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Journal of Virology, December 1999, p. 10540-10545, Vol. 73, No. 12
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Characterization of the Phosphorylated Forms and
the Phosphorylated Residues of Hepatitis Delta Virus Delta
Antigens
Jung-Jung
Mu,1
Hui-Lin
Wu,2
Bor-Luen
Chiang,3
Ruo-Ping
Chang,3
Ding-Shinn
Chen,2 and
Pei-Jer
Chen1,2,3,*
Graduate Institute of
Microbiology,1 Hepatitis Research
Center,2 and Graduate Institute of
Clinical Medicine,3 College of Medicine,
National Taiwan University, Taipei, Taiwan
Received 26 May 1999/Accepted 23 August 1999
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ABSTRACT |
Hepatitis delta virus (HDV) replication requires both the cellular
RNA polymerase and one virus-encoded protein, small delta antigen
(S-HDAg). S-HDAg has been shown to be a phosphoprotein, but its
phosphorylation status is not yet clear. In this study, we employed
three methods to address this question. A special two-dimensional gel
electrophoresis, namely, nonequilibrium pH gradient electrophoresis,
was used to separate the very basic S-HDAg. By carefully adjusting the
pH of solubilization solution, the ampholyte composition, and the
appropriate electrophoresis time periods, we were able to clearly
resolve S-HDAg into two phosphorylated isoforms and one
unphosphorylated form. In contrast, the viral large delta antigen
(L-HDAg) can only be separated into one phosphorylated and one
unphosphorylated form. By metabolic 32P labeling, both
immunoprecipitated S-HDAg and L-HDAg were found to incorporate
radioactive phosphate. The extent of S-HDAg phosphorylation was
increased upon 12-O-tetradecanoylphorbol-13-acetate
treatment, while that of L-HDAg was not affected. Finally, phosphoamino
acid analysis identified serine and threonine as the phospho residues in the labeled S-HDAg and only serine in the L-HDAg. Therefore, HDV S-
and L-HDAgs differ in their phosphorylation patterns, which may account
for their distinct biological functions.
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TEXT |
Hepatitis delta virus (HDV) is the
smallest member of negative-strand RNA viruses and possesses a circular
genome of 1.7 kb (19, 21, 29). Replication of HDV is
entirely through RNA intermediates and proposed to proceed by a double
rolling-circle mechanism (19, 29). Hepatitis delta antigen
(HDAg) is the only known protein encoded by HDV and exists as two major
forms: small HDAg (S-HDAg; 195 amino acids) and large HDAg (L-HDAg; 214 amino acids) (29, 30, 31). Both forms of HDAg are translated from the same initiation codon of one open reading frame (ORF), but
L-HDAg contains an additional 19 amino acids at the C terminus of
S-HDAg (31) because of RNA editing of the original
termination codon during replication. Hence, they have different
biological functions: S-HDAg is essential for viral replication
(18); however, L-HDAg exerts a dominant-negative role in HDV
replication and is involved in viral assembly (5, 10).
L-HDAg was first shown as a nuclear phosphoprotein when expressed in
mammalian cells, and a previous result indicated that L-HDAg is
phosphorylated only at the serine residue(s) (6). In a
subsequent study, both L- and S-HDAg were found to be phosphorylated in
insect cells (16). S-HDAg was further demonstrated to be a
phosphoprotein in mammalian cells (35), and the
phosphorylation level of S-HDAg is reported to be downregulated by
casein kinase (CK II)- and protein kinase C (PKC)-specific inhibitors,
whereas that of L-HDAg is downregulated only by CK II inhibitor
(35). A recent study (2) tried to distinguish the
phosphorylated from the unphosphorylated delta antigens by using an
NEPHGE (nonequilibrium pH gradient electrophoresis)-sodium dodecyl
sulfate (SDS) system instead of the conventional isoelectric focusing
(IEF)-SDS system. The results of that study revealed ca. 20 to 40% of
L-HDAg to be phosphorylated. However, in contrast to previous studies,
hardly any phosphorylated S-HDAg was detected (2).
The different results concerning the phosphorylation status of S-HDAg
may be reconciled by at least two factors. First, because of its
nonequilibrium nature, separation of the isoforms of basic protein by
NEPHGE has been empirical. Careful optimization of running conditions
is required to achieve the best resolution. Second, protein
phosphorylation is subjected to modulation by many signals; thus,
different cells and culture conditions may influence the extent of
S-HDAg phosphorylation. In this study, we tried to address the question
by optimizing the conditions for NEPHGE by carefully adjusting many
parameters in order to separate the phosphorylated forms of both HDAgs
from the unphosphorylated forms.
Separation of HDAg isoforms of different pI by fine-tuning the
NEPHGE.
Because the IEF allows a clear separation of proteins and
their isoforms of different pIs, it is usually used in separating the
unphosphorylated from the phosphorylated forms of a protein (32,
34). However, it has been noted that the standard IEF would not
give satisfactory resolution in separating very basic or acidic
proteins (23, 33). According to the predicted pI values of
S-HDAg and L-HDAg (10.2 and 9.9, respectively), both HDAgs seemed to be
very basic, and it was difficult to reach pH equilibrium in
conventional IEF. Therefore, we adapted an alternative approach,
namely, NEPHGE, which was developed to resolve very basic protein by
using a nonequilibrium condition rather than true focusing (1, 23,
33). To investigate S- and L-HDAgs in the NEPHGE system, two
plasmids, pCDAg-S and pCDAg-L, which express S- and L-HDAg,
respectively, were cotransfected into HuH-7 cells. pCDAg-S
contained the S-HDAg ORF of HDV (nucleotides 46 to 781) under the
control of human cytomegalovirus immediate-early promoter. To construct
pCDAg-L, the site-directed mutagenesis (Transformer Site-Directed
Mutagenesis Kit; Clontech Laboratories, Inc.) was employed to change
the stop codon (UAG) in the S-HDAg ORF of pCDAg-S to UAA (encoding
Trp). On day 3 posttransfection, cells were washed twice with cold TBS
(150 mM NaCl, 20 mM Tris; pH 7.5) and lysed in radioimmunoprecipitation
assay (RIPA) buffer (150 mM NaCl; 50 mM Tris, pH 8.0; 5 mM EDTA; 0.2%
NP-40; 1% Triton X-100; 0.1% SDS). The cell lysates were centrifuged
at 12,000 rpm for 20 min to remove debris. For preclearing, the
supernatant was incubated with a 1/10 volume of normal mouse serum (50 mg/ml; Jackson ImmunoResearch Laboratories, Inc.) for 1 h at 4°C
and then with 25 µl of protein G-agarose beads (Boehringer Mannheim) for another 1 h at 4°C. After centrifugation at 12,000 rpm for 1 min, precleared supernatant was reacted for 1 h at 4°C with a
mouse monoclonal anti-HDAg antibody (5 µg/ml), D9-3, followed by
another 1 h at 4°C with 25 µl of protein G-agarose. D9-3 was raised in BALB/c mice by inoculation of S-HDAg expressed in
Escherichia coli. After centrifugation at 12,000 rpm for 1 min, the pellet was washed with 1 ml of RIPA buffer once, 1 ml of
high-salt buffer (25 mM HEPES, pH 7.5; 1% Triton X-100; 1%
deoxycholate; 0.1% SDS; 500 mM NaCl; 5 mM EDTA) twice, and then
low-salt buffer (25 mM HEPES, pH 7.5; 0.2% Triton X-100; 1 mM EDTA) once.
The immune complex was resuspended in 50 µl of urea-NP-40
solubilizer (9 M urea, 4% NP-40; 1.6% Pharmalyte [pH 3.5 to 10; Amersham Pharmacia Biotech AB], 0.4% Servalyte [pH 9 to 11; Serva FeinBiochemica GmbH and Co.], and 1% dithiothreitol [adjusted to pH
3.0]) and held at room temperature for 10 min. After dissolution, the
supernatant was loaded onto the well of one cylindrical gel (12 cm
[length] by 4 mm [diameter]) and covered with 100 µl of 4 M urea
overlay solution. The gel was made with 3.3% acrylamide (30%
acrylamide and 1.8% bisacrylamide) containing 9 M urea, 2% NP-40,
1.6% Pharmalyte (pH 3.5 to 10) and 0.4% Servalyte (pH 9 to 11).
Finally, the gels were installed vertically in electrophoresis apparatus (Desaga GmbH, D-6900 Heidelberg, Germany) with 20 mM NaOH in
the lower chamber and 10 mM H3PO4 in the upper
chamber. Lysozyme and trypsinogen (Sigma, St. Louis, Mo.) were used as the reference for pI markers and run in parallel cylindrical gels.
In NEPHGE, proteins migrate in nonequilibrium pH gradient; therefore,
it is important to find the critical window (defined
by the running
volt-hour) at which the proteins are best focused
(
23,
33).
Proteins did not enter gels completely if the electrophoresis
time
period was insufficient. On the other hand, prolonged period
of
electrophoresis made proteins in acidic ends migrate backwards
off the
gel and caused the collapse of the pH gradient in the
basic ends.
Therefore, outside the critical window, proteins will
clump together,
spread into a broader pattern, or intermingle
with others, and this
results in poor separation (
23). In order
to fine-tune the
NEPHGE, gels were electrophoresed at four different
volt-hour levels:
600, 800, 1,300, and 1,600 V · h, respectively.
Gels were
electrophoresed at 400 V for 1 h and then run at 800
V at
different periods of time with the current reversed. After
electrophoresis, the cylindrical gels were extruded from the glass
tubes by syringes, and each one was soaked in 10 ml of equilibration
buffer (10% glycerol, 4.9 mM dithiothreitol, 2% SDS, 0.125 M Tris;
adjusted to pH 6.8) for 10 min. Then, the tube gel was laid on
top of
the second-dimension gel. The second dimension was the
standard
discontinuous SDS-polyacrylamide gel electrophoresis
(PAGE) employing a
20-by-20-by-0.15-cm gel (12% separation gel
and 5% stacking
gel) with a beveled plate to assemble the glass-plate
sandwich
(BRL Vertical Gel Electrophoresis System, model V16-2).
This plate
provided a wider space to accommodate the thick cylindrical
gel from
the NEPHGE. After equilibration, the extruded tube gel
was laid on top
of the stacking gel and sealed with 0.5% agarose.
Electrophoresis was
carried out overnight (70 V, 16 to 18 h),
and gels were
electrotransferred onto nitrocellulose membrane
(Hybond-c Super;
Amersham Pharmacia Biotech). Both HDAgs were
detected by Western blot
probing with human polyclonal anti-HDAg
antibody.
Based upon the migration of the molecular weight marker and recognition
of HDAgs by specific antibody in a subsequent Western
blot, we
identified both HDAgs in the NEPHGE-SDS gel (Fig.
1).
In the shorter electrophoresis
periods (600 and 800 V · h), both
the S- and L-HDAgs migrated as
a large, clumped spot without separation,
probably due to insufficient
time for electrophoresis. Because
of a higher pI, the S-HDAg is noted
moving farther right than
the L-HDAg (Fig.
1B). Notably, in the
appropriate volt-hour (in
the case of 1,300 V · h), the L-HDAg
separates into two spots
(Fig.
1C), and the S-HDAg resolves into three
spots. However,
with a longer electrophoresis period, the L-HDAg is
still separated
into two spots (Fig.
1D), but the S-HDAg now becomes
broader again.
This clumped signal may result from the S-HDAg isoforms
residing
in the collapsed pH gradient at the basic end of gel after
prolonged
electrophoresis. The results suggested that the
electrophoresis
period needs to be experimentally titrated, so
different pI isoforms
of one basic protein can be resolved.
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FIG. 1.
Effect of different electrophoresis time periods
(NEPHGE) in separating isoforms of S- and L-HDAgs in a two-dimensional
system. L-HDAg- and S-HDAg-expressing constructs (pCDAg-L and pCDAg-S)
were cotransfected into HuH-7 cells. On day 3 posttransfection, cell
lysates were immunoprecipitated with the anti-HDAg monoclonal antibody
D9-3. The immune complex was resuspended in 50 µl of solubilizer and
electrophoresed in the NEPHGE gel for different periods of time: 600 (A), 800 (B), 1,300 (C), and 1,600 (D) V · h. Electrophoresis in
the first dimension migrated from the acidic to the basic end of the
NEPHGE gel and in the second dimension separated proteins by their
different molecular weights. The isoforms of both HDAgs were detected
by Western blot with polyclonal anti-HDAg antibody. Lysozyme and
trypsinogen (Sigma) were used as the references for pI markers and were
run in parallel NEPHGE experiments. The positions of these markers were
identified after Coomassie blue staining. The asterisk above each panel
indicates the position of trypsinogen with a known pI value of 9.3, and
the star indicates the position of lysozyme with a pI value of 10.5 to
11 as pH markers.
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Effect of protein dephosphorylation on pI isoforms of both
HDAgs.
If the observed pI isoforms of both HDAgs were generated by
differential phosphorylation, then the stepwise decrease in pI value
should be caused by the incorporation of phosphates. The incorporation
with one phosphate into the protein is noted to decrease its pI, which
varies from 0.04 to 0.46 pI units (11, 12, 32, 34).
Phosphorylation on more residues will further reduce its pI and
generate more isoforms. However, treatment with phosphatase will remove
the phosphates, and then the dephosphorylated protein should become a
single species with the same pI. To test this, immunoprecipitated HDAgs
were treated with alkaline phosphatase before the NEPHGE-SDS-PAGE and
detected by Western blot as described above. The immunoprecipitated
HDAgs were resuspended in 90 µl 1× dephosphorylation buffer (50 mM
Tris, 0.1 mM EDTA; pH 8.8) for 10 min at 30°C. Then, 10 µl of calf
intestinal alkaline phosphatase (Boehringer Mannheim) was added into
reaction buffer (100 U/ml) for 1 h at 37°C, followed by another
1 h at 42°C. The sample was washed with 1 ml of high-salt buffer
once and 1 ml of low-salt buffer once and then dissolved in the
solubilizer for NEPHGE-SDS-PAGE. In the untreated samples, the L-HDAgs
are separated into two spots and the S-HDAgs are separated into three
spots (Fig. 2A). After alkaline
phosphatase treatment, both the L- and S- HDAgs were reduced
to only one spot (Fig. 2B). This spot migrated to the same distance as
the isoform with the highest pI in untreated HDAgs (Fig. 2A, indicated
by open triangles). It supported that the isoform with the highest pI
value is the unphosphorylated HDAg. Those isoforms with lower pI values
were phosphorylated S- or L-HDAgs, which could be dephosphorylated by
alkaline phosphatase treatment (Fig. 2A, indicated by solid triangles).
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FIG. 2.
Effect of alkaline phosphatase treatment on the mobility
of delta antigen isoforms. The immunoprecipitated HDAgs were prepared
as described in Fig. 1. Prior to resuspension in solubilizer, the
immune complex was treated with (B) or without (A) alkaline phosphatase
and then subjected to NEPHGE for 1,300 V · h, followed by
SDS-PAGE. The open and solid triangles represent unphosphorylated and
phosphorylated isoforms of both HDAgs, respectively, as identified by
Western blot.
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These data demonstrated that the L-HDAg contains two isoforms, one
unphosphorylated and the other perhaps monophosphorylated.
For S-HDAgs,
other than the unphosphorylated one, there exist
two obvious
phosphorylated isoforms (probably mono- and diphosphorylated).
However,
there could be small amounts of S-HDAg with higher phosphorylation,
since minor spots with even lower pIs sometimes were seen (data
not
shown). Although there was another posttranslational modification
for
L-HDAg farnesylation (
13,
14,
24), farnesylated L-HDAg
could
not be separated from unfarnesylated isoforms, presuming
that
farnesylation does not change the pI value (
2). By
establishing
this two-dimensional system to separate phosphorylated
isoforms
of HDAgs, we concluded that there are two major
phosphorylation
forms for S-HDAg but only one for L-HDAg.
Metabolic labeling of both HDAgs.
The results described above
suggested that the S-HDAg is phosphorylated differently from that of
L-HDAg. To further investigate this, the in vivo-labeling experiment
was performed. On day 3 posttransfection, cells were washed twice with
TBS and starved in 0.8 ml of phosphate-free DMEM (Gibco BRL/Life
Technologies) for 2 h. Then, 0.5 mCi of
[32P]orthophosphate (PBS-13; Amersham Pharmacia Biotech)
was added into medium for 4 h to label the cellular
phosphoprotein. Cells were then harvested and lysed for
immunoprecipitation as described above. The L-HDAg was shown to be a
phosphoprotein in previous reports (2, 6, 35, 36). It was
also labeled in our experiments (Fig. 3A,
lane 3). For S-HDAg, there was a major phospholabeled protein of the
expected molecular weight being immunoprecipitated (Fig. 3A, lane 1).
This labeled protein presumably represented the S-HDAg, since it was
not detected in the cells transfected with L-HDAg expressing plasmid
(Fig. 3A, lane 3). The metabolically labeled L-HDAg and S-HDAg detected
in Fig. 3A were confirmed by a Western blot of the same sample (Fig.
3B, lanes 1 and 3).
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FIG. 3.
In vivo phosphate labeling of both HDAgs and the effect
of TPA stimulation on HDAg phosphorylation. S-HDAg (lanes 1 and 2) and
L-HDAg (lanes 3 and 4) were transiently expressed in HuH-7 cells and
metabolically labeled with [32P]orthophosphate on day 3 posttransfection. After immunoprecipitation with monoclonal anti-HDAg
antibody, seven-eighths of the immunoprecipitates was analyzed by
SDS-PAGE and autoradiography (A), and the remainder was detected by
Western blot analysis with human polyclonal anti-HDAg antibody (B).
Where indicated (lanes 2 and 4), 100 ng of TPA per ml was added to the
labeled cells 15 min before harvest (+). The positions of S-HDAg and
L-HDAg were marked.
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It has been demonstrated that PKC inhibitor, H7, reduces the
phosphorylation level of S-HDAg but not that of L-HDAg (
35).
As the phorbol ester TPA
(12-
O-tetradecanoylphorbol-13-acetate)
is known to activate
PKC, mimicking the physiological activator
diacylglycerol, to mediate
TPA-induced biological effects (
22).
Therefore,
metabolically labeled S-HDAg or L-HDAg in transfected
cells were
exposed to 100 ng of TPA (Sigma) per ml for 15 min
before harvest to
observe any effect. As shown in Fig.
3A, the
phosphorylation level of
S-HDAg increased by ca. threefold after
TPA treatment (Fig.
3A,
comparing lanes 1 and 2). The parallel
immunoprecipitates analyzed by
Western blot showed that the amount
of precipitated S-HDAg was
equivalent with or without TPA treatment
(Fig.
3B, lanes 1 and 2).
Therefore, TPA stimulates the phosphorylation
level instead of the
expression level of S-HDAg. However, it was
noted that the
phosphorylation of L-HDAg was not affected by TPA
treatment (Fig.
3A,
lanes 3 and 4). The stimulation of S-HDAg
phosphorylation may be
through a TPA signaling pathway, and the
phosphorylation pathway of
S-HDAg at least partly differs from
that of L-HDAg.
S-HDAg is phosphorylated at both serine and threonine
residues.
We have shown that phosphorylation of S-HDAg may be
different from that of the L-HDAgs, in terms of the number of
phosphorylation residues and possible kinases. We then tried to
identify the phosphorylated residues of the S-HDAg by performing
two-dimensional phosphoamino acid analysis (PAA). The
32P-labeled S-HDAg in Fig. 3A, which was separated by
SDS-12% PAGE, was electrotransferred onto a polyvinylidene difluoride
(PVDF) membrane and identified by autoradiography. The labeled S-HDAg was excised from the membrane and acid hydrolyzed in 300 µl of 6 N
HCl at 110°C for 90 min. After a lyophilizing and resuspending step
in 10 µl of pH 1.9 buffer (0.58 M formic acid and 1.36 M glacial
acetic acid), the sample was applied to a thin-layer chromatography (TLC) plate (20 by 20 cm; Merck). Electrophoresis was carried out in pH
1.9 buffer at 1,200 V for 20 min for the first dimension and the run
for second dimension in pH 3.5 buffer (0.87 M glacial acetic acid,
0.5% pyridine, 0.5 mM EDTA) at 1,600 V for 25 min (Hunter Thin-Layer
Peptide Mapping System, model HTLE-7000; C.B.S. Scientific Co.). After
electrophoresis, the plate was dried in a 65°C oven for 20 to 30 min,
and phosphoamino acid standards (Sigma) were visualized by spraying
them with 0.25% ninhydrin solution in acetone. The labeled signals
were subjected to autoradiography by a radioanalytic imaging system
(Fujix BAS 1000). As shown in Fig. 4, the
hydrolyzed, 32P-labeled amino acids from S-HDAg were found
comigrating with both authentic phosphoserine and phosphothreonine
standards, but not with phosphotyrosine, indicating that S-HDAg is
phosphorylated at both serine and threonine residues. We also carried
out PAA to determine the phosphorylated residue(s) of L-HDAg as a
control, and the data obtained were consistent with the former report
(6) that L-HDAg is phosphorylated at only the serine residue
(Fig. 4). Since the phosphorylated residues of S-HDAg were identified as both serine and threonine, there were at least two phosphorylation sites in S-HDAg, thus corresponding with previous data that showed in
an NEPHGE-SDS system that S-HDAg contained two major phospho isoforms.
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FIG. 4.
PAA of both HDAgs. The labeled immune complex as
described in Fig. 3 was separated by SDS-PAGE and then
electrotransferred onto PVDF membrane. The membrane slice containing
labeled delta antigens was cut out and acid hydrolyzed in 6 N HCl. The
phosphorylated amino acids were then separated by two-dimensional
electrophoresis on TLC plates. The regions circled with dashed lines
indicated the authentic phosphoserine (P-Ser), phosphothreonine
(P-Thr), and phosphotyrosine (P-Tyr) forms identified by use of
ninhydrin. Phospho residues of S-HDAg were shown by autoradiography in
the left panel, and that of L-HDAg is shown in the right panel.
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For HDV delta antigens, it is generally accepted that the L-HDAg is a
phosphoprotein. However, the phosphorylation of S-HDAg
has not yet been
settled. Though S-HDAg could be labeled by
[
32P]orthophosphate in both mammalian and insect cells
(
15,
35),
the data from a previous two-dimensional gel
analysis of S-HDAg
showed very little phosphorylation (
2).
To reconcile the difference
in the S-HDAgs, we tried to determine
whether the resolution of
NEPHGE could be improved by careful
adjustments. By optimizing
several parameters for NEPHGE, especially
the running time for
electrophoresis, we finally succeeded in
separating the phosphorylated
forms of S-HDAg expressed in transfected
cells. It has been noted
that the separation by NEPHGE was achieved
long before approaching
pH gradient equilibrium and that more basic
proteins need shorter
periods of electrophoresis for separation
(
23,
33). So the
optimum electrophoresis time is very
important. As shown in former
reports, NEPHGE was able to resolve the
phospho isoform of L-HDAg
at 1,600 V · h (
2). We
obtained the same results (Fig.
1D).
However, this electrophoresis time
period was not suitable for
separating the phospho isoforms for the
S-HDAg (Fig.
1D). Probably
the S-HDAg is a more basic protein than
L-HDAg and should be run
in a shorter time period before the collapse
of the basic end
in NEPHGE. Actually, our results showed that 1,300 V · h is optimal
for resolving different phospho isoforms of
S-HDAg (Fig.
1C).
However, with time periods shorter than 1,300 V
· h, the resolution
failed again (Fig.
1A and B). Therefore, the
electrophoresis time
period is critical for the successful separation
of S-HDAg. Our
results explained the poor separation of phospho
isoforms of S-HDAg
by NEPHGE obtained in the previous study, which
employed an inappropriate
condition (
2).
The phosphorylation of S-HDAg apparently differed from that of L-HDAg.
There were two major phospho isoforms and one unphosphorylated
form for
S-HDAg. (We noted that, after a longer exposure, there
were minor
phospho isoforms with lower pIs.) Phosphoamino acid
analysis also
showed that S-HDAg is phosphorylated at both serine
and threonine. This
further confirmed that more than one phosphorylation
occurred on
S-HDAg. L-HDAg has one phosphorylated form and is
phosphorylated only
at serine residues. In metabolically labeling
experiments, the
phosphorylation level of S-HDAg was enhanced
three times after TPA
stimulation, but that of L-HDAg was not
affected. These findings
indicated that the phosphorylation pathways
for both HDAgs are quite
different. Although these two HDAgs shared
the same ORF, except that
L-HDAg has an extra 19 amino acids at
the C terminus; however, they
have different protein conformations
(
13,
14). Their
phosphorylation properties may account for
such a
difference.
Our results only indicated the S-HDAg to be a phosphoprotein but did
not yet identify the exact phosphorylation sites. There
are 10 serine
residues and 5 threonine in S-HDAg coding sequences
of the specific HDV
strain used in this study (Fig.
5, strain
I). Among these 10 serine residues, three (Ser2, Ser4, and Ser123)
have
been studied for their biochemical and biological properties
related to
phosphorylation by site-directed mutagenesis (
35).
Both CK
II and PKC have been demonstrated to modulate the function
and
phosphorylation of S-HDAg, and Ser2 was proposed as one of
the
candidates for S-HDAg phosphorylation affected by CK II. As
for Ser4
and Ser123, both were not affected by CK II inhibitors,
even though
they also resided in the predicted consensus sequences
for CK II. A
comparison of deduced primary sequences of HDAgs
among HDV variants
revealed that, in addition to Ser2 and Ser123,
there is another
completely conserved serine residue, Ser177 (Fig.
5, including all
three genotypes), which resides in a consensus
sequence conforming the
motif of mitogen-activated protein (MAP)
kinase substrate (PESP)
(
25,
26). Since PKC has been demonstrated
to be involved in
the phosphorylation of S-HDAg, the activation
of PKC could lead to the
phosphorylation of MAP kinase (
3,
7). Therefore, the effect
of PKC on S-HDAg may mediate the
phosphorylation of Ser177 through
PKC-MAP kinase pathway. Though
Ser2 and Ser177 may be the
phosphorylated residues for S-HDAg,
this speculation awaits further
investigation.
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FIG. 5.
Conserved serine and threonine residues among HDV
strains. The boldface characters in the left lane represented
geographically distinct HDV isolates: A, American (21); I, Italian
(30); N, Nauru (8); F, French (27); L, Lebanon (20); T, Taiwan (9); C,
Central African (28); J, Japan-2 (16); E, Ethiopia (37); S, Somalia
(37); J', Japan-1 (17); P, Peru (4). The numbers indicate completely or
highly conserved Ser and Thr residues.
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Besides serine, we discovered that threonine is also a phosphorylation
acceptor in S-HDAg. Amino acid 95 is the only completely
conserved Ser
or Thr residue among all the HDV variants (Fig.
5; amino acid 95 is
threonine in the strain we used). Thr134 and
Thr182 are highly but not
completely conserved (Fig.
5, amino
acid 134 as Ala in the S and P
isolates and amino acid 182 as
Arg in the S isolate and as His in the E
and L isolates, respectively).
The sequences encompassing Ser95/Thr95
and Thr134 were similar,
with predicted consensus sequence for CK II or
PKC (
25). Phosphorylations
in S-HDAg might be more likely to
occur on these highly conserved
serine and threonine residues (Ser2 and
Ser177 and Thr95, -134,
and -182). However, the functional involvement
of the less-conserved
ones (Ser6, -83, -116, -159, -170, and -180 and
Thr37 and -76)
could not be completely
excluded.
The biological function of S-HDAg is to assist the replication of HDV
RNA, and the phosphorylation of S-HDAg may modulate
this replication
(
35). In our preliminary data, phosphorylated
S-HDAg could
only be detected inside the cells but not in viral
particles
(unpublished data). It appears that the phosphorylation
of S-HDAg is
relevant for HDV RNA
replication.
Phosphoamino acid analysis indicated that L-HDAg is phosphorylated only
at the serine residue. Completely conserved serines
(Ser2, Ser123, and
Ser177) and highly conserved serines (Ser4,
Ser207, and Ser210) have
been studied by using site-directed mutagenesis,
and none of their
mutants seemed to affect the biological and
biochemical significance
for L-HDAg (
2,
35,
36). Besides,
CK II inhibitor decreased
the phosphorylation level of L-HDAg,
but it does not affect both
particle assembly and the
trans-dominant
function of L-HDAg
(
36). However, mutation at the farnesylation
site (Cys211)
has been reported to eliminate the phosphorylation
on L-HDAg, though
its biological significance has not yet been
investigated
(
2). Therefore, the function of L-HDAg phosphorylation
is
still not
clear.
Despite the speculation on the possible phosphorylation sites in S- or
L-HDAg, a more solid basis is required. It is necessary
to identify the
exact amino acid residues to be phosphorylated
by biochemical
procedures, such as two-dimensional peptide mapping
or liquid
chromatography-mass spectrometry. With this information,
we can address
the biological significance of each phosphorylation
of delta antigens
in the viral life cycle. Finally, the results
also help in searching
for the kinases responsible for phosphorylating
the delta
antigens.
| |
ACKNOWLEDGMENTS |
This work was supported by grants (NSC88-2314-B002-028) from
National Science Council, Executive Yuan, Taiwan.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Graduate
Institute of Clinical Medicine, National Taiwan University Hospital,
No. 7 Chung-Shan South Rd., Taipei, Taiwan. Phone: 886-2-23970800, ext. 7072. Fax: 886-2-23317624. E-mail:
peijer{at}ha.mc.ntu.edu.tw.
| |
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