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J. Biol. Chem., Vol. 277, Issue 36, 33099-33104, September 6, 2002
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From the Institut für Virologie der
Philipps-Universität Marburg, Robert-Koch-Strasse 17, Marburg
35037, Germany
Received for publication, April 18, 2002, and in revised form, June 4, 2002
Transcription of the highly pathogenic Ebola
virus (EBOV) is dependent on VP30, a constituent of the viral
nucleocapsid complex. Here we present evidence that phosphorylation of
VP30, which takes place at six N-terminal serine residues and one
threonine residue, is of functional significance. Replacement of the
phosphoserines by alanines resulted in an only slightly phosphorylated
VP30 (VP306A) that is still able to activate
EBOV-specific transcription in a plasmid-based minigenome system.
VP306A, however, did not bind to inclusions that are
induced by the major nucleocapsid protein NP. Three intracellular
phosphatases (PP1, PP2A, and PP2C) have been determined to
dephosphorylate VP30. The presence of okadaic acid (OA), an inhibitor
of PP1 and PP2A, had the same negative effect on transcription
activation by VP30 as the substitution of the six phosphoserines for
aspartate residues. OA, however, did not impair transcription when VP30
was replaced by VP306A. In EBOV-infected cells, OA blocked
virus growth dose-dependently. The block was mediated by
the extensive phosphorylation of VP30, which is evidenced by the result
that expression of VP306A, in trans, led to the
progression of EBOV infection in the presence of OA. In conclusion,
phosphorylation of VP30 was shown to regulate negatively transcription
activation and positively binding to the NP inclusions.
Ebola virus (EBOV),1 a
filovirus, is notorious for its unpredictable sporadic outbreaks of a
fatal hemorrhagic fever in Africa (1-4). To date, neither a vaccine
nor a treatment of the EBOV infection is available.
The enveloped EBOV particles are composed of seven structural proteins
and the negative sense RNA genome. Four viral proteins NP, VP35, L, and
VP30 are the constituents of the nucleocapsid. The main component of
the nucleocapsid complex is NP, a heavily phosphorylated protein, that
encapsidates the genomic RNA and forms intracellular inclusions upon
recombinant expression (5-7). The NP inclusions are morphologically
highly similar to the inclusions formed during EBOV infection of target
cells. VP35 and L are the components of the viral polymerase. VP30
represents an EBOV-specific transcription activation factor (8).
Most viruses of the order Mononegavirales contain three proteins, N
(NP), P, and L, that drive the processes of replication (synthesis of
genomic RNA) and transcription (synthesis of viral mRNAs). These
proteins also constitute the respective viral nucleocapsid complex. N
(or NP) represents the major nucleocapsid protein that encapsidates the
viral genome. The encapsidated genome serves as a template for the
viral polymerase complex, which is constituted by the catalytic subunit
L and the cofactor P (9).
A recently established minigenome-based reverse genetic system revealed
that EBOV follows another strategy to synthesize the different RNA
species. NP, VP35 (the P analogue), and L were sufficient for viral
replication, similar to the other Mononegavirales. The fourth
nucleocapsid protein VP30, although not influencing replication, dramatically activated the synthesis of the viral mRNAs (8). The
mechanism, i.e. how the phosphoprotein VP30 activates
transcription, is still unclear.
The phosphorylation state of a cellular or viral protein is determined
by the coordinated action of kinases and phosphatases. Although the
significance of several cellular kinases for the phosphorylation state
of viral proteins of the order Mononegavirales is well established,
only a few proteins are described whose function is influenced by
phosphorylation (10, 11). Even less is known about the impact of
cellular phosphatases on the viral replication cycle. However, for
papovae- and adenoviruses as well as for the human immunodeficiency
virus types 1 and 2, the activity of the ubiquitous phosphatase PP2A
has been shown to be of functional significance (12, 13).
In this report, we have determined the phosphorylation sites of VP30
and found that phosphorylation of two N-terminal serine clusters
positively regulated binding of VP30 to NP-induced inclusions and
negatively regulated the transcription activation function of the
protein. We further show that VP30 is a target for cellular protein
phosphatases PP1 and PP2A. In a reconstituted minigenome system,
EBOV-specific transcription was blocked by okadaic acid (OA) that is
known to inhibit PP1 and PP2A. The effect of OA on the transcription
could be attributed to an extensive phosphorylation of VP30. Moreover,
the treatment of EBOV-infected cells with OA inhibited EBOV growth,
which could be restored by the expression of a nonphosphorylatable VP30
in trans. Taken together, our results show for the first time that VP30
phosphorylation is a regulatory factor in the replication cycle of EBOV
that might be a suitable target for the development of antiviral drugs.
Viruses and Cell Lines
EBOV-Zaire strain Mayinga was grown and passaged as described
elsewhere (14). MVA-T7 was grown and titered in chicken embryo fibroblasts (15). HeLa cells were cultured as described by
Mühlberger et al. (8). Monolayer cultures of HeLa
cells were used for all experiments with the recombinant vaccinia virus
MVA-T7. BSR T7/5 cells (a BHK-21 cell clone), which constitutively
expressed T7 RNA polymerase, were cultured as described by Buchholz
et al. (16). For transfection experiments, cells were grown
in six-well plates (7 cm2).
Molecular Cloning of VP30 and VP30 Mutants
For expression of VP30 and VP30 mutants the respective genes
were cloned into the plasmid pTM1 under the control of the T7 RNA
polymerase promoter (17). VP30 and all VP30 mutants contained a
FLAG epitope at the C terminus.
Cloning of pT-VP30F--
For the construction of a
FLAG-tagged VP30, the plasmid pT-VP30EBO was used as
template (8). Using the primers #408 (5'-ACC GGA TCC ATG
GAA GCT TCA TAT GAG AGA-3') and #409 (5'-AGA CTC GAG TTA
CTT GTC ATC GTC GTC CTT GTA GTC AGG GGT ACC CTC ATC AGA CCA TGA GCA TG-3'), which contained either a BamHI or an
XhoI restriction site (underlined), and the sequence coding
for the FLAG epitope (italics), the VP30 gene was amplified
by PCR, gel-purified, and cloned into the BamHI site and
XhoI sites of the vector pTM1. The final plasmid was
verified by sequencing and designated as pT-VP30F.
The influence of the FLAG epitope on intracellular localization and on
transcription activation of VP30 was checked by immunofluorescence and
transcription analysis. This pilot study revealed that neither the
intracellular distribution nor the transactivating function of VP30 was
altered (data not shown).
Cloning of the Mutant pT-VP30 Cloning of VP30 Substitution Mutants--
Cloning of the VP30
substitution mutants was performed using the QuikChange site-directed
mutagenesis kit (Stratagene), with pT-VP30F as template.
All mutants were verified by sequencing.
Ebola Virus Transcription Analysis
BSR T7/5 cells (5 × 105 in a 7-cm2
well) were transfected with plasmids encoding the EBOV nucleocapsid
proteins NP, VP35, VP30, and L, and an EBOV-specific minigenome
containing the CAT reporter gene (8) using FuGENE6 (Roche Molecular
Biochemicals) according to the supplier's prescription. After an
incubation period of 8 h, cells were washed two times with
Dulbecco's modified Eagle's medium and further incubated for 12-36 h
at 37 °C. Subsequently, cells were harvested and CAT activity as a
readout for transcription activity was determined. When the impact of
OA on transcription was investigated, the drug, diluted in
Me2SO, was added 8 h post transfection. The
final concentration of Me2SO was 0.2%.
CAT Assay
CAT activity was determined using a standard protocol (18).
Quantification of the radioactivity was done with a Fuji BAS-1000 Bio-Imaging Analyzer (Fujifilm) by using the TINA software (Raytest).
Transfection of EBOV-infected Cells
5 × 105 BSR T7/5 cells on glass coverslips in
a 7-cm2 dish were infected with EBOV at an multiplicity of
infection of ~1 plaque forming unit per milliliter. At 1 h post
infection (p.i.), inoculum was removed and cells were transfected with
1.5 µg of pT-VP30EBO or pT-VP306A as
described above. At 3 h p.i., 40 and 80 nM OA, respectively, were added. Cells were fixed at 24 h p.i. with 4% paraformaldehyde, and NP expression was analyzed by immunofluorescence using a monoclonal anti-NP antibody and a rhodamine-coupled anti-mouse antibody.
VP30 Dephosphorylation by Protein Phosphatases
VP30 was expressed in HeLa cells using the vaccinia virus-T7
system, metabolically labeled with 32Pi and
immunoprecipitated using an anti-FLAG monoclonal antibody M2 (Sigma,
Deisenhofen, Germany) (19). Immune complexes were washed five times in
the precipitation buffer without SDS, three times in buffer E (20 mM Tris/HCl, pH 7.5, 50 mM NaCl) and finally resuspended in five volumes buffer E. This suspension was used for
incubation with the respective phosphatases. 300 ng of either PP1,
PP2A, PP2C, or alkaline phosphatase (AP) were mixed with 1.5 µl of
10× concentrated phosphatase buffer (PP1/PP2A: 500 mM Tris/HCl, pH 7.5, 10% glycerol, 1% mercaptoethanol, 50 mM
MnCl2; PP2C: 250 mM Tris/HCl, pH 7.5, 10%
glycerol, 1% mercaptoethanol, 100 mM MgCl2;
alkaline phosphatase: 500 mM Tris/HCl, pH 7.5, 50 mM MgCl2), and 10 µl of VP30 suspension.
Finally, the total volume was filled up with dH2O to 15 µl, and the samples were incubated for 30 min at 30 °C.
Other Methods
Infection and transfection of HeLa cells, metabolic labeling
with [32P]orthophosphate or 35S-Promix,
immunoprecipitation, Western blot analysis, immunofluorescence analysis, and formic acid treatment were carried out as described by
Modrof et al. (19).
Phosphorylation State Analysis of VP30 Substitution
Mutants--
Proteolytic digestion and phosphoamino acid analyses of
32Pi-labeled recombinant VP30 revealed that the
phosphate acceptor sites are represented by serine and threonine
residues within the 60 N-terminal amino acids (data not shown).
Analysis of the amino acid sequence in this particular region revealed
two serine clusters (amino acids 29-31 and 42-46) each containing
three serine residues that seemed to represent suitable targets for
cellular protein kinases (Fig. 1,
top). To examine whether these amino acids are, indeed,
phosphorylated, we constructed FLAG-tagged full-length mutants of VP30
by substituting the respective serine residues and threonine 52 with
alanines and expressed the mutants in HeLa cells using the vaccinia
virus-T7 system. Cells were metabolically labeled with
32Pi, immunoprecipitated, separated by
SDS-PAGE, and analyzed by autoradiography. The substitution of one
serine cluster, either 29-31 or 42-46, resulted in a reduction of the
phosphorylation to 59% or 44% of the wild-type VP30, respectively
(Fig. 1, lanes 1, 3, and 4). When all
six serine residues between amino acids 29 and 46 were substituted
(VP306A), 16% of wild-type VP30 phosphorylation was
detected (lane 5). The additional exchange of threonine 52 (VP30S29-46T52A) further decreased the phosphorylation
signal to 6% in comparison with the phosphorylation of wild-type VP30 (lane 6). Exchange of single serine residues did not
impair phosphorylation significantly (data not shown). To confirm
that phosphorylation occurred mainly within the N terminus, the
phosphorylation state of a VP30 deletion mutant was investigated that
lacked the 68 N-terminal amino acids. This mutant was phosphorylated to
the same extent as the substitution mutant
VP30S29-46T52A (lanes 2 and 6),
indicating that the major phosphate acceptor sites in VP30 have been
identified. Moreover, these results point to the fact that most if not
all of the serine residues in the amino acid region 29-46 and
threonine 52 are targets for cellular kinases.
Influence of the Phosphorylation of VP30 on the Association with NP
Inclusions--
VP30 is a nucleocapsid-associated protein, which could
be detected in EBOV-infected cells in intracytoplasmic inclusions
together with the major nucleocapsid protein NP (7) (Fig.
2A). Ultrastructural analyses
revealed that the inclusions contained viral nucleocapsids (20). Single
expression of NP resulted in the formation of cytoplasmic inclusions
similar to the inclusions in EBOV-infected cells (Fig. 2B).
To determine whether VP30 was able to associate with the NP inclusions,
VP30 was coexpressed together with NP in HeLa cells, and cells were
subjected to immunofluorescence analysis at 12 h post
transfection. While solitarily expressed VP30 was homogeneously distributed (Fig. 2B, middle panel) coexpression
with NP resulted in a redistribution of the protein into the NP-induced
inclusion bodies (Fig. 2C). This result suggested that the
connection of VP30 to the inclusions is directly or indirectly mediated
by NP.
It was now of interest whether phosphorylation of VP30 has impact on
the interaction of VP30 with the NP-induced inclusions. Immunofluorescence analyses of cells coexpressing VP30 mutants and NP
revealed that VP306A was impaired in its ability to
associate with the NP-induced inclusions. Although NP was still found
in clusters, VP306A was homogeneously distributed (Fig.
2D). Substitution of either serines 29-31 or serines 42-46
to alanines changed the intracellular distribution of VP30 only
slightly (Fig. 2, E and F). It was therefore
concluded that phosphorylation of at least one of the serine clusters
was essential for the interaction between NP inclusions and VP30.
To mimic the negative charges of the phosphate groups at serines
29-46, we replaced the six amino acids by aspartate residues (21, 22)
and coexpressed the mutant (VP306D) with NP.
Immunofluorescence analysis showed that VP306D was able to
interact with the inclusions as the wild-type VP30 (Fig.
2G). This result indicated that the phosphorylation-induced
negative charges at the serine residues rather than the serine residues
itself were crucial for the interaction of VP30 with the NP-induced inclusions.
Influence of Phosphorylation of VP30 on Transcription
Activation--
To address the question of whether the phosphorylation
of VP30 influenced the transcription activation function of VP30, the wild-type VP30 was replaced by phosphorylation-deficient VP30 mutants
in an EBOV-specific transcription system (8). BSR T7/5 cells were
transfected with plasmids encoding an artificial EBOV minigenome that
contained the CAT reporter gene, and the EBOV nucleocapsid proteins NP,
VP35, L, and VP30 or the VP30 substitution mutants, respectively (8).
As expected, EBOV-specific transcription was dramatically enhanced in
the presence of VP30 (Fig. 3, lanes
1 and 2). Interestingly, the phosphorylation-deficient
VP30 mutants were still able to support transcription, irrespectively,
whether single or both serine clusters were exchanged for alanines
(lanes 3-5). When one of the serine clusters was
substituted with aspartates, VP30 was functional as well (lanes
6 and 7). However, substitution of all six
phosphorylated serine residues with aspartates resulted in an inactive
VP30 (lane 8). These results suggested that phosphorylation of VP30 plays a key role in regulating the activity of the EBOV transcription in a way that the critical serine residues are partly nonphosphorylated.
Identification and Inhibition of VP30-dephosphorylating
Phosphatases--
To confirm that phosphorylation is critical for VP30
function, it was checked whether VP30-specific phosphatases could be identified. Inhibition of such phosphatases would lead to an
extensively phosphorylated VP30, which, like VP306D, is not
able to mediate transcription.
VP30 was expressed using the vaccinia virus-T7 system, metabolically
labeled with 32Pi, and purified by
immunoprecipitation. The precipitate was then incubated with the
catalytic subunits of protein phosphatases PP1, PP2A, PP2C, and
alkaline phosphatase, and the phosphorylation of VP30 was checked by
SDS-PAGE and autoradiography. It is shown in Fig.
4A that PP1 and PP2A almost
completely dephosphorylated VP30 (Fig. 4, lanes 4 and
5). PP2C dephosphorylated VP30 as well, but to a lesser
extent (Fig. 4, lane 3). Alkaline phosphatase did not
recognize VP30 as a substrate (Fig. 4, lane 1). This result was confirmed by the finding that the VP30-dephosphorylating activity of HeLa cell lysates could be inhibited by OA, which is known to
inhibit PP1 and PP2A (Refs. 23 and 24; data not shown). We concluded
that VP30 was dephosphorylated in vitro and in
vivo by OA-sensitive phosphatases, most likely PP1 and/or
PP2A.
Using the above-described minigenome-based transcription/replication
system (8), the influence of OA on EBOV-specific transcription was
investigated. To control possible side effects of OA on the cellular
protein synthesis (25), we used VP306A, whose transcription activation function is independent of phosphorylation (see above). An
inhibitory effect of OA on transcription in the presence of VP30
wild-type and no effect in the presence of VP306A would
indicate that inhibition of PP1 and/or PP2A specifically interferes
with EBOV-specific transcription by inducing a highly phosphorylated VP30. A transcription assay was set up in BSR T7/5 cells as described above using either VP30 wild-type or VP306A. OA was added
in different concentrations after transfection. Transcription activity
was determined, and the values gained for VP306A at the
respective concentrations of OA were set to 100%. Fig. 4B
shows that increasing amounts of OA concomitantly inhibited the ability
of VP30 wild-type to activate transcription in a saturable manner
reaching a plateau at ~200 nM OA. The IC50
was determined to be 130 nM OA. These results underlined
that transcription activation function of VP30 is inhibited by
extensive phosphorylation of the N-terminal phosphate acceptor sites.
Block of Ebola Virus Growth by Okadaic Acid Is Released by the
Nonphosphorylatable VP306A--
Because OA strongly
inhibited EBOV-specific transcription in the artificial minigenome
system, it was investigated whether EBOV reproduction in target cells
could be inhibited by OA, as well. BSR T7/5 cells were infected with
EBOV and treated with different concentrations of OA. We found that the
number of infected cells at 24 h p.i. was significantly decreased
concomitantly with increasing amounts of the inhibitor (Fig.
5, gray columns). To confirm
that this effect was due to a specific inactivation of VP30 by
hyperphosphorylation, VP306A was provided by plasmid-based expression in the EBOV-infected cells in the presence of OA. Under these conditions, OA had only minute effects on EBOV infection (Fig. 5,
black columns). This result indicated that the OA-induced inhibition of EBOV growth reflects, indeed, the
phosphorylation-dependent down-regulation of VP30-mediated
viral transcription.
We have mapped the phosphorylation sites of VP30 to two N-terminal
serine clusters and threonine 52. This pattern of phosphorylation is
similar to that of Marburg virus VP30 (19).
When functional significance of VP30 phosphorylation was investigated
it was found that phosphorylated VP30 was located inside NP inclusions;
nonphosphorylated VP30, however, was evenly distributed throughout the
cytoplasm. It is reasonable to presume that the NP inclusions, like
their Marburg virus counterparts, consist of NP-induced helical
structures representing the core structures of the nucleocapsid (26).
The colocalization of NP and VP30 inside the inclusions is either
mediated by a direct interaction between the NP helices and VP30 or by
another component, e.g. RNA, as it has been shown recently
for M2-1 and N of human respiratory syncytial virus (27).
In contrast to the positive effect on the interaction with NP
inclusions, phosphorylation of VP30 negatively regulated its transcription activation function. Although nonphosphorylated VP30
activated transcription as the wild-type, a completely phosphorylated VP30 was inactive. These results were supported by the negative effect
of the phosphatase inhibitor OA on viral transcription. OA inhibits PP1
and PP2A (23, 28) so that both are not able to dephosphorylate
VP30. It is presumed that inhibition of the phosphatases leads to an
increase of VP30 phosphorylation and consequently to an inactive
protein as in the case of the pseudophosphorylated VP306D.
This presumption is strongly supported by the result that the effect of
OA on EBOV-specific transcription was overcome when VP30 was replaced
by the nonphosphorylatable mutant VP306A.
Taken together, phosphorylation inversely influenced the two known
functions of VP30. A completely phosphorylated form of VP30 was capable
of interacting with NP inclusions, but was restricted in mediating
viral transcription. In contrast, a phosphorylation-deficient VP30 was
inhibited in its interaction with the NP inclusions but supported
EBOV-specific transcription. An intermediately phosphorylated VP30
enabled both viral transcription and assembly. Phosphorylation of VP30
is therefore presumed to represent a molecular switch for the different
functions of the protein. It is hypothesized that, depending on the
respective demands, VP30 enables either viral transcription or assembly
or both. Possibly, the non- or weakly phosphorylated VP30 supports
transcription until it is removed from the general pool by the
phosphorylation-induced binding to the nucleocapsid.
The only other viruses in the order Mononegavirales containing a
structural protein of similar characteristics as filoviral VP30 belong
to the pneumovirinae subfamily of Paramyxoviridae. Here, M2-1 has been
shown to be phosphorylated in its N terminus and to interact with the
nucleoprotein of human respiratory syncytial virus in intracellular
inclusions (29-31). M2-1 is essential for respiratory syncytial virus
transcription elongation and antitermination (32-34). The
phosphorylation of M2-1 was shown to influence the RNA binding
specificity (29) and to be essential for the transcription antitermination activity (27). Thus, phosphorylation of M2-1 is of
functional significance, but the detected functions differ from that of
VP30 phosphorylation.
Interestingly, our experiments revealed that OA was able to
specifically inhibit the multiplication of EBOV in target cells. Because virus growth was only slightly affected by OA when
VP306A was expressed in trans, it is concluded
that inhibition was, indeed, caused by an inactive, hence
hyperphosphorylated, VP30.
It is reasonable to consider whether antiviral drugs could be
developed, based on the detected phosphorylation dependence of
VP30-mediated EBOV transcription. The severe side effects of OA,
i.e. promotion of tumor growth and genetic instability (35, 36), prohibit its employment as a drug to block EBOV infection. For
future antivirals it is necessary to separate the cytotoxic and the
VP30-directed effects of phosphatase inhibition. Because the
specificities of most known phosphatases are determined by their
subunit composition, i.e. regulatory and scaffolding units (37-39), it can be expected that the identification and subsequent inhibition of the VP30-specific regulatory subunit(s) of PP1 and PP2A
could lead to promising tools for antagonizing EBOV infection.
We thank Karl-Klaus Conzelmann for kindly
providing the BSR T7/5 cells and Angelika Lander for expert technical
assistance. For helpful discussion and critical reading of the
manuscript we thank Stephan Ludwig, Susanne Klumpp, and Dagmar Selke.
The used phosphatases have been provided by Susanne Klumpp. Finally, we
thank Viktor Volchkov, Lyon, for supporting work under BSL-4 conditions.
*
The work was supported by the Deutsche
Forschungsgemeinschaft (Sonderforschungsbereich 286, TP A6,
Sonderforschungsbereich 535, TP A9) and the Fazit Stiftung (to J. M.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Published, JBC Papers in Press, June 6, 2002, DOI 10.1074/jbc.M203775200
The abbreviations used are:
EBOV, Ebola virus;
OA, okadaic acid;
CAT, chloramphenicol acetyltransferase;
p.i., post
infection.
Phosphorylation of VP30 Impairs Ebola Virus Transcription*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
68--
The coding
region for amino acids 69-288 of VP30 gene was amplified by
PCR using pT-VP30F as template. The forward primer #833
(5'-ACC GGA TCC ATG CCT AAA GAC ATA TGT CCG-3') contained a
BamHI restriction site (underlined) followed by the
nucleotides 8710-8727 of the VP30 gene sequence (the
sequence number has been assigned to GenBankTM accession
number AF086833). As reverse primer, primer #409 was used. The
resulting DNA fragment was cloned into the plasmid pTM1, and the
construct was designated pT-VP3068.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Phosphorylation state analysis of VP30 and
VP30 mutants. Top panel, amino acid sequence of
position 26-55 of VP30. Serine and threonine residues, which were
mutated, are printed in boldface letters. Middle
panel, 5 × 105 HeLa cells were infected with
MVA-T7 and subsequently transfected with 1 µg of DNA plasmids
encoding either wild-type VP30 or mutants of VP30. Proteins were
metabolically labeled with 32Pi and
immunoprecipitated from the lysate using a monoclonal anti-FLAG
antibody (dilution 1:500). Immune complexes were separated on SDS-PAGE,
blotted onto polyvinylidene difluoride membranes and exposed to an
Imaging Plate. Radioactive signals were quantified using the Raytest
TINA software and normalized to the expression level of the respective
mutant, which has been checked by Western blotting. As primary antibody
a monoclonal anti-FLAG antibody (dilution 1:3,000) and as secondary
antibody, an POD-coupled sheep anti-mouse antibody (dilution 1:20,000)
was used. Lower panel, quantification of the phosphorylation
state analysis. Each mutant was tested in three independent
experiments.
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Fig. 2.
Influence of serine residues 29-46 on
colocalization of VP30 with NP inclusions. A, Vero
cells, grown on glass coverslips were infected with EBOV. At
24 h p.i., cells were fixed, permeabilized using 0.2% Triton
X-100, and subjected to immunofluorescence analysis using a monoclonal
anti-NP IgG (1:20) and a monoclonal anti-VP30 IgM (1:10). Bound
antibodies were detected with a µ-chain-specific fluorescein
isothiocyanate-conjugated F(ab')2 fragment goat anti-mouse
IgM (1:100, Dianova) and a rhodamine-conjugated goat anti-mouse IgG
(1:100, Dianova) and subjected to immunofluorescence analysis.
B-G, HeLa cells, grown on glass coverslips, were infected
with MVA-T7 and cotransfected with 200 ng of DNA plasmids encoding the
wild-type VP30 or the VP30 substitution mutants together with 1 µg
of pT-NPEBO, encoding EBOV NP. At 12 h p.i., cells
were fixed and subjected to immunofluorescence as described above.
B, single expression of NP (left panel) and VP30
(right panel). C, cotransfection of NP and VP30;
D, cotransfection of NP and VP306A;
E, cotransfection of NP and VP30S29-31A;
F, cotransfection of NP and VP30S42-46A;
G, cotransfection of NP and VP306D (all six
phosphoserines were mutated to aspartates). Staining of NP
(left-hand panel), staining of VP30 (middle
panel). Merge image (right-hand panel).
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Fig. 3.
Transcription activation by EBOV VP30 and
VP30 substitution mutants. Approximately 5 × 105
BSR T7/5 cells were transfected with DNA plasmids encoding EBOV
nucleocapsid proteins NP, VP35, L, and VP30 or VP30 mutants,
respectively, together with a DNA plasmid encoding the EBOV-specific
artificial minigenome 3E-5E, which contained the leader and trailer
regions of the EBOV genome flanking the CAT reporter gene. At 2 days
post transfection, cells were lysed and CAT activity was determined.
Lane 1, control without VP30; lane 2, wild-type
VP30; lanes 3-8, VP30 substitution mutants (the respective
mutant is given at the top of the panel).
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Fig. 4.
Identification and inhibition of VP30
dephosphorylating protein phosphatases. A, VP30 is
recognized by PP1, PP2A, and PP2C. VP30 was expressed in HeLa cells,
labeled with 32Pi, and immunoprecipitated as
described under Fig. 1. Immune complexes were incubated for 30 min at
30 °C with the catalytic subunits of PP1, PP2A, PP2C, and alkaline
phosphatase (AP). The immune complexes were then separated
by SDS-PAGE, and the gel was exposed to an Imaging Plate. Radioactive
signals were quantified using the Raytest TINA software. B,
OA inhibits VP30-mediated EBOV-specific transcription. The
transcription assay was performed as described under Fig. 3 with
wild-type VP30 or VP306A. OA was added in increasing
concentrations at 8 h post transfection. Cells were lysed at
24-36 h post transfection, and CAT activity was determined. The
inhibition of transcription activation mediated by wild-type VP30 is
given in a percentage of the transcription activation by
VP306A. Inset, CAT assay representing selected
points of the curve: Absence of OA (lanes 1 and
2), presence of 300 nM OA (lanes 3 and 4). Lanes 1 and 3, wild-type VP30;
lanes 2 and 4, VP306A.
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Fig. 5.
OA inhibits EBOV multiplication by inducing
extensive phosphorylation of VP30. BSR T7/5 cells on glass
coverslips were infected with EBOV at a multiplicity of infection of 1 plaque forming unit per cell. At 1-h p.i., cells were transfected with
a plasmid encoding either VP30 wild-type (gray columns) or
VP306A (black columns). At 24-h p.i., cells were
fixed and analyzed for the presence of NP by immunofluorescence
analysis. The number of infected cells was counted and given as the
percentage of the total cell number (determined by
4',6-diamidino-2-phenyl-indole staining).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Tel.: 64-21-286-5433;
Fax: 64-21-286-5482; E-mail: becker@mailer.uni-marburg.de.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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