CsCl gradient
fractions were analyzed for HBV-specific DNA, core protein, and HBsAg
as indicated. Fraction densities are given at the bottom.
The DNA peak at around 1.25 g/ml corresponds to enveloped virions, that
around 1.35 g/ml to naked cores. The presence of core protein in the
intermediate fractions is probably due to the presence of empty cores.
HBsAg appeared in fractions 8 to 13. B, southern blot of DNA
in the virion and naked core fractions. DNA present in the indicated
fractions was isolated, separated by agarose gel electrophoresis and
detected using an HBV-specific 32P-labeled probe. The
positions of full-length double-stranded 3.2 kb and single-stranded
(ss) HBV DNA, and of marker DNAs of the indicated sizes in
kb are shown. The panels marked 10x and 4x are
10- and 4-fold longer exposures to visualize the small amounts of DNA
remaining upon co-transfection with coreSN.
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Fig. 5.
The core domain in coreSN is essential for
antiviral activity. A, lack of effect on nonviral (GFP)
and viral (core protein) gene expression by free SN. Huh7 cells were
transfected with combinations of plasmids encoding the indicated
products. Equal aliquots from cytoplasmic lysates were analyzed by
Western blotting using a polyclonal serum against denatured coreSN
(upper panel), or a mixture of monoclonal antibodies against
core protein and GFP (lower panel). Specific bands were
visualized using appropriate secondary antibody-peroxidase conjugates
and a chemiluminescent substrate. Wild-type (wt) and
inactive (mut) SN proteins from vector pcDNA6-SN contain
the DPTVYS propeptide and are slightly larger than than those from
pCS1-SN; the specific products are marked with white
arrowheads. The weak bands marked with an asterisk were
present irrespective of which plasmid was transfected and are therefore
nonspecific. M2, linear 3.2-kb HBV fragment (ds);
M1, same but heat-denatured (ss).
Numbers on the right indicate the size in kb of
DNA marker fragments. B, lack of effect on DNA containing
enveloped HB virions by free SN. Particles contained in cell culture
supernatants from cells transfected with the HBV vector plus effector
plasmids encoding the indicated products were separated on CsCl density
gradients. DNA was analyzed as described in the legend to Fig. 4,
except that fractions covering the density range for enveloped virions
(left), and from naked cores (right) were pooled,
and that a digoxygenin-labeled HBV probe was used for detection.
coreA80K-SN led to a similar reduction as coreSN whereas no significant
effect was observed with free SN or SNmut.
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Fig. 6.
CoreSN does not affect intracellular
nucleocapsid formation and RNA packaging but reduces DNA content.
A and B, native agarose gel electrophoresis.
Lysates from cells transfected with HBV DNA only (lanes 0)
or co-transfected with coreSN (lanes cSN) or coreSNmut
(cSNmut) were subjected to electrophoresis, blotted, and HBV
core protein (A) and DNA (B) were sequentially
detected on the same membrane. Densitometric scanning of the DNA
versus core protein signals revealed a reduction in nucleic
acid content by coreSN to about 30%. C, Northern blot.
Total and encapsidated RNAs from cells transfected as indicated were
separated by agarose gel electrophoresis and probed for HBV and
cellular GAPDH. Lanes marked mock are derived from
nontransfected cells.
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Fig. 7.
CoreSN reduces the template quality of
encapsidated HBV nucleic acids. A and B,
DNAs from intracellular cores were separated by agarose gel
electrophoresis either without ( AMV), or with prior
fill-in reaction (+AMV) with exogenous AMV-RT and detected
using a digitonin-labeled HBV probe. M denotes
digoxygenin-labeled marker fragments of the indicated sizes;
ds refers to a double-stranded 3.2-kb HBV fragment present
at 100 pg (lane 4) and 400 pg (lane 7); lane
ss contains a heat-denatured, single-stranded aliquot of the
same fragment. To visualize the weak bands obtained in the presence of
coreSN, overexposures of lanes 1 plus 2, and
8 plus 9 are shown in B. Note the only
partial shift toward longer products by AMV-RT.
DISCUSSION
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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)-strand DNA, would prevent formation of mature
double-stranded DNA. The failure to efficiently extend by an
exogenously added polymerase the replicative intermediates produced in
the presence of coreSN, but not of coreSNmut, supports this view.
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ACKNOWLEDGEMENTS |
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We thank Johanna Römmelt and Anja Wahl-Feuerstein for excellent technical assistance, and Hubert E. Blum for providing a stimulating research environment.
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FOOTNOTES |
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* This work was supported by Deutsche Forschungsgemeinschaft Grant DFG Na154/5-1, Bundesministerium für Bildung und Forschung BMBF Grant 01KV9804, Center for Clinical Research I Grant ZKF-B7, and the Fonds der Chemischen Industrie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: University Hospital
Freiburg, Dept. of Internal Medicine II/Molecular Biology, Hugstetter
Str. 55, D-79106 Freiburg, Germany. Tel./Fax: 49-761-270-3507; E-mail:
nassal2@ukl.uni-freiburg.de.
Published, JBC Papers in Press, December 21, 2000, DOI 10.1074/jbc.M006335200
2 J. Vorreiter and M. Nassal, unpublished data.
3 J. Beck and M. Nassal, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: HBV, hepatitis B virus; CTVI, capsid-targeted viral inactivation; HBcAg, hepatitis B core antigen; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; SN, Staphylococcus aureus nuclease; kb, kilobase(s); pgRNA, pregenomic RNA; aa, amino acid(s); CMV-IE, cytomegalovirus immediate early; GFP, green fluorescent protein; AMV-RT, avian myoblastosis virus reverse transcriptase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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