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Journal of Virology, November 1998, p. 9359-9364, Vol. 72, No. 11
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Metabolic Labeling of Woodchuck Hepatitis B Virus X Protein in
Naturally Infected Hepatocytes Reveals a Bimodal Half-Life and
Association with the Nuclear Framework
Maura
Dandri,1,2
Joerg
Petersen,1,2,3
Richard
J.
Stockert,1
Thomas M.
Harris,1 and
Charles
E.
Rogler1,*
Marion Bessin Liver Research Center,
Department of Medicine, Jack and Pearl Resnick Campus of the Albert
Einstein College of Medicine, Bronx, New York
10461,1 and
Heinrich-Pette Institut
for Experimental Virology and Immunology at the University of
Hamburg2 and
Department of Medicine,
University of Hamburg,3 Hamburg, Germany
Received 10 April 1998/Accepted 22 July 1998
 |
ABSTRACT |
In order to identify potential sites of
hepadnavirus X protein action, we have investigated the
subcellular distribution and the stability of woodchuck hepatitis virus
(WHV) X protein (WHx) in primary hepatocytes isolated from
woodchucks with persistent WHV infection. In vivo cell labeling and
cell fractionation studies showed that the majority of WHx is a soluble
cytoplasmic protein while a minor part of newly synthesized WHx is
associated with a nuclear framework fraction (20%) and with
cytoskeletal components (5 to 10%). Pulse-chase experiments revealed
that cytoplasmic WHx has a short half-life and decays with bimodal
kinetics (approximately 20 min and 3 h). The rates of
association and turnover of nucleus-associated WHx suggest that
compartmentalization may be responsible for the bimodal turnover
observed in the cytoplasm.
| |
TEXT |
Human hepatitis B virus is the
prototype member of the hepadnavirus family, which
includes viruses that infect woodchucks, ground squirrels, Pekin ducks,
and other avian species (14). All mammalian
hepadnaviruses can cause both acute and persistent infection, and persistent infection is a recognized risk factor in the
development of primary hepatocellular carcinoma (4, 29, 33,
46).
The genetic organization of all the members of the
hepadnavirus family is highly conserved. Each of the
viruses has a small (approximately 3-kb) circular DNA genome which
encodes the envelope, the nucleocapsid, and the polymerase genes in a
very compact, overlapping arrangement which utilizes all three reading
frames of the DNA (14). However, the mammalian
hepadnaviruses have a fourth major open reading frame,
which was originally designated the X gene because its function was
unknown (48). The fact that the three most pathogenic
mammalian hepadnaviruses, human hepatitis B virus,
woodchuck hepatitis virus (WHV), and ground squirrel hepatitis virus,
contained the X open reading frame and that the less pathogenic avian
viruses did not led to the speculation that the X gene might contribute
to the pathogenicity of the mammalian viruses. In an effort to
elucidate the role of the X gene in viral infection and
hepatocarcinogenesis, DNA transfection experiments have demonstrated
that overexpression of the human hepatitis B virus X protein (HBx)
alters the activity of various endogenous transcription factors and
causes transactivation of a wide range of viral elements and cellular
promoters (34, 48). The evidence that HBx-responsive
enhancers and promoters do not share any DNA sequence and that HBx does
not bind double-stranded DNA suggested that HBx might exert its
transactivating activity indirectly, through protein-protein
interactions. In favor of this idea, several transfection studies have
shown that HBx can affect cytoplasmic signal transduction pathways by
activating both the Ras-Raf-mitogen-activated protein kinase cascade
(3, 9, 10, 12, 27) and the protein kinase C pathway
(21). Moreover, in vitro binding studies have shown that HBx
has the capability to bind both nuclear (8, 26, 30) and
cytoplasmic (19, 45) proteins.
In vivo studies using the woodchuck system as an animal model of
hepadnavirus infection now have demonstrated that WHV X
protein (WHx) is expressed in liver during infection (11,
20) and that a functional X gene is required for infectivity
(7, 49), suggesting an essential role for WHx in viral
replication. Furthermore, WHx expression during persistent infection
leaves open the possibility that it could affect cellular control
mechanisms associated with premalignant changes in hepatocytes. In this
regard, transgenic-mouse studies of WHx and HBx also support a role for
X protein as a carcinogenic cofactor (11, 22, 25, 39), and
studies with immortalized cell lines suggest a possible transforming
function (18, 38).
In light of the potential actions of hepadnaviral X protein in
cytoplasmic and nuclear compartments of hepatocytes, we
have now undertaken a study to determine the distribution and the
stability of metabolically labeled WHx in the subcellular fractions of
naturally infected woodchuck hepatocytes, where all of the viral
protein is expressed at natural levels and the viral life cycle is
complete.
Identification and stability of WHx in the cytosol of WHV-infected
hepatocytes.
Our previous study utilized immunoprecipitation in
combination with a Western blot-enhanced chemiluminescence
(ECL) detection method to identify steady-state levels of WHx in
the cytoplasmic fraction of hepatocytes isolated from woodchucks
persistently infected with WHV (11). In an effort to
increase the sensitivity of our detection system and to determine the
turnover of WHx in naturally infected hepatocytes, cell fractionation
procedures followed by immunoprecipitation of metabolically
radiolabeled WHx were performed in this study. Primary hepatocytes were
isolated by collagenase perfusion from adult woodchucks with persistent WHV infection and from uninfected animals (11, 17,
28). Hepatocytes (>90% viability) were seeded onto 10-cm
tissue culture plates (4 × 106 cells/dish) and
maintained for 2 to 3 weeks, at 37°C in a 5% CO2
atmosphere, in a previously described L-15 modified medium (1) supplemented with 5% fetal bovine serum. For metabolic radiolabeling, cells were preincubated for 30 min (37°C) in
methionine- and cysteine-free RPMI 1640 (Gibco/BRL) supplemented
with 5% dialyzed fetal bovine serum (Sigma), and then 300 µCi of
35S-Promix (Amersham) per ml was added. After 2 h, the
labeling medium was removed and the cells were chased with
nonradioactive medium for various times. After the chase,
hepatocytes were washed in ice-cold phosphate-buffered saline,
harvested by scraping, and collected by low-speed centrifugation
(50 × g, 5 min).
To answer the question of whether WHx is a soluble cytoplasmic protein
or whether it is associated with other cytoplasmic constituents,
35S-labeled hepatocytes were resuspended in 10 volumes of
hypotonic TKM buffer (10 mM Tris [pH 7.5], 1 mM MgCl2, 5 mM KCl, 2 mM phenylmethylsulfonyl fluoride [PMSF], 1 mg of leupeptin
per ml, 1 mg of pepstatin A per ml) and disrupted by Dounce
homogenization. Hepatocyte breakage was monitored by phase-contrast
microscopy. Nuclei, plasma membrane sheets, and remaining whole cells
were first removed by low-speed centrifugation (1,000 × g, 10 min), and then the postnuclear supernatants were
fractionated by high-speed centrifugation (100,000 × g, 2 h) into a soluble S100 fraction (cytosolic
fraction) and a particulate P100 fraction (microsomal fraction). After
the S100 supernatants were removed, the P100 pellets (microsomal
fractions) were dissolved in detergent buffer (TKM buffer supplemented
with 150 mM NaCl, 1% Nonidet P-40, and 1% sodium dodecyl sulfate
[SDS]), passed through a 26-gauge needle, and then used for the
immunoprecipitation with WHx-antiserum or normal rabbit serum as
previously described (11). Similarly, equivalent amounts of
S100 cytosolic proteins were used for the immunoprecipitation.
Immunoprecipitates were finally solubilized in SDS-Laemmli
sample buffer, resolved on a 13% SDS-polyacrylamide gel
electrophoresis (PAGE), and visualized by autoradiography.
As shown in Fig.
1A,
35S-labeled WHx was specifically immunoprecipitated with
the rabbit WHx antiserum from the S100 cytosolic
fraction of
WHV-positive hepatocytes (S100 lanes 3 and 4), while
no protein of
similar size (15.5 kDa) was immunoprecipitated from
uninfected
woodchuck hepatocytes (lane 1). Normal rabbit serum
also did not
immunoprecipitate a 15.5-kDa protein from the same
WHV-infected cell
extracts (lane 2). A quantitative evaluation
of the amount of labeled
WHx present in the S100 fraction after
0 and 30 min of chase (Fig.
1A,
S100 lanes 3 and 4, respectively)
indicated that cytosolic WHx has a
short half-life in chronically
infected hepatocytes, since its amount
was reduced to 45% after
30 min of chase.
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FIG. 1.
Localization and turnover of WHx in the cytosol of
hepatocytes chronically infected with WHV (A) Woodchuck hepatocytes
were metabolically radiolabeled with 35S-Promix for 2 h and then chased with cold medium. After 0 (lanes 1 to 3) or 30 (lanes
4) min of chase, cell-equivalent amounts (6 × 106
hepatocytes/assay) were fractionated into soluble (S100) and
particulate (P100) cytoplasmic fractions, and fractions were
immunoprecipitated with WHx antiserum (lanes 1, 3, and 4) or normal
rabbit serum (lanes 2). Immunoprecipitates from uninfected (lanes 1)
and from WHV-positive hepatocytes (lanes 2, 3, and 4) were then
analyzed by SDS-PAGE (12%) and detected by autoradiography (2 weeks' exposure). WHx was not detected in P100 fractions even after
longer exposure of the gel (5 weeks). (B) Cells were labeled for 1 h and chased for the indicated periods (in minutes). Equivalent amounts
of total soluble proteins (S100 fractions) per time point were
immunoprecipitated with WHx antiserum (lanes 2 to 8) or with normal
rabbit serum (lane 1) and fractionated by SDS-PAGE (13%). (C) Kinetics
of turnover of cytosolic WHx. The amount of labeled WHx present in the
S100 fraction at each time point was quantitated by scanning
densitometry and normalized to the amount present at time zero
(arbitrary scale).
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|
35S-labeled WHx was not detected in immunoprecipitates from
P100 particulate fractions (Fig.
1A), which contained fragments
of
plasma membrane, intracellular membranes such as the endoplasmic
reticulum and the Golgi, and organelles such as lysosomes and
mitochondria (
32). The distribution of protein disulfide
isomerase
(
43), a specific marker for the endoplasmic
reticulum, was analyzed
to confirm the purity of the cytosolic and
microsomal fractions.
Protein disulfide isomerase was exclusively
detected by immunoblotting
in the P100 microsomal fraction (data not
shown). These findings
indicate that WHx is not tightly associated with
these subcellular
compartments.
To determine the kinetics of turnover of cytosolic WHx, cells were
metabolically radiolabeled for 1 h and chased up to 60
min (Fig.
1B). The S100 cytosolic fractions, containing equal
amounts of total
proteins, were immunoprecipitated with WHx antiserum
(Fig.
1B, lanes 2 to 8). Densitometric analysis of the fluorographs
of immunoprecipitated
WHx revealed a bimodal half-life of cytosolic
WHx in the first hour of
chase. In fact, approximately 50% of
labeled WHx was no longer
detected at 15 to 20 min of chase. In
contrast, the remaining WHx
turned over more slowly, since 40%
of metabolically radiolabeled
WHx was still detected after 1 h
of chase (Fig.
1C). Longer chase
experiments, extending up to
6 h, also confirmed the
presence of the more stable WHx component
(see below).
Subcellular distribution of labeled WHx.
A bimodal degradation
of newly synthesized proteins suggested that compartmentalization and/
or posttranslational modifications of WHx might have occurred. To
investigate whether a different compartmentalization of WHx could
account for the bimodal turnover observed in the cytoplasm, we
next used a differential detergent extraction procedure to fractionate
hepatocytes chronically infected with WHV (36, 42). Cells
were labeled for 1 h with 35S-Promix (300 µCi/ml), harvested as described above, and then resuspended in TX
buffer (10 mM Tris [pH 7.5], 250 mM sucrose, 100 mM NaCl, 3 mM
MgCl2, 2 mM CaCl2, 0.5% Triton X-100, 2 mM
PMSF, 1 mg of leupeptin per ml, 1 mg of pepstatin A per ml). After an 8-min incubation on ice, the cells were centrifuged (600 × g, 3 min) to pellet the Triton-insoluble fraction. As
highlighted in Fig. 2A, this procedure
first separated soluble cytoplasmic and nucleoplasmic proteins (the
TX-sol fraction) from Triton-insoluble cellular components (P1). Next,
the P1 pellet was resuspended in CSK buffer (10 mM Tris [pH 7.4], 10 mM NaCl, 1.5 mM MgCl2, 1% Tween 40, 0.5% Na deoxycholate,
2 mM PMSF, 1 mg of leupeptin per ml, 1 mg of pepstatin per ml) to
solubilize the cytoskeletal components. The solution was passed
through a 26-gauge needle 20 times, and then the CSK fraction was
separated by centrifugation (1,000 × g for 5 min) from
the insoluble nuclear framework fraction (P2). Finally, the proteins in
the nuclear framework pellet, which is mainly composed of nuclear
matrix and chromatin (5, 42), were resuspended in CSK buffer
supplemented with 1% SDS and passed through a 26-gauge needle to be
solubilized (NM fraction).
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FIG. 2.
Subcellular distribution of labeled WHx protein in the
TX-sol, CSK, and NM fractions. (A) Outline of the differential
detergent extraction procedure used to investigate the subcellular
distribution of WHx protein in primary hepatocytes isolated from
woodchucks persistently infected with WHV. (B) Hepatocytes (6 × 106) from a WHV-positive woodchuck were metabolically
radiolabeled with 35S-Promix for 1 h, harvested, and
fractionated by differential detergent extraction. Fifty percent of
each fraction was then immunoprecipitated with normal rabbit serum
(lanes 1) or with WHx antiserum (lanes 2), and the immunoprecipitates
were resolved on a SDS-PAGE (12%). (C) The amount of labeled WHx
recovered from each fraction was quantitated by densitometry scanning
of fluorographs. Each column represents the relative amount of labeled
WHx detected in each fraction. These values have been reproduced in
three independent experiments.
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|
To determine the subcellular distribution of labeled WHx in the TX-sol,
CSK, and NM fractions, we conducted immunoprecipitation-PAGE
with
either WHx antiserum (Fig.
2B, lanes 2) or rabbit normal
serum (Fig.
2B, lanes 1). Quantitative analysis of the amount
of labeled WHx
recovered from each fraction revealed 70 to 75%
of labeled WHx in the
TX-sol fraction, 5 to 10% in the CSK fraction,
and, to our surprise,
approximately 20% in the NM fraction (Fig.
2C).
To check the efficiency of this cell fractionation procedure, the NM
fraction was assayed for the presence of contaminant
cytoplasmic
proteins. Figure
3A shows that ligandin,
a cytosolic
protein involved in bilirubin intracellular transport
(
50),
was completely extracted into the TX-sol fraction, as
determined
by SDS-PAGE and ECL-Western blotting with specific
antiligandin
antibodies (gift from I. Listowsky, Albert Einstein
College of
Medicine, Bronx, N.Y.). Furthermore, aliquots from
each cell fraction
were analyzed by SDS-PAGE and Coomassie blue
staining. As shown
in Fig.
3B, the histone proteins were retained
entirely in the
NM fraction. Protein concentration analyses
and Coomassie blue
staining (Fig.
3B) also demonstrated that the CSK
and the NM fractions
contained much smaller amounts of total proteins
(about 15 and
10%, respectively) than the TX-sol fraction
(approximately 75
to 78%). Therefore, the Triton-insoluble
fractions were considerably
enriched for WHx.
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FIG. 3.
Distribution of cellular marker proteins in the TX-sol,
CSK, and NM fractions. (A) Aliquots representing 0.5% (vol/vol) of the
TX-sol and 2% (vol/vol) of the NM fractions were analyzed by SDS-PAGE
(13%) and subjected to ECL-Western blot analysis with an antiligandin
rabbit antiserum. (B) Total proteins from the TX-sol (0.5%), CSK
(1%), and NM (1%) fractions were also subjected to SDS-PAGE (13%)
and Coomassie blue staining.
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Our results are consistent with those of Schek et al. (
36),
who previously described a similar subcellular distribution
and bimodal
half-life of HBx in HepG2 cells when vaccinia virus
vectors were used
to induce transient overexpression of HBx protein.
A similar partition
of WHx between soluble and cytoskeletal fractions
has also been
observed recently in hepatocytes isolated from acutely
WHV-infected woodchucks, although nucleus-associated WHx was not
detected under the conditions used in that study (
20).
However,
another report by Doria et al. (
12) provided
evidence, by confocal
laser microscopy, for the presence of a small
fraction of HBx
within the nuclei of transfected cells. It is possible
that some
discrepancies in terms of X-protein distribution and
solubility
might be due to differences in the techniques used as well
as
differences in the expression levels of X protein obtained after
infection with recombinant systems (
12,
36,
44) versus the
low steady-state levels of WHx actually present in naturally
infected
hepatocytes.
Half-life of TX-sol WHx and kinetics of association of labeled WHx
with the nuclear framework.
To examine the rate of association of
WHx with the NM fraction and to determine the half-lives of WHx in the
TX-sol and Triton-insoluble fractions, WHV-positive hepatocytes
were pulse-labeled for a short time (10 min) with 600 µCi of
35S-Promix per ml at 37°C. Two 10-cm plates of confluent
woodchuck hepatocytes were used per time point. After the chase, cells
were washed in ice-cold phosphate-buffered saline and fractionated by
using the differential detergent extraction procedure outlined in Fig.
2A. TX-sol fractions containing the same amount of total proteins per
chase point were immunoprecipitated with WHx antiserum, and
immunoprecipitates were analyzed by SDS-PAGE. Quantitative analyses of the autoradiographs shown in Fig.
4A (chase up to 1 h) and Fig.
4B (chase up to 6 h) demonstrated that the turnover of TX-sol WHx
was bimodal. In fact, within 20 min of chase, 50% of TX-sol WHx turned
over while the remaining 50% decayed with a half-life of 3 to 4 h
(Fig. 4C).
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FIG. 4.
Kinetics of the turnover of WHx protein in TX-sol and NM
fractions. (A) WHV-infected hepatocytes were pulse-labeled with
35S-Promix for 10 min and chased with cold medium for
various times as indicated (in minutes). After 0 min and each chase
time, 6 × 106 cells per time point were fractionated
as described in the text and in Fig. 2. Fractions were
immunoprecipitated with WHx-antiserum (lanes 2 to 5) or with normal
rabbit serum (lanes 1), subjected to SDS-PAGE (12%), and visualized by
fluorography. (B) Metabolically radiolabeled WHV-positive hepatocytes
were chased for longer periods, and immunoprecipitates from TX-sol
fractions were analyzed by SDS-PAGE and autoradiography. (C) The amount
of WHx present in the TX-sol fraction at each time point was
quantitated by scanning densitometry and normalized (on an
arbitrary scale) to the amount present at time zero. (D) The amount of
labeled WHx associated with the NM fraction in the first hour of
chase was normalized to the amount of labeled WHx present at time
zero.
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Densitometric scanning of the NM data showed that newly synthesized WHx
rapidly associated with the NM fraction (Fig.
4A,
NM lane 2), since it
reached a maximum immediately after the pulse
(0 min of chase).
Densitometry was difficult to analyze because
of the weakness of the
signal after only 10 min of pulse, and
the amount of labeled WHx in the
cytoskeletal fractions was below
the limit of detection (data not
shown). However, results of several
experiments (one of which is shown
in Fig.
4A) showed that WHx
did not accumulate within the NM fraction,
where it appeared to
have a short half-life (approximately 30 min).
Therefore, our
data support the hypothesis that subcellular
distribution of WHx
may be responsible, at least in part, for the
bimodal kinetics
of WHx observed in the cytoplasm.
The cytoplasmic cytoskeleton is contiguous with the nuclear matrix
(
5), and several studies have shown that these structures
may support the transport of viral components to the nucleus (
31,
35,
42). The presence of small amounts of WHx associated with
the
CSK fraction would suggest this possibility. However, it is
also
possible that WHx could enter the nucleus by diffusion, since
the X
protein is smaller than the exclusion limit of the nuclear
pore.
Nuclear WHx could then be retained in the nucleus by binding
to a
nondiffusible nuclear component, like the nuclear matrix.
Our finding
suggests that a fraction of newly synthesized WHx
actually associates
with components of the nuclear framework and
that the transit
from the soluble fraction to the insoluble framework
fraction is rapid.
This would also explain why we did not detect
WHx in
nucleoplasmic extracts when standard procedures to detect
unlabeled WHx
were used (
11).
Overall, our subcellular fractionation data indicate that the great
majority of WHx is a soluble cytoplasmic protein and that
only a minor
fraction of WHx is associated with nuclear components
in vivo. An
accumulating body of evidence (
3,
9,
10,
12,
21,
24,
27)
suggests that the cytosol is an important site
for X-protein
transactivating function. Thus, the presence of
WHx in the cytosol of
chronically infected hepatocytes might lead
to a moderate but
constitutive activation of several components
of the cytoplasmic
signaling cascades.
A short half-life is a typical feature of many viral and cellular
proteins that have transcriptional regulatory functions,
such as the
Tax protein of human T-cell leukemia virus type 1
(
40), the
E1A protein of adenovirus (
41), and the c-Myc protein
(
47). Furthermore, the presence of newly synthesized WHx
associated
with the NM fraction gives rise to the idea of a possible
functional
interaction between nuclear transcription factors and
structural
components of the nuclear matrix. It is known that the
nuclear
matrix plays an important role in regulating gene expression,
and it has been shown that several transcription factors, like
the
ubiquitous transcription factor YY1 (
15), ATF1
(
16), and
Oct-1 (
23), as well as the
retinoblastoma gene product Rb (
2),
the glucocorticoid
receptor (
13), the simian virus 40 large
T antigen
(
37), and the E1A herpesvirus protein (
6), are
partially sequestered in the nuclear matrix. Studies targeted
towards investigating such interactions may shed further light
on the
natural mechanism of action of WHx in hepatocytes.
| |
ACKNOWLEDGMENTS |
We thank L. Johnson and B. Tennant for providing woodchucks from
the experimental woodchuck colony at Cornell University, Ithaca, N.Y., William Mason for providing WHx antiserum;
M. L. Schilsky for the PDI antiserum; and I. Listowsky for the
ligandin antiserum. Many thanks to S. Gupta and members of his
laboratory for their assistance during woodchuck liver perfusions and
to H. Will for helpful discussions.
This work was supported by Public Health Service grants CA37232 and DK
46952, Center grants P30CA13330 and P30DK41296, and a grant from the
Council for Tobacco Research. C.E.R. is the recipient of an Irma T. Hirschl-Weiler career scientific award. M.D. was supported in part by a
stipend from the Deutsche Krebshilfe. J.P. was supported by the
Deutsche Forschungsgemeinschaft (Pe/608 2-1), Bonn, Germany.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: M. Bessin Liver
Research Center, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Phone: (718) 430-2607. Fax: (718) 430-8975. E-mail: crogler{at}aecom.yu.edu.
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Journal of Virology, November 1998, p. 9359-9364, Vol. 72, No. 11
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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