Copyright © 1999, American Society for Microbiology. All Rights Reserved.
Properties of Monoclonal Antibodies Directed against
Hepatitis B Virus Polymerase Protein
JASPER
ZUPUTLITZ,
1† ROBERT E. LANFORD,
2ROLF I. CARLSON,
1LENA NOTVALL,
2SUZANNE M.
DE LAMONTE,
1ANDJACK R. WANDS
1*
Molecular Hepatology Laboratory, Massachusetts General Hospital Cancer Center and Harvard Medical School,
Boston, Massachusetts 02129,
1and Department of Virology and Immunology, Southwest Foundation
for Biomedical Research, San Antonio, Texas 78227
2Received 30 April 1998/Accepted 9 February 1999
Hepadnavirus polymerases are multifunctional enzymes that play critical roles during the viral life cycle but
have been difficult to study due to a lack of a well-defined panel of monoclonal antibodies (MAbs). We have
used recombinant human hepatitis B virus (HBV) polymerase (Pol) expressed in and purified from
baculo-virus-infected insect cells to generate a panel of six MAbs directed against HBV Pol protein. Such MAbs were
subsequently characterized with respect to their isotypes and functions in analytical and preparative assays.
Using these MAbs as probes together with various deletion mutants of Pol expressed in insect cells, we mapped
the B-cell epitopes of Pol recognized by these MAbs to amino acids (aa) 8 to 20 and 20 to 30 in the terminal
protein (TP) region of Pol, to aa 225 to 250 in the spacer region, and to aa 800 to 832 in the RNase H domain.
Confocal microscopy and immunocytochemical studies using various Pol-specific MAbs revealed that the
protein itself appears to be exclusively localized to the cytoplasm. Finally, MAbs specific for the TP domain,
but not MAbs specific for the spacer or RNase H regions of Pol, appeared to inhibit Pol function in the in vitro
priming assay, suggesting that antibody-mediated interference with TP may now be assessed in the context of
HBV replication.
Hepadnaviruses are a group of small, enveloped DNA
vi-ruses that cause acute and chronic hepatitis and strongly
pre-dispose to the development of hepatocellular carcinoma (11).
The prototype member of this virus family is the human
hep-atitis B virus (HBV). Despite containing a small (3 to 3.3 kb)
encapsidated DNA genome, hepadnaviruses are classified as
viral retroelements, because the central step in their
replica-tion cycle is the reverse transcripreplica-tion of an RNA intermediate
(called a pregenome) (57) by virtue of a protein-primed
reac-tion (3, 31, 63). Reverse transcripreac-tion occurs within the
nu-cleocapsid (core particle) composed of the nunu-cleocapsid
pro-tein, the reverse transcriptase (RT)-polymerase (Pol), and the
pregenome which is used as an RNA template. Pol is
com-posed of four domains (44). From the amino terminus, the
domains are (i) the terminal protein (TP), which becomes
covalently linked to negative-strand DNA through the
protein-primed initiation of reverse transcription, (ii) the spacer, which
is tolerant of mutations, (iii) the RT, which contains the
YMDD consensus motif for RT, and (iv) the RNase H.
The mechanism of genome replication for hepadnaviruses
has been determined in detail. The initial step appears to be
the recognition of the pregenomic RNA by Pol. This
recogni-tion occurs best in cis, appears to be cotranslarecogni-tional (2, 18, 19,
22, 25, 43), and is mediated by an RNA sequence (designated
ε
) that is present at both ends of the terminally redundant
pregenomic RNA. However, only the 59
copy of
ε
appears to
function in packaging, and the
ε
sequence in itself is sufficient
to induce the packaging of foreign RNA by HBV Pol (19, 22).
The packaging of Pol is dependent upon an RNA molecule
possessing a 59
copy of
ε
(4). Thus, neither Pol nor pregenomic
RNA can be packaged in the absence of the other. The second
critical event in genome replication involves a priming reaction
in which a nucleotide becomes covalently attached to Pol (3, 5,
38, 58, 63). The addition of the first four nucleotides is
tem-plated by a sequence in a bulge in the 59
copy of
ε
(42, 59, 62).
The primed Pol complex is then translocated to a
complemen-tary sequence present in the 39
copy of a genetic element
termed direct repeat (DR) 1, where the synthesis of
minus-strand DNA resumes (8, 33, 39, 47–49, 59, 62, 67). The
syn-thesis of minus-strand DNA terminates at the 59
end of
pre-genomic RNA (47, 67). The RNA template is degraded by the
RNase H activity of Pol. Only a short terminal
oligoribonu-cleotide remains, which is then translocated to a homologous
site, DR 2, on minus-strand DNA where it serves as the primer
for plus-strand DNA (32, 35, 50, 55). Once plus-strand DNA
synthesis has reached the 59
end of minus-strand DNA, a final
translocation to the 39
end of minus-strand DNA occurs,
re-sulting in a noncovalently closed, partially double-stranded,
circular DNA molecule.
Hepadnavirus Pol proteins play a central role in the viral life
cycle. Recently it was demonstrated that the formation of the
Pol-pregenomic RNA ribonucleoprotein complex in the avian
hepadnavirus duck hepatitis B virus depends on host cellular
factors including the heat shock protein 90 (Hsp-90) and p23,
a chaperone partner of Hsp-90 (21). This chaperone complex
also appeared to be incorporated into viral nucleocapsids.
These findings lend support to the concept that interactions of
molecular chaperones with Pol play a critical role in the
main-tenance of the enzyme in a conformational state that renders it
competent for its various functions.
Several systems which permit the direct analysis of Pol
func-tion in the absence of viral replicafunc-tion and other viral proteins
have been described (20, 29, 30, 51, 58, 63). The Pol system
* Corresponding author. Present address: The Liver Research
Cen-ter, Rhode Island Hospital and Brown University School of Medicine,
55 Claverick St., 4th floor, Providence, RI 02903. Phone: (401)
444-2795. Fax: (401) 444-2939. E-mail: [email protected].
† Present address: Department of Internal Medicine II, University
of Freiburg, 79106 Freiburg, Germany.
4188
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utilizing purified HBV Pol from baculovirus-infected insect
cells has been employed to dissect protein and
protein-RNA interactions involving Pol (30).
The baculovirus system has enabled us to obtain large
amounts of purified Pol protein which we have used in the
present study to raise monoclonal antibodies (MAbs) against
HBV Pol by using the entire protein as the antigen. Such
reagents have been difficult to generate because antigen
prep-arations of sufficient purity did not exist, and Pol appears to be
poorly immunogenic at the B-cell level. We have characterized
these MAbs in detail and have used them as probes for the
mapping of B-cell epitopes of Pol, as well as for studies
ad-dressing the intracellular localization of the protein. In
addi-tion, TP-specific MAbs appeared to inhibit the in vitro priming
reaction.
MATERIALS AND METHODS
Production and purification of recombinant Pol.Recombinant HBV Pol pro-tein carrying a FLAG epitope at the N terminus was produced in baculovirus-infected insect cells as previously described (29, 30). The immunoaffinity purifi-cation of Pol with the M2 MAb (International Biotechnologies Inc., New Haven, Conn.) has been described previously (29, 30). To obtain large quantities of gel-purified Pol for immunizations, Pol was produced in the High Five
Tricho-plusia ni cell line (Invitrogen, Carlsbad, Calif.). High Five cells were infected with
the recombinant baculovirus feline panleukopenia virus (FPL)-Pol (29), and 48 h postinfection the cells were scraped into a TNM buffer (100 mM Tris-HCl, pH 7.5; 30 mM NaCl; 10 mM MgCl2) and sonicated. The cell lysate was clarified, and the insoluble pellet was solubilized by sonication in TNM buffer containing 6 M urea. Pol was separated on sodium dodecyl sulfate (SDS)–8% polyacrylamide preparative gels (26), localized by staining with Coomassie brilliant blue (0.25%) in H2O, and excised from the gel. The gel fragments were homogenized, and Pol was eluted by shaking in 0.1% SDS. Pol was concentrated in a Centricon 30 microconcentrator (Millipore Co., Bedford, Mass.).
Establishment of MAbs against Pol.BALB/c mice were immunized intraperi-toneally with purified Pol protein, and serum from immunized animals was periodically analyzed for reactivity against Pol by Western blotting. After a final intravenous boost with antigen 3 days prior to fusion, spleen cells were fused with the Sp2/O-Ag14 myeloma cell line (American Type Culture Collection, Rock-ville, Md.) as described previously (61). Hybridomas were selected and main-tained as described previously (16, 61). The screening procedure was as follows. Preparations of purified Pol were separated by SDS–8% polyacrylamide gel electrophoresis (PAGE) and transferred to an Immobilon-P membrane (Milli-pore Co.). Undiluted supernatants from hybridoma colonies were applied as the primary antibody with a Miniblotter model 45 (Immunetics, Cambridge, Mass.), which allowed the testing of 45 supernatants on one 13- by 13-cm membrane. Antibodies that bound to Pol were visualized after incubation with a horseradish peroxidase-conjugated sheep anti-mouse antiserum (NA 931; Amersham Life Sciences Inc., Arlington Heights, Ill.) and subsequent chemiluminescence detec-tion with the ECL system (Amersham Life Sciences Inc.). Hybridomas that were immunoreactive with recombinant Pol were cloned by limiting dilution. The MAb isotype was determined with the IsoStrip mouse MAb isotyping kit (Boehr-inger Mannheim, Indianapolis, Ind.). A protein G column (Pharmacia, Piscat-away, N.J.) was used for the affinity purification of MAbs from ascites fluid.
EIA and immunoprecipitation.Recombinant Pol (200 ng/well) was coated onto enzyme immunoassay (EIA) plates (Corning Costar Co., Cambridge, Mass.) for 12 to 16 h at room temperature and incubated for 1 h at room temperature with various MAbs (final concentration, 1mg/ml), followed by incubation for 1 h at room temperature with a 1:5,000 dilution of a horseradish peroxidase-conjugated sheep anti-mouse antiserum (NA 931; Amersham Life Sciences Inc.). Bound antibodies were visualized with the OPD (o-phenylenedi-amine-2–HCl) reagent (Abbott Diagnostics, North Chicago, Ill.). For the immu-noprecipitation of recombinant Pol with MAbs, Sf9 insect cells infected with FPL-Pol were labeled 42 to 46 h postinfection with 200mCi of [35S]methionine (NEN, Boston, Mass.) per ml. Cells were washed twice in phosphate-buffered saline (PBS) and lysed in an extraction buffer (EB) (50 mM Tris-HCl, pH 9.0; 100 mM NaCl; 1% Nonidet P-40), supplemented with protease inhibitors. Clarified lysates were incubated for 4 h at 4°C with MAbs against Pol prebound to protein G affinity beads (Life Technologies, Gaithersburg, Md.), followed by three washes with buffer WB (EB plus 0.5% sodium deoxycholate and 0.1% SDS). Proteins bound to pelleted beads were eluted with SDS-gel sample buffer con-taining 2% SDS and 2%b-mercaptoethanol and separated by SDS–12% PAGE as described previously (27).
Cells, transfections, and infections. The human hepatocellular carcinoma (HCC) cell line HuH-7 (41) was grown in modified Eagle minimal essential medium (Cellgro Mediatech, Washington, D.C.), supplemented with 10% fetal calf serum, 1% nonessential amino acid solution (Life Technologies), and 1% penicillin-streptomycin stock solution (Cellgro Mediatech). Transfections were performed by using a modified calcium phosphate precipitation protocol (7)
routinely with 20mg of DNA plus 1mg of reporter plasmid pTKGH (52) per 100-mm-diameter plate seeded with 73106cells. HuH-7 cells were transfected either with the construct pMT-HBVpol (the kind gift of Heinz Schaller) (44) in which Pol expression is driven by the ubiquitously active human metallothionein IIApromoter (15), or with the construct pCH3142 (22) (the kind gift of Michael Nassal). pCH3142 bears a 1.1 HBV genome-length HBV DNA sequence under the control of the cytomegalovirus immediate-early promoter but carries a 42-nucleotide (nt) deletion from nt 1818 to 1859 (numbering according to reference 10). Transcription from this construct yields pregenomic RNA species carrying a short deletion in the lower stem of the 59εsignal that renders these transcripts noncompetent for encapsidation. As a consequence, pregenomic RNA, the core protein, and Pol are not assembled into nucleocapsids that support viral DNA synthesis, and Pol protein is expected to be present intracellularly in a nonen-capsidated state. For some immunofluorescence experiments, HuH-7 HCC cells were infected with a recombinant vaccinia virus that allowed for the inducible expression of Pol (30a). The FPL-Pol insert was cloned into the pVOTE-2 vector, and a recombinant vaccinia virus was generated as described previously (64).
Protein analysis.The reactivity patterns of the MAbs against Pol and Pol degradation products were determined by Western blot analysis with purified Pol. Pol was separated on a preparative minigel (SDS–12% PAGE) and trans-ferred to a Sequiblot polyvinylidene fluoride membrane (Bio-Rad Laboratories, Hercules, Calif.). Antibodies were incubated with the membrane in individual lanes with a PR-150 Mini decaprobe (Hoefer Scientific Instruments, San Fran-cisco, Calif.) at a final concentration of 5mg/ml, followed by rabbit anti-mouse immunoglobulin G (IgG) (final concentration) and125I-labeled protein A (NEN).
Immunofluorescence and immunocytochemistry studies. HuH-7 cells were grown on sterile glass slides and either transfected by the standard calcium phosphate precipitation protocol (7) or infected with recombinant vaccinia virus. Cells were washed once with PBS and fixed with HistoChoice (Amresco, Solon, Ohio) tissue fixative for 30 min at room temperature. After one wash with PBS, cells were permeabilized with 0.05% Saponin in PBS for 10 min at room tem-perature. After blocking for 1 h at room temperature in PBS–1% bovine serum albumin, MAbs directed against Pol (final concentration, 1mg/ml) were added, and the solution was incubated 12 to 16 h at 4°C. After being washed three times with PBS, slides were incubated for 30 min with a 1:250 dilution of a biotinylated horse anti-mouse antiserum (Vector Laboratories, Burlingame, Calif.). For im-munofluorescence, cells were equilibrated in 0.1 M NaHCO3–1.5 M NaCl, pH 8.2, for 5 min, and the final incubation was performed with an avidin-fluorescein isothiocyanate conjugate (Vector Laboratories) at a 1:500 dilution. Cover slides were mounted in Vectashield (Vector Laboratories) and examined with a Nikon Labophot photomicroscope equipped with the epifluorescence attachment EF-D (Nikon, Garden City, N.Y.). Confocal microscopy was performed with a Leica TCS4D confocal scanner (Leitz, Wetzlar, Germany). For immunocytochemistry, cells were incubated with the Vectastain Elite ABC reagent and stained by using a 3,39-diaminobenzidine substrate kit (both from Vector Laboratories) according to the instructions of the manufacturer.
Epitope mapping.A set of deletion mutants of Pol produced in and purified from baculovirus-infected insect cells was used to test the reactivity of MAbs against Pol by Western blotting. The constructs represented a series of amino-and carboxy-terminal deletion mutants of the TP amino-and RT domains amino-and permitted the mapping of epitopes to within 10 to 32 amino acids. The details of the construction of these vectors are described elsewhere (28).
Pol assays.Pol reactions were performed with Pol polypeptides immunopre-cipitated by MAbs against Pol and still bound to the affinity beads. The beads were suspended in TNM (100 mM Tris HCl, pH 7.5; 30 mM NaCl; 10 mM MgCl2) containing 100mM concentrations of unlabeled deoxyribonucleoside triphosphates (dATP, dGTP, and dCTP) and 5mCi of [a-32P]TTP (3,000 Ci/ mmol; NEN). Assays were routinely performed at 30°C for 30 min. Densitometry of gels was performed using the NIH Image 1.60 software (42a).
RESULTS
Generation of MAbs against Pol protein.
Initially, animals
were immunized with Pol purified by the M2 MAb affinity
column. When analyzed by Coomassie blue-stained
SDS-PAGE, this material derived from insect cells contained
sev-eral additional bands that copurified with Pol (29). Such
pro-teins could not be removed from Pol without denaturing the
protein. Mice immunized with this material showed a
predom-inant immune response against a protein with an apparent
molecular weight (MW of 70,000), but no reactivity against Pol
was detectable. Pol appeared to be less immunogenic than one
or several of the contaminating bands. Therefore, mice were
repeatedly immunized with gel-purified Pol protein over the
time course of 1 year. Finally, the serum of one animal that had
been immunized intraperitoneally seven times exhibited a
strong reactivity against Pol at a serum dilution of 1:5,000. This
animal was used for the cell fusion with Sp2/O-Ag14 myeloma
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cells. The screening of hybridoma supernatants was performed
by Western blotting. Six hybridomas producing Pol-specific
MAbs were obtained from this fusion. Table 1 summarizes the
characteristics of these MAbs. All MAbs functioned well in a
Western blot format, and all except the IgM MAb 10B9
rec-ognized Pol in an EIA format. MAbs 2C8, 8D5, and 9F9 were
able to detect endogenously synthesized Pol by indirect
immu-nofluorescence. These three antibodies also functioned well in
immunoprecipitation studies as shown below.
Epitope mapping.
Figure 1 shows the results from epitope
mapping studies performed by using all six MAbs as primary
antibodies in immunoblots against deletion mutants of Pol.
MAbs 1B4, 7C3, and 10B9 all recognized an epitope within
amino acid (aa) positions 20 to 30 of Pol in the TP region.
MAb 2C8 recognized an epitope between aa positions 8 and 20
within the TP region. An epitope between aa positions 225 and
250 in the spacer region of Pol was recognized by MAb 8D5.
The MAb 9F9 recognized an epitope between aa positions 800
and 832 within the RNase H domain of Pol. Thus, murine
B-cell epitopes of HBV Pol appeared to be positioned at the
very N and C termini of the protein and within the Pol spacer
region.
Detection of endogenously synthesized Pol by indirect
im-munofluorescence.
HuH-7 HCC cells were transfected with the
construct pMT-HBVpol in which Pol expression is driven by
the human metallothionein IIA
promoter (Fig. 2A to F). The
expression level of Pol from this construct was estimated to be
at least 10 times higher than the level obtained from the
en-dogenous Pol promoter. Alternatively, Pol was also inducibly
expressed in HuH-7 cells after infection with a recombinant
vaccinia virus coding for Pol (VVPol) (Fig. 2G to K). The
expression levels of Pol reached with VVPol
were at least 10
times higher than those with pMT-HBVpol. Cells were fixed 2
days after transfection or 4 h after vaccinia virus infection. The
results shown in Fig. 2 illustrate intracellular staining patterns
obtained with the MAb 2C8, specific for an epitope in the TP
domain, and 8D5, specific for an epitope in the spacer region.
Staining with the MAb 9F9 yielded similar results (data not
shown). As demonstrated in Fig. 2A and B, a fine granular,
cytoplasmic staining pattern (compare Nomarski images in
panels D and E) was observed with both MAbs when Pol was
expressed from the construct pMT-HBVpol. Control
transfec-tions with the HBV L protein expression construct pApLHBs
(13) and incubation with Pol MAb 8D5 (Fig. 2C and F) or 2C8
(data not shown) did not result in specific signals. All
VVPol-infected HuH-7 cells exhibited a very strong cytoplasmic
stain-ing pattern (Fig. 2G). When such cells were incubated with the
HBV L protein-specific MAb 18/7 (17), no specific signals were
visible (Fig. 2H). Nuclear staining was not detectable in all
cases. Similar results were obtained when murine BALB/3T3
fibroblasts were infected with VVPol
(data not shown). These
data illustrated that MAbs 2C8, 8D5, and 9F9 were capable of
detecting endogenously synthesized Pol protein in transfected
or vaccinia virus-infected HCC cells. Pol expressed in HuH-7
HCC cells appeared to be exclusively localized in the
cyto-plasm.
[image:3.612.55.555.84.182.2]Immunocytochemistry was used to detect HBV Pol
intracel-lularly in the presence of the other HBV proteins. When cells
TABLE 1. Characteristics of MAbs
Characteristic or test MAb
1B4 2C8 7C3 8D5 9F9 10B9
Isotype
IgG
1k
IgG
1k
IgG
2ak
IgG
1k
IgG
1k
IgM
k
Immunoblot
1
1
1
1
1
1
EIA
1
1
1
1
1
2
Indirect immunofluorescence
2
1
2
1
1
2
Immunoprecipitation
(
1
)
a1
(
1
)
a1
1
2
Epitope
aa 20–30
aa 8–20
aa 20–30
aa 225–250
aa 800–832
aa 20–30
Pol domain
TP
TP
TP
Spacer
RNase H
TP
a(1), weak reactivity.
FIG. 1. Epitope mapping of Pol MAbs. MAbs 1B4, 2C8, 7C3, 8D5, 9F9, and 10B9 were used as primary antibodies in immunoblots against various deletion mutants of Pol expressed in and purified from baculovirus-infected insect cells (see Materials and Methods). MAbs 1B4, 7C3, and 10B9 recognize an epitope within aa 20 to 30 of Pol in the TP region of HBV subtype ayw. MAb 2C8 recognizes an epitope between aa 8 and 20 within the TP region. An epitope between aa 250 and 275 in the spacer region of Pol is recognized by MAb 8D5. The MAb 9F9 recognizes an epitope between aa 800 and 832 within the RNase H domain of Pol.
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[image:3.612.107.508.540.689.2]FIG. 2. Confocal microscopy studies of endogenously synthesized Pol in HuH-7 HCC cells with Pol MAbs. Immunofluorescence (A, B, C, G, and H) and Nomarski images (D, E, F, J, and K) are illustrated. MAbs used for the staining are indicated on the lower right of each immunofluorescence image. (A to F) HuH-7 HCC cells transfected with the construct pMT-HBVpol in which Pol expression is driven by the human metallothionein IIApromoter. (G to K) Expression of Pol in HuH-7 cells after infection with recombinant VVPol. Cells were fixed 2 days after transfection or 4 h after vaccinia virus infection. The intracellular staining patterns obtained with the MAbs 2C8 (specific for an epitope in the TP region) and 8D5 (specific for an epitope in the spacer region) are illustrated. MAb 9F9 yielded similar results (data not shown). A fine granular, exclusively cytoplasmic staining pattern (compare panel A with D, B with E, and G with J) is observed. Control transfections with the HBV L protein expression construct pApLHBs (13) and staining with Pol MAb 2C8 (data not shown) 8D5 do not result in specific signals (C and F). VVPol-infected cells are negative when stained with the HBV L protein-specific MAb 18/7 (17) (H and K). Similar results were obtained when murine BALB/3T3 fibroblasts were infected with VVPol(data not shown).
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transfected by HBV constructs (of more than one genome
length) that allow for viral DNA synthesis were analyzed with
MAb 2C8, 8D5, or 9F9, no signals corresponding to HBV Pol
could be detected, while viral core and envelope proteins were
readily detectable (data not shown). Similar results were
ob-tained when the cell line HepG2-2.2.15 (53), stably expressing
HBV proteins and replicating the virus, was analyzed (data not
shown). When liver tissue sections from mice transgenic for
HBV were subjected to immunohistochemistry with the
Pol-specific MAbs, no signal was detected (data not shown;
anal-ysis kindly performed by Luca Guidotti, Scripps Research
In-stitute). Some possible reasons for the inability to detect Pol in
these experimental settings included (i) low intracellular levels
of Pol and/or (ii) the inaccessibility of Pol due to encapsidation
of the protein into core particles. To test the latter hypothesis,
we used the construct pCH3142 (22), coding for mutant
pre-genomic RNA species with a deletion in the 59
ε
signal
ren-dering these transcripts noncompetent for encapsidation. After
the transfection of cells with pCH3142, pregenomic RNA, core
protein, and Pol were not expected to be assembled into
nu-cleocapsids supporting viral DNA synthesis, and Pol protein
was likely present intracellularly in a nonencapsidated state.
HuH-7 HCC cells were transfected with either pMT-HBVpol
or pCH3142, and Pol was detected with MAb 2C8 (Fig. 3). As
a control, HBV core protein was detected with a polyclonal
rabbit anti-HBcAg antiserum (DAKO, Carpinteria, Calif.). A
faint cytoplasmic signal corresponding to HBV Pol was
detect-able in cells transfected with pCH3142 (Fig. 3B), which was at
least 10 times weaker than the signal obtained after the
trans-fection of pMT-HBVpol (Fig. 3A). The HBV core protein was
readily detectable in the nuclei and cytoplasms of cells
trans-fected with pCH3142 (Fig. 3D), while it was not observed in
cells transfected with pMT-HBVpol (Fig. 3C). In addition,
HBV envelope proteins were detectable after the transfection
of pCH3142 (data not shown). These data suggested that HBV
Pol was predominantly localized in the cytoplasms of
trans-fected cells in the presence of other HBV proteins.
Western blots and immunoprecipitations.
All Pol MAbs
ex-cept 10B9 were analyzed for their staining patterns of Pol and
Pol degradation products on Western blots. The M2 MAb
served as a positive control. This MAb is known to bind to the
N terminus of Pol. As demonstrated in Fig. 4, lane 6, M2
recognized full-length Pol as well as degradation products
con-taining the N terminus with apparent MWs down to ca. 30,000.
MAbs 1B4, 2C8, and 7C3 exhibited the same staining pattern
as M2 (Fig. 4, lanes 1 to 3). These data are consistent with the
observation that these Pol MAbs recognize epitopes at the N
terminus of Pol. MAb 8D5 (Fig. 4, lane 4) stained all
Pol-associated bands except the smallest one, which is consistent
with its epitope being located in the spacer region. Finally,
MAb 9F9 (Fig. 4, lane 5) identified only full-length Pol and a
very minor degradation product with an apparent MW of
68,000. This pattern is consistent with the observation that
MAb 9F9 recognizes an epitope at the C terminus of Pol.
[image:5.612.98.497.69.373.2]Pol MAbs were analyzed for their potential to
immunopre-cipitate metabolically labeled Pol from cellular extracts of Sf9
insect cells. As demonstrated in Fig. 5, lanes 3, 5, and 6, MAbs
2C8, 8D5, and 9F9 were able to immunoprecipitate Pol well.
FIG. 3. Detection of HBV Pol in the presence of other HBV proteins. The immunocytochemistry of cells transfected with pMT-HBVpol (A and C) or pCH3142 (B and D), a construct that allows for the intracellular expression of Pol in a nonencapsidated state in the presence of core and envelope proteins (22), is shown. The staining was performed with MAb 2C8 (A and B) or a polyclonal rabbit anti-HBcAg antiserum (DAKO) (C and D). A faint cytoplasmic signal corresponding to HBV Pol is detectable in cells transfected with pCH3142 (B). In addition, HBcAg is detectable in the cytoplasms and nuclei of pCH3142-transfected cells (D). pMT-HBVpol-transfected cells exhibit the previously detected, strong cytoplasmic staining pattern for Pol (A) (see Fig. 2), while no HBcAg is detectable (C).on November 9, 2019 by guest
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The M2 MAb (Fig. 5, lane 10) served as a positive control.
MAbs 1B4 and 7C3 yielded only very small amounts of Pol,
whereas the IgM MAb 10B9 did not immunoprecipitate Pol
(Fig. 5, lanes 2, 4, and 7). Several additional signals with lower
apparent MWs than that of Pol were visible after
immunopre-cipitations with MAbs 2C8, 8D5, and 9F9. It is currently
un-clear which proteins correspond to the observed signals, but it
is unlikely that they represent the Pol degradation products
observed by Western blotting, since 2C8 and 9F9 would not be
expected to recognize the same degradation products of Pol.
Of note, the MAb 9F9 coimmunoprecipitated a protein that
was visible as a band with an apparent MW of 27,000. This
band did not appear to correspond to a degradation product of
Pol since it was not recognized by Western blotting with the
same antibody.
[image:6.612.91.257.74.199.2]Inhibition of in vitro priming with Pol MAbs.
All Pol MAbs
that were capable of immunoprecipitating Pol were tested for
their potential to inhibit the in vitro priming activity of the
enzyme. For this purpose, Pol reactions were performed with
Pol proteins immunoprecipitated by Pol MAbs. The
Coomas-sie blue-stained protein gel (Fig. 6, top panel) illustrates the
amounts of Pol precipitated with the various MAbs, and the
lower panel in Fig. 6 shows the result from the priming
reac-tion of the same samples with Pol bound to the protein G
beads by the respective antibodies. Signals present on the gels
were quantified by densitometry. Immunoprecipitation and in
vitro priming with the M2 MAb (Fig. 6, lane 8) served as a
positive control. No immunoprecipitation of Pol was observed
with the negative-control antibodies C7-57, specific for the
bacterial glutathione S-transferase protein (40) (Fig. 6, lane 6),
and 12CA5 directed against an influenza virus hemagglutinin
FIG. 4. Detection of recombinant Pol by MAbs on Western blots. Stainingwith the FLAG epitope-specific M2 MAb (lane 6) serves as a positive control. M2 recognizes full-length Pol (position indicated on the left) as well as C-terminal degradation products with apparent MWs down to ca. 30,000. MAbs 1B4 (lane 1), 2C8 (lane 2), and 7C3 (lane 3) exhibit the same staining pattern as M2, consistent with the observation that these Pol MAbs recognize epitopes at the N terminus of Pol. MAb 8D5 (lane 4) stains all Pol-associated bands except the smallest one, which is consistent with its epitope being located in the spacer region. MAb 9F9 (lane 5) stains only full-length Pol and a minor degradation product with an apparent MW of 68,000. Positions of molecular mass markers are indicated on the right.
[image:6.612.312.549.76.312.2]FIG. 5. Immunoprecipitation of metabolically labeled Pol from cellular ex-tracts of Sf9 insect cells. MAbs 2C8 (lane 3), 8D5 (lane 5), and 9F9 (lane 6) immunoprecipitate Pol well. The M2 MAb (lane 10) serves as a positive control. MAbs 1B4 (lane 2) and 7C3 (lane 4) yield only very small amounts of Pol, whereas the IgM MAb 10B9 (lane 7) does not immunoprecipitate Pol at all. Controls with protein G alone (lane 8) and an irrelevant antibody (lane 9) are negative. MAb 9F9 coimmunoprecipitates a protein that is visible as a band with an apparent MW of 27,000. The position of full-length Pol protein is indicated on the right. Lane 1, molecular mass markers.
FIG. 6. Inhibition of in vitro priming with TP-specific MAbs. (Top panel) Immunoprecipitation (IP) of Pol protein by various MAbs and subsequent de-tection by Coomassie blue-stained SDS-PAGE. (Bottom panel) In vitro priming assay of the same samples (Pol protein radiolabeled by nascent HBV minus-strand DNA; see Materials and Methods). The Coomassie blue-stained protein gel shows the amounts of Pol precipitated with the various MAbs. Immunopre-cipitation and in vitro priming with the M2 MAb (lane 8) serve as a positive control. No immunoprecipitation of Pol is observed with the negative-control antibodies C7-57 (specific for the bacterial glutathione S-transferase protein) (lane 6) and 12CA5 (directed against an influenza virus hemagglutinin peptide sequence) (lane 7), and consequently, no in vitro priming is detectable. The MAb 2C8 (bottom panel, lane 4) inhibits priming by 86% (analysis by densitometry) when compared with M2 (bottom panel, lane 8). In contrast, the MAb 9F9 (bottom panel, lane 5) does not inhibit priming. All three MAbs immunopre-cipitate equal amounts of Pol (top panel, lanes 4, 5, and 8). MAbs 1B4, 7C3, and 8D5 immunoprecipitate less Pol than M2 (top panel, lanes 1, 2, 3, and 8). MAbs 1B4 and 7C3 strongly reduce priming (1B4, 98% inhibition; 7C3, 81% inhibition) when compared with MAbs 8D5 and M2 (bottom panel, lanes 1, 2, 3, and 8). The positions of mouse Ig heavy chains (HC) and light chains (LC) are indicated on the right. Positions of molecular mass markers are indicated on the left.
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[image:6.612.79.268.431.648.2]peptide sequence (Fig. 6, lane 7), and consequently, no in vitro
priming was detectable. When Pol was immunoprecipitated
with the MAb 9F9, no inhibition of priming activity was
ob-served (Fig. 6, lane 5). In contrast, the MAb 2C8
immunopre-cipitated equal amounts of Pol protein (Fig. 6, lane 4), but the
extent of in vitro priming was inhibited by 86%. A 14%
inhi-bition of priming was observed in the case of the MAb 8D5
(Fig. 6, bottom panel, lane 3). MAbs 1B4 (Fig. 6, top panel,
lane 1) and 7C3 (top panel, lane 2) immunoprecipitated equal
amounts of Pol when compared with MAb 8D5, but the in vitro
priming obtained with MAbs 1B4 and 7C3 was strongly
re-duced (1B4, 98% inhibition; 7C3, 81% inhibition) when
com-pared with MAb 8D5 (Fig. 6, bottom panel; compare lanes 1
and 2 with lane 3). These observations suggested that
TP-specific MAbs were capable of inhibiting in vitro priming by
Pol. However, the possibility existed that these MAbs
recog-nized and immunoprecipitated an inactive fraction of the Pol
expressed in insect cells. To examine this possibility, Pol was
immunoprecipitated with M2 to ensure that the
immunopre-cipitated protein represented the active fraction, and then Pol
still bound to the M2 beads was exposed to purified MAb 2C8
or 9F9 or to the buffer without antibodies. The beads were
washed to remove excess antibody, and priming reactions were
conducted with the bound Pol. The buffer control and
9F9-exposed Pol exhibited similar priming activities, while the
priming reaction for 2C8-exposed Pol was reduced by more
than 50% (data not shown). These observations confirmed that
the MAbs directed to the TP domain were capable of
inhibit-ing Pol in vitro priminhibit-ing activity.
DISCUSSION
This report describes the generation and characterization of
MAbs against the full-length HBV Pol protein and their value
for study of this protein which plays a central role in the viral
life cycle. A panel of six MAbs against Pol was generated from
a mouse immunized seven times over the time course of 1 year.
Several attempts to obtain Pol MAbs from animals that had
been immunized fewer times over shorter time periods were
unsuccessful. In addition, the purity of the Pol antigen used for
immunizations turned out to be a critical factor. Standard
purified Pol preparations from baculovirus-infected insect cells
(29) still contained ample amounts of several additional
pro-teins of which at least one contaminant was found to be very
immunogenic. Eventually, only gel-purified material was able
to elicit an immune response that was sufficient for the
gener-ation of Pol-specific MAbs. The recombinant Pol used in this
study was poorly immunogenic with respect to eliciting a
hu-moral immune response in mice. Interestingly, a fusion
per-formed with spleen cells from an animal that had been
immu-nized four times over half a year yielded only MAbs of the
isotype IgM (data not shown), suggesting that the affinity
mat-uration and isotype switching in mice during the humoral
im-mune response to recombinant Pol occur slowly. In contrast,
Rehermann et al. (45) found Pol to be quite immunogenic at
the cytotoxic T-lymphocyte level. These authors also noted a
rapid degradation of Pol from its C terminus, which also was
detectable with the various MAbs used in this study. Of note,
one other murine MAb produced against a recombinant Pol
polypeptide derived from the TP region has been described
(14).
Epitope mapping studies presented here demonstrated that
the newly established MAbs recognized four different epitopes
on Pol. Two of these epitopes are positioned adjacently to each
other at the N terminus: one is in the spacer region and the
fourth is located at the C terminus of Pol. It has been
previ-ously demonstrated that certain patients infected with HBV
exhibit humoral immune responses against Pol (6, 9, 24, 56, 65,
66, 69). Most of these studies have identified antigenic regions
of the Pol protein at the N and C termini, whereas the immune
responses to central regions of the Pol protein were
repre-sented to a lesser extent. Our study shows that murine B-cell
epitopes appear to be located in Pol regions that have
previ-ously been demonstrated to elicit humoral immune responses
in HBV-infected individuals.
Indirect immunofluorescence and confocal microscopy as
well as immunocytochemistry studies using the newly
estab-lished MAbs demonstrated that full-length Pol appeared to be
exclusively localized in the cytoplasms of HuH-7 HCC cells,
even in the presence of other HBV proteins. In no case was
nuclear staining detectable, neither by MAb 2C8, which binds
to the terminal protein region, nor by MAb 8D5, reactive with
the spacer region, or MAb 9F9, directed against the RNase H
domain. Similar observations were made after the infection of
HuH-7 HCC cells or BALB/3T3 fibroblasts with VVPol.
Vac-cinia virus-infected cells overexpressed Pol, and strong staining
throughout the cytoplasm was observed. However, the
pres-ence of Pol in the nucleus of transfected or infected cells at
very low levels that were not detectable by the MAbs cannot be
excluded. Our observations suggest that the full-length HBV
Pol protein alone does not contain a nuclear localization signal
that is efficiently recognized by the cell types used in this study.
This finding is relevant because Pol protein is covalently bound
to the minus DNA strand of the virion-encapsidated form of
the genome. Therefore, it has been suggested that Pol may play
a role in the intracellular amplification of viral covalently
closed circular DNA by facilitating the entry of viral genomes
(68) from mature core particles that are located in the
cyto-plasm into the nucleus. Kann and coworkers (23) have
ad-dressed this question by analyzing the intracellular trafficking
of viral components and complexes in digitonin-permeabilized
HuH-7 HCC cells, whose cytosol was substituted by rabbit
reticulocyte lysate and an ATP-generating system. They found
that a woodchuck hepatitis B virus-derived Pol-DNA complex
was efficiently transported into the nuclei of HuH-7 cells by an
ATP-dependent mechanism, whereas deproteinized viral DNA
remained completely outside the nucleus, suggesting that the
viral Pol is sufficient for mediating the transport of the viral
genome into the nucleus. However, a possible contribution of
core protein subunits associated with Pol (3) to the transport of
viral genomes into the nucleus could not be excluded.
An immunohistochemical study performed with liver
speci-mens from patients chronically infected with HBV
demon-strated that polyclonal antisera raised against portions of the
TP regions of Pol stained hepatocytes predominantly in the
nucleus (37). These observations suggested that either the
en-tire Pol protein or the portion encoding the TP is translocated
to the nucleus during the course of natural infection, although
the mechanism of transport remained unclear. Our
observa-tion that the MAb 2C8, which is specific for an epitope within
the TP region of Pol, did not exhibit a nuclear staining pattern
is not necessarily contradictory to these findings, because we
expressed Pol in transfected cells, either in the presence or
absence of other viral proteins. Our observation that Pol alone
remains in the cytoplasms of transfected cells even when
strongly overexpressed makes it possible that the putative
sig-nal that mediates the entry of Pol or Pol subdomains into the
nuclei of naturally infected cells consists of multiple
compo-nents and may be active only during certain stages of the viral
life cycle. Thus, the intracellular localization of Pol may be
determined by viral and/or cellular factors that are associated
with it. It will be of interest to reassess the intracellular
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ization of Pol and Pol subdomains in the infected liver by using
the MAbs described here.
The MAbs developed in our study were used to investigate
whether the inhibition of in vitro priming by Pol could be
achieved with these reagents. We used the various MAbs to
immunoprecipitate Pol and then performed priming-reverse
transcription reactions. These experiments revealed that the
TP-specific MAbs 1B4, 2C8, and 7C3 were able to inhibit
priming and reverse transcription, whereas the spacer-specific
MAb 8D5 and the RNase H-specific MAb 9F9 were not. These
data suggest that the TP-specific MAbs were able to interfere
with the TP function in this assay and point to the importance
of the conservation of structural features within the TP region
of Pol for the proper function of the enzyme. While the
ex-perimental system used for these studies is quite different with
respect to in vivo viral replication, a further assessment of the
potential of the MAbs described here to interfere with Pol
functions within the cell will be of interest.
Considerable efforts are being made to develop compounds
that block HBV replication. One present focus is to find
chem-ical compounds that selectively interfere with essential steps of
the viral life cycle and replication without significantly affecting
host cell metabolism. HBV Pol is a candidate target protein,
because it is essential for viral multiplication. Inhibitors that
target Pol fall within two broad categories, nucleoside analogs
and nonnucleoside derivatives. However, prolonged
chemo-therapy may result in the emergence of resistant viruses. Such
resistance phenomena may be due to specific changes in the
gene encoding Pol (1, 34, 60). Therefore, alternative strategies
against HBV based on gene therapy approaches are being
actively studied (70). One such strategy involves the expression
of virus-specific recombinant antibodies targeted to
intracellu-lar compartments of infected cells in order to interfere in a
specific manner with the corresponding viral antigen (46). This
approach has been demonstrated to be effective against human
immunodeficiency virus type 1 in experimentally infected cells.
For example, intracellularly expressed engineered antibodies
against human immunodeficiency virus type 1 RT were able to
confer protection against viral infection and replication (36,
54). An important requirement for this approach is the
avail-ability of recombinant forms of a high-affinity antibody specific
for the target antigen (12) and the characterization of the
antibody with respect to its possible neutralization of essential
viral functions. In this context, the development of HBV
Pol-specific MAbs and the study of their impact on Pol function in
the in vitro priming assay represent an important first step
towards the further exploration of the intracellular antibody
strategy against HBV. In the infected cell, Pol is encapsidated
together with pregenomic RNA and possibly other host
cell-derived proteins into nucleocapsids. In addition to the possible
interference of Pol-specific MAbs with the priming of
minus-strand DNA, it is likely that recombinant antibody fragments
bound to Pol in the cell will reduce the efficiency with which
the enzyme is packaged into nucleocapsids. If so, MAbs that
bind to Pol regions other than TP may also interfere with Pol
function intracellularly.
ACKNOWLEDGMENTS
J.Z.P. and R.E.L. contributed equally to these studies.
This work was supported by grants CA-35711 and AA-02169 from
the National Institutes of Health. J.Z.P. is supported by the
Stipen-dienprogramm “Infektionsforschung” of the German Cancer Research
Center, Heidelberg, Germany.
We thank Luca Guidotti, The Scripps Research Institute, for
immu-nohistochemical analysis of transgenic mouse livers. We also thank
Patricia Mora for helpful discussions on immunocytochemistry. We are
indebted to Heinz Schaller, Zentrum fu¨r Molekulare Biologie
(ZMBH), University of Heidelberg, for the construct pMT-HBVpol,
to Michael Nassal and Peter Kratz, University of Freiburg, for the
construct pCH3142, and to Shuping Tong and Jisu Li for helpful
discussions. We also thank Norman G. Jones and Christian Brander,
AIDS Research Center, Massachusetts General Hospital, for help with
the vaccinia virus system. J.Z.P. thanks Ed Harlow and Chidi
Ezuma-Ngwu for many helpful discussions and advice. We are grateful to
Yimin Ge, Cutaneous Biology Research Center, Massachusetts
Gen-eral Hospital, for help with confocal microscopy.
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