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(Received for publication, February 7, 1995; and in revised form, August 9,
1995) From the
The phosphorylation of the P protein of vesicular stomatitis
virus by cellular casein kinase II (CKII) is essential for its activity
in viral transcription. Recent in vitro studies have
demonstrated that CKII converts the inactive unphosphorylated form of P
(P0) to an active phosphorylated form P1, after phosphorylation at two
serine residues, Ser-59 and Ser-61. To gain insight into the role of
CKII-mediated phosphorylation in the structure and function of the P
protein, we have carried out circular dichroism (CD) and biochemical
analyses of both P0 and P1. The results of CD analyses reveal that
phosphorylation of P0 to P1 significantly increases the predicted
The RNA-dependent RNA polymerase of vesicular stomatitis virus
(VSV) ( The role of cellular
CKII in the phosphorylation of P protein was demonstrated in vitro primarily by using the unphosphorylated form of P protein obtained
by expression of the P gene in Escherichia coli(4) .
Two forms of P protein (NJ serotype) were shown to be involved in the
activation process: a partially phosphorylated intermediate (P1) and a
fully phosphorylated form (P2). Cellular CKII phosphorylated
bacterially expressed P0 and converted it into P1, but not to P2,
demonstrating that P1 is the end product of cell kinase-mediated
phosphorylation. A highly purified L protein preparation failed to
phosphorylate P0 but phosphorylated P1 to produce P2. Thus, a cascade
phosphorylation pathway was proposed in which a sequential
phosphorylation step occurred as P0 In an
attempt to understand the phophorylation pathway and the role of
phosphorylation in P function, we have carried out structure-function
analyses of the P protein in more detail using various P mutants. Here,
we demonstrate that phosphorylation by cellular CKII induces a profound
increase in the predicted
The high salt fraction
containing L and P was dialyzed against phosphocellulose buffer (20
mM Tris-HCl (pH 7.5), 10% glycerol, 1 mM DTT) and
loaded onto a 2.0-ml phosphocellulose column pre-equilibrated with the
same buffer. The column was washed with phosphocellulose buffer, and
the bound L protein was eluted with a 0-1.0 M NaCl
gradient (12 ml) in the same buffer. Fractions in which L was
completely free of P protein (as identified by silver staining) and
devoid of cellular kinase (as checked by phosphorylation of bacterially
expressed VSV P protein as substrate) were pooled. If necessary, the L
protein was further rechromatographed onto a second phosphocellulose
column to remove any contaminating viral P protein and cellular kinase.
Figure 1:
CD
spectra of the P proteins. The CD spectra of P0 (trace 1), P1 (trace 2), and (trace 3) P59/61 were collected as
described under ``Experimental Procedures.'' The secondary
structure predictions were deduced from the spectra by the variable
selection method. The inset shows the spectra resulting from
the subtraction of P0 from P1 (A) and P59/61 (B). Any
differences in the protein concentrations were eliminated by
multiplying the P0 spectrum by a factor that made the P0 ellipticity
equal to that of P1 or P59/61 at 205 nm.
Figure 3:
Schematic representation of VSV(NJ) P
protein. The entire P protein containing all the domains is shown. The solid dots in domain I and II represent phosphorylation sites
in these regions. An enlargement of a part of domain I shows the
locations of serine residues 59 and 61 that are phosphorylated by CKII.
The different mutant P proteins are also shown. In the case of P59/61,
both Ser-59 and Ser-61 are mutated as indicated by asterisks,
whereas in the case of single mutants, either Ser-59 or Ser-61 is
unaltered, but four other possible sites are
mutated(18) .
Figure 2:
Elution profile of the P protein from gel
filtration column. Wild-type P protein was fractionated through a
Sephadex G-100 column as described under ``Experimental
Procedures.'' Positions of the two standard markers (66k and 29k) are shown on the top. Top
panel, fractionation of unphosphorylated P protein. P0 peak was
monitored either by silver staining or by labeling the protein with
[
Next, the P0 protein was phosphorylated by CKII to form P1
in the presence of [
Figure 4:
Elution profile of single serine mutant P
protein from gel filtration column. The P mutant protein was
fractionated through Sephadex G-100 column as described in the legend
to Fig. 2. The top panel represents the fractionation
of P4+61 as identified by silver staining (
To test this possibility, we used
bacterially expressed P mutant proteins and repeated our previous
experiments. Purified CsCl-banded N-RNA template and kinase-free L
protein from purified virions were prepared as described under
``Experimental Procedures'' and used in transcription
reconstitution reaction using bacterially expressed wild-type as well
as P4+59 and P4+61 mutant proteins. As shown in Fig. 5, both P4+61 and P4+59 mutant proteins were
unable to support viral transcription in the presence of N-RNA and L
protein. The P0 (denoted Pwt, Fig. 5), on the other
hand, showed the expected transcriptional activity under the same
experimental conditions. As noted earlier, the trace quantity of CKII
present in the purified N-RNA template efficiently activated P0 (Pwt)
but failed to do so for the single serine mutants. However, when
similar transcription reactions were performed in the presence of
excess recombinant CKII, both the mutant proteins were
transcriptionally active almost to the same extent as P0 (Pwt) (Fig. 6). These results indicate that P4+61 and P4+59
potentially need increased concentrations of CKII for their activation.
Mutation in a single Ser residue has increased its requirement for CKII
for phosphorylation leading to activation. To confirm whether CKII
present in the N-RNA template was indeed unable to phosphorylate
P4+61 and P4+59, we performed an in vitro phosphorylation reaction using the same amount of N-RNA template
(0.5 µg in 25 µl) as the source of CKII and 100 ng each of the
P proteins as substrate in the transcription reaction. The results
shown in Fig. 7indicate that the amount of CKII present in the
N-RNA template was indeed unable to phosphorylate P4+61 and
p4+59. In contrast, when excess recombinant CKII was added to the
reaction mixture, both mutant proteins were phosphorylated completely.
These results strongly suggest that P4+59 and P4+61 have
lower affinity for CKII, but once phosphorylated by excess CKII can
support VSV transcription. Thus, we conclude that phosphorylation of
either Ser-59 or Ser-61 is necessary and sufficient to transactivate
L-polymerase, although alteration of one serine residue significantly
decreases its affinity for CKII. To further demonstrate that complete
phosphorylation of P4+59 or P4+61 by CKII brings about
similar conformational change as observed for P1, we carried out CD
analyses of the mutant proteins. As shown in Fig. 8and Table 2, phosphorylated P4+61, as expected, exhibited
similar increases in
Figure 5:
Transcription of N-RNA template by P
proteins. In vitro transcriptions were reconstituted using
CsCl-banded N-RNA template and cellular kinase free L protein along
with various bacterially expressed mutant P proteins as described in
the text. The corresponding viral mRNAs are indicated by G, N,
P, and M. + indicates presence and - indicates
absence. Note that Pwt signifies the bacterially expressed
unphosphorylated P0 form.
Figure 6:
Transcription reconstitution with the P
proteins in the presence of CKII. Identical reactions as described in Fig. 5were performed with the exception that a saturating
concentration of CKII (0.01 milliunits) was included into each
reconstitution reaction. + indicates presence and -
indicates absence.
Figure 7:
Phosphorylation of P proteins during
transcription reaction. Identical reactions as described in the legends
to Fig. 5and Fig. 6were performed with minor
modifications. Instead of using four NTPs, 100 µM unlabeled ATP and 10 µCi of
[
Figure 8:
CD spectra of single serine mutant P
protein. The CD spectra of P1 (trace 1) and P4+61 (trace 2) were collected and analyzed as in Fig. 1. The inset shows the subtraction of P1 from
P4+61.
Figure 9:
Phosphorylation of P proteins by
L-associated kinase. The unphosphorylated (P0) and phosphorylated (P1)
forms of indicated P proteins were incubated with highly purified L
protein (
Figure 10:
Binding of P proteins to the N-RNA
template. Indicated amount of P protein was incubated with 2 µg of
N-RNA template in a 50-µl reaction mixture containing VSV
transcription buffer for 1 h at 30 °C as described under
``Experimental Procedures.'' The amount of P protein bound to
the template was determined from the autoradiogram of the gel using a
Bio-Rad densitometer scanner, and the relative value was plotted
against the amount of P protein added. We used
Figure 11:
Inhibitory effect of P59/61 in VSV
transcription. An indicated amount of P59/61 mutant protein was added
to the transcription reaction containing a constant amount of N-RNA
template (500 ng) and wild-type P protein (100 ng). The transcription
reactions were processed as described under ``Experimental
Procedures.'' Solid line, indicates transcription with
wild-type P only that is considered as 100% transcription.
It is becoming increasingly apparent that cellular protein
kinases play important role in the life cycle of several nonsegmented
negative strand RNA viruses(33, 34, 35) . It
appears that the virus structural protein P, which is a transcription
factor for such groups of viruses, needs to be phosphorylated by a
specific cellular protein kinase for transactivation of the
RNA-dependent RNA polymerase (L). Recent work from our laboratory
indicates that a cascade phosphorylation is operative for the
activation of the P protein of VSV (4, 5, 6) . First, the cellular CKII
phosphorylates two serine residues 59 and 61 within the acidic domain I
of unphosphorylated P0 rendering it biologically active (P1
form)(18) . The P1 form is then phosphorylated within the
C-terminal domain II to P2 form by L-associated protein kinase during
transcription in vitro and presumably in vivo.
However, the exact role played by the phosphorylated serine residues 59
and 61 to activate P protein and imparting the transactivation property
remains unclear. It is conceivable that phosphorylation of serine
residues brings about a change in the secondary structure of the P
protein that facilitates its binding to the L protein as well as the
N-RNA template. To probe into this possible structural alteration of
the P protein, we performed CD analyses of various P mutant proteins,
and the results obtained from such analyses strongly support the
contention that the phosphorylation by CKII indeed imparts a
significant effect in the secondary structure of the P protein. The
phosphorylated P1 form is predicted to have a high degree of
Using two single serine mutants, i.e.
mutant P4+61 or P4+59, we have shown that these mutants have
low affinity for CKII for phosphorylation such that at low
concentration of CKII, these mutants are poorly phosphorylated and
accordingly transcriptionally ineffective. However, in the presence of
a high concentration of CKII, the single mutants are not only
completely phosphorylated but also dimerized apparently and
transcriptionally active as the wild-type P1. Therefore, a single
serine phosphorylation is necessary and sufficient to activate the P
protein. Thus, two serine residues are strategically located within the
acidic domain of the P protein in such a way that phosphorylation by a
low amount of CKII allows it to rapidly fold into its proper structure.
It seems that the proper structural alteration of the P protein is a
prerequisite for its activation. In this respect, it would be
interesting to find out whether the chimeric protein in which the
acidic domain I has been replaced by apparently unrelated acidic
polypeptide, such as tubulin, possesses a structure similar to that of
P1. Earlier studies have shown that such chimeric P protein was
transcriptionally active in an in vitro transcription
reconstitution reaction(32) . Our results further
demonstrate that subsequent phosphorylation of the P protein by
L-associated kinase and its efficient binding to the N-RNA template are
all dependent on prior phosphorylation by CKII. In this connection, it
is interesting to note that genetic complementation data and the
presence of excess P protein over L protein in the viral transcription
complex have led to the suggestion that the functional VSV polymerase
may consists of one polypeptide of L and two of P, i.e. L(P) Finally, it seems that the
P protein behaves as an RNA virus transcription factor where
phosphorylation causes a major structural change which mediates
efficient binding of the P protein to the N-RNA template and presumably
L protein. In this respect, the P protein behaves like several
eucaryotic transcription factors where phosphorylation promotes
dimerization and binding to the DNA template (19, 24) . Detailed structural studies of the P
protein, e.g. crystallography, NMR studies, etc., would
certainly aid us understanding its three-dimensional structure as it
relates to its function. The knowledge derived from the above studies
would certainly provide the opportunity to understand whether the
phosphorylation pathway as demonstrated in VSV is unique to other
viruses like rabies, measles, mumps, respiratory syncytial virus (RSV),
human parainfluenza virus (HPIV), etc., which employ the same strategy
as VSV to invade cells. Recently, the cellular protein kinases that
phosphorylate the P proteins of several RNA viruses have been
identified. Similar to VSV, both RSV P (35, 36) and
measles P (37) proteins have been shown to be phosphorylated by
the same cellular enzyme, CKII. In contrast, the cellular protein
kinase that phosphorylates HPIV-3 P protein was found to be
indistinguishable from cellular protein kinase C subtype
Volume 270,
Number 41,
Issue of October 13, 1995 pp. 24100-24107
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
-helical structure of the P1 protein from 27 to 48%. The
phosphorylation defective double serine mutant (P59/61), which is
transcriptionally inactive, possesses a secondary structure similar to
that of P0. P1, at a protein concentration of 50 µg/ml, elutes from
a gel filtration column apparently as a dimer, whereas both P0 and the
double serine mutant elute as a monomer at the same concentration.
Interestingly, unlike wild-type P1 protein, the P mutants in which
either Ser-59 or Ser-61 is altered to alanine required a high
concentration of CKII for optimal phosphorylation. We demonstrate here
that phosphorylation of either Ser-59 or Ser-61 is necessary and
sufficient to transactivate L polymerase although alteration of one
serine residue significantly decreases its affinity for CKII. We have
also shown that P1 binds to the N-RNA template more efficiently than P0
and the formation of P1 is a prerequisite for the subsequent
phosphorylation by L protein-associated kinase. In addition, mutant
P59/61 acts as a transdominant negative mutant when used in a
transcription reconstitution assay in the presence of wild-type P
protein.
)consists of two proteins: the large protein L (241
kDa) and the phosphoprotein P (29 kDa). Together, these proteins are
needed to transcribe the linear, single-stranded viral RNA genome of
negative polarity, which is tightly wrapped with the nucleocapsid N
protein (N-RNA template)(1, 3) . Genetic and
biochemical studies have suggested that the L protein encodes all the
basic transcription activities, whereas the P protein appears to be an
RNA virus transcription factor (1, 2, 7) with
properties similar to many well studied eucaryotic transcription
factors/activators(31) . The P protein contains -helical
coiled structure and is highly acidic, with Asp and Glu residues
constituting one-third of the first 100 amino acid residues in the
N-terminal half (domain I) of the
polypeptide(17, 18) . The acidic domain is also
phosphorylated by cellular protein kinase(14, 15) .
The possible contribution of the N-terminal acidic domain I in the
function of P protein seems to transactivate the L protein for
transcription similar to those observed for eucaryotic acidic
transactivators(30, 31, 32) . The C-terminal
end, on the other hand, serves as the binding site for the L protein
(domain II) and the N-RNA template (domain
III)(8, 13) . Initial studies of P protein isolated
from virions or infected cell extract indicated that it exists in a
variety of phosphorylated states and that this phosphorylation event
was important for the transcriptional activity of
L(10, 11, 12) . Recently we have shown that
cellular protein kinase, casein kinase II (CKII), is directly involved
in phosphorylating the P protein at serine residues 59 and 61 in domain
I(6) ; activation of P protein occurs following this initial
phosphorylation event. Two additional sites at the C-terminal domain
(domain II) are also phosphorylated by an L protein-associated kinase
at serine residues 236 and 242, as determined previously by mutational
analyses of recombinant P protein(16) . P1
P2, leading to the
activation of the P protein(5, 6) . Thus, it seems
that phosphorylation of the P protein by CKII is the first biosynthetic
event in the infected cell that possibly leads to a conformational
change in the P protein such that domain II becomes accessible to
L-kinase. However, the precise role of CKII-mediated phosphorylation in
the structure and function of the P protein remains unclear.
-helical structure and in the apparent
dimerization of the P protein as determined by gel filtration analysis.
In addition, phosphorylation facilitates the binding of P to the N-RNA
template as well as subsequent phosphorylation by L-associated kinase.
We have characterized two P phosphorylation mutants which require
higher concentration of CKII for their optimal phosphorylation leading
to activation and concomitant alteration of structure of the P protein.
Materials
All enzymes and biochemicals were
obtained from Boehringer Mannheim and Sigma. Tissue culture reagents
and media were purchased from Life Technologies, Inc.Cell Cultures and Virus
VSV New Jersey serotype,
Ogden strain was purified as described previously (4, 22) from baby hamster kidney cells (BHK-21: ATCC
CCL 10) by inoculating with virus at a multiplicity of infection of
0.05. BHK cells were maintained in Eagle's minimal essential
media supplemented with 7% fetal bovine serum.Purification of Recombinant P Proteins from E.
coli
Various recombinant P mutant proteins were expressed in E. coli and purified essentially as described
previously(4) . After purification by phosphocellulose and DE52
column chromatography, the P proteins were more than 90% pure.CD Spectroscopy of P Protein
CD spectra of various
P mutant proteins were measured at 25 °C on a Jasco J-600
spectropolarimeter interfaced to a computer for data collection and
manipulation. The instrument was calibrated with d-10 camphorsulfonic
acid(25) . Spectra were recorded between 180 and 260 nm using a
0.01-mm quartz cell. All spectra, averages of at least 10 scans, are
correlated for the spectral contributions of the buffer. The
measurements were performed at varying protein concentrations in a
solution containing 20 mM Hepes (pH 7.5), 50 mM NaCl.
The protein concentration was determined according to Beaven and
Holiday(28) . The mean residue mass was calculated from the
amino acid composition(26) . The spectra were analyzed to
estimate the distribution of secondary structural elements by the
variable selection method developed by Manavalan and
Johnson(27) .Gel Filtration Analysis of P Protein
Bacterially
expressed, purified P protein at various concentrations was
chromatographed through a Sephadex G-100 gel filtration column. An
18-ml bed volume column was first equilibrated with VSV transcription
buffer (50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 5
mM MgCl
, 2 mM DTT) and subsequently
calibrated with standard proteins of known molecular size. We used
bovine serum albumin (66 kDa), ovalbumin (45 kDa), and carbonic
anhydrase (29 kDa) (purchased from Sigma) as known protein standards. P
protein (5 µg) in a 100-µl VSV transcription buffer was
fractionated, and the alternate fractions were monitored by SDS-PAGE,
followed by silver staining and autoradiography where applicable. For
unphosphorylated and P59/61 mutant P proteins, the fractions were
analyzed either by silver stain or by autoradiography of S-labeled proteins. On the other hand, for phosphorylated
P proteins, bacterially expressed P0 and P4+61 mutant proteins
were first phosphorylated with recombinant CKII and
[
-
P]ATP in vitro in a VSV
transcription buffer and chromatographed through Sephadex G-100 in the
same way as described above. The P protein band in the SDS gel was
identified by its characteristic mobility (molecular mass
46 kDa)
as well as by Western blot analyses with specific antibody.
Protein Kinase Assay
In the standard protein
kinase assay, a varying amount (0.1-1 µg) of the desired form
of P protein (P0, P1, or mutant P proteins) was incubated in the
presence of CKII (0.01 milliunits), or 1 µg of L, or 0.5 µg of
CsCl-banded N-RNA template as the source of protein kinases in a
20-µl reaction mixture containing VSV transcription buffer and 100
µM ATP in the presence of 10 µCi of
[-
P]ATP. Reactions were incubated for
1-2 h at 30 °C and either immediately stopped by adding
Laemmli buffer or subjected to chromatography on DE52 to resolve and
purify the product as described earlier(5) .
Binding of P Protein to the N-RNA Template in
Vitro
N-RNA (2 µg) was incubated with increasing amount of P
proteins in a reaction volume of 50 µl containing VSV transcription
buffer for 1 h at 30 °C. In the case of P0 and P59/61 mutant, we
used [S]methionine-labeled proteins, whereas in
the case of P1 and phosphorylated mutant proteins, we labeled the
proteins with [
-
P]ATP in the presence of
CKII. Reactions were placed on ice and layered onto 600 µl of same
buffer containing 30% glycerol and centrifuged for 3 h at 45 K in the
SW50.1 rotor in 0.7-ml centrifuge tubes. The glycerol was gently
removed with a long tip micropipette, and the sides of the tubes were
dried using tissue paper. The N-RNA pellet and any associated P
proteins were resuspended in Laemmli loading buffer and analyzed on 10%
SDS-PAGE. Binding of the P proteins to the N-RNA template was
quantified by desitometric scanning of the P band in the autoradiogram.
We also performed control experiments where P proteins were pelleted
without the N-RNA template, and this background count was subtracted
from each experiment. Each binding assay was performed independently,
in duplicate, and the results presented are the average of these
experiments.
Purification of VSV Transcription Components
The
isolation and purification of N-RNA template and L protein was
performed essentially as described (23) with a number of
modifications. 10 mg of purified VSV was initially disrupted in a
buffer containing 0.4 M NaCl, 10 mM Tris-HCl (pH
8.0), 5% glycerol, 2% Triton X-100, 1 mM DTT, by incubation on
ice for 90 min with continuous gentle rocking. The viral
ribonucleoprotein (i.e. the genomic RNA associated with N, P,
and L proteins) was then purified by centrifugation onto a 100%
glycerol cushion through 30% glycerol containing 10 mM Tris-HCl (pH 8.0), 10 mM NaCl, 2 mM MgCl, 1 mM DTT for 2 h at 45 K in an SW60
rotor at 4 °C. Ribonucleoprotein was collected from the top of a
100% glycerol cushion in 2 ml of Tris-EDTA and was disrupted again as
described above, but in buffer containing 1 M NaCl and 0.5%
Triton X-100 to dissociate the L and the P proteins from the N-RNA
template. The N-RNA was purified by centrifugation through 30% glycerol
onto a 100% glycerol cushion in the same manner as described above. The
high salt supernatant containing L and P proteins was stored at
-80 °C until further use. N-RNA was further purified by an
additional high salt wash, centrifuged through 15% Renografin onto a
76% Renografin cushion(23) , followed by two serial recoveries
from CsCl gradients (0.35 mg of CsCl/ml of Tris-EDTA final
concentration) by centrifugation at 45 K for 16 h in an SW60 rotor at
20 °C. N-RNA was finally dialyzed against Tris-EDTA. The purity of
the N-RNA template was determined initially by silver staining of gels
after SDS-PAGE and finally by reconstitution of transcription in
vitro with recombinant (29) or viral L protein and
bacterially expressed P protein(4) .Reconstitution of VSV Transcription in Vitro
VSV
transcription in vitro was carried out essentially as
described earlier (23) except that E. coli expressed,
recombinant P proteins were used instead of viral P protein. 500 ng of
N-RNA, approximately 50 ng of L, and 100 ng of P were used in a
25-µl reaction mixture which also contained VSV transcription
buffer, 0.5 mM each of ATP, CTP, GTP, 100 µM UTP,
10 µCi of [-P]UTP and 1 unit/µl
RNasin. Reactions were incubated for 2 h at 30 °C, and poly(A)
tails were removed from the viral messages by treating with 100 ng of
oligo(dT) and 1 unit of RNase H at 37 °C for 15 min. Reactions were
terminated by extraction with phenol-chloroform and precipitated with
ethanol in the presence of 5 µg of carrier tRNA. Viral RNA products
were analyzed by electrophoresis on 5% polyacrylamide gel containing 7 M urea as described earlier(23) .
Other Procedures
Quantitative silver staining of
polyacrylamide gels was done by using Bio-Rad reagents and protocols.
Radioactivity of labeled protein fractions was measured in a model LS
1701 Beckman liquid scintillation counter. Where needed, densitometric
scanning of stained protein bands or autoradiograms were performed in a
model 620 Bio-Rad video densitometer.
CD Spectroscopy of P Protein
The cascade
phosphorylation pathway proposed by Barik and Banerjee (5) strongly suggests that the phosphorylation of P0 to P1 must
initiate a conformational change in the protein which presumably
imparts its transactivation property. Since the prediction of the
secondary structure for globular proteins from circular dichroism
spectroscopic data is a well established method in recent
years(27) , we were interested in comparing the secondary
structures of P0 and P1 proteins using far UV circular dichroism
spectroscopy. As shown in Fig. 1and Table 1, CD analysis
of P0 predicts a secondary structure distribution of 27% -helix
and 23% 
-structure. Interestingly, a dramatic change from
-structure to -helical structure is predicted when the P
protein is phosphorylated by CKII. The predicted secondary structure of
P1 consists of 48% -helix and only 7% -structure (Table 1), i.e. an increase of approximately 2-fold in
helical content over unphosphorylated P0. We have shown previously that
Ser-59 and Ser-61 residues of the P protein are phosphorylated by CKII,
and when both serine residues are altered to alanine (Fig. 3,
P59/61), the mutant P protein became inactive in
transcription(18) . When the structure of the P mutant was
analyzed by CD, the mutant protein showed similar overall secondary
structure as that of P0 protein (Fig. 1, Table 1). These
results strongly suggest that there is a significant difference in the
secondary structures between P0 and P1 proteins. The conformation of P1
is more -helical in nature similar to many well studied eucaryotic
transcription factors.
CKII-mediated Phosphorylation of Domain I Facilitates the
Apparent Dimerization of P Protein
Since the activities of many
transcription factors have also been shown to depend on their ability
to form homodimers(24) , we were interested to examine whether
phosphorylated P protein (P1) can oligomerize in vitro. To do
so, we expressed various P mutants in bacteria and used the purified
proteins for such studies. The P0 at a concentration of 50 µg/ml
was subjected to chromatography in Sephadex G-100 gel filtration column
along with standard proteins of known molecular size as detailed under
``Experimental Procedures.'' Fractions were analyzed by
SDS-PAGE, and the P protein band was identified by its characteristic
mobility (M
46,000) after silver staining or
autoradiography (in the case of
S-labeled protein). A
densitometric scan of a representative fraction (Fig. 2, top
panel) shows that essentially all of the P protein eluted at a
position consistent with a 29-kDa monomeric polypeptide, suggesting
that in the unphosphorylated form, P protein exists as a monomer at 50
µg/ml concentration. However, at a high protein concentration, i.e. 200 µg/ml, P0 eluted at a position higher than the
66-kDa marker, indicating that the unphosphorylated P protein has a
natural propensity to form oligomer at high concentration (data not
shown).
S]methionine (
). Middle panel,
fractionation of a mixture of unphosphorylated (P0) and phosphorylated
P1 protein. The P1 peak was monitored by silver staining () as
well as P counting (
), arbitrary scale not shown. Bottom panel, the fractionation of P59/61 (phosphorylation
defective mutant).
-
P]ATP in vitro and tested its size estimated in the same manner as described
above. Interestingly, P1 eluted at a position consistent with its being
a dimer at the same protein concentration, i.e. 50 µg/ml,
at which P0 fractionated as a monomer (Fig. 2, middle
panel). A small amount of remaining unphosphorylated P0 was
fractionated as a monomer. When phosphorylation-defective P59/61 double
mutant was subjected to a similar gel filtration analysis, as expected,
it fractionated as a monomer at the same protein concentration as P0 (Fig. 2, lower panel). These results strongly suggest
that CKII-mediated phosphorylation brings about changes in the
secondary structure of the P protein and perhaps facilitates apparent
dimerization of P monomer.
Role of Single Phosphorylation in P Function
We
were especially interested in two P mutants (Fig. 3), e.g. P4+59 (where Ser-59 is unaltered but four other possible
phosphorylation sites are mutated to alanine) or P4+61 (where
Ser-61 is unaltered and four other sites are mutated to alanine), which
when expressed in COS cells, were phosphorylated to the same extent as
that of wild-type P protein(18) . However, these mutants were
transcriptionally inactive in vitro, when
transcription-reconstitution was performed using wheat germ-translated
proteins(18) . To resolve the discrepancy between the in
vivo and in vitro data, we expressed these single serine
mutant P proteins in E. coli and examined their elution
profile in Sephadex G-100 column chromatography as described above.
Unexpectedly, when these mutant proteins were phosphorylated by CKII in vitro and subjected to gel filtration analysis, they eluted
in the same manner as that of wild-type P1 (Fig. 4, top
panel), suggesting that they form apparent dimers at low protein
concentration (50 µg/ml). We reasoned that the inability of
P4+61 and P4+59 to transactivate L protein as observed before
may be due to incomplete phosphorylation of the single serine residues
by CKII present in wheat germ extract during their translation in
vitro due possibly to their low affinity for CKII(18) .
Whereas, when these mutant proteins were completely phosphorylated by
recombinant CKII in vitro, they were able to form dimers,
thus, transcriptionally active.
) and P counting (
), whereas the bottom panel shows the result for P0, for
comparison.
-helical structure as the wild-type P1.
-
P]ATP were used in each reaction. After
incubation at 30 °C for 2 h, the samples were analyzed by 10%
SDS-PAGE followed by autoradiography. + indicates presence and
- indicates absence.
Phosphorylation of P Mutants by L-kinase
Next, we
were interested to study whether CKII-mediated phosphorylation is a
prerequisite to the subsequent phosphorylation by L-associated kinase (5) . To do so, we performed standard protein kinase assay by
using CKII-free L protein purified from viral ribonucleoprotein complex
as described under ``Experimental Procedures.'' As shown in Fig. 9, the L protein failed to phosphorylate P0, confirming
that P0 is not the substrate for L-kinase(5) . Similarly,
L-kinase was unable to phosphorylate the P mutant where both Ser-59 and
Ser-61 were altered. In contrast, when P1 was used as the substrate in
the L-kinase reaction, L protein could effectively phosphorylate P1 to
produce P2 as observed earlier(5) . As expected, the
phosphorylated single P mutants (either P4+61 or P4+59),
which are transcriptionally active, were further phosphorylated by
L-kinase. These results indicate that the P mutants, which can attain
the similar structure as that of P1 after phosphorylation by CKII, are
the proper substrates for L-kinase, suggesting that L protein
recognizes only the phosphorylated form of the P protein.
1 µg) in a standard protein kinase reaction as
described under ``Experimental Procedures.'' In case of P0, 1
µg of P protein was used in a 20-µl kinase reaction. However,
in the case of P1, 5 µg of bacterially expressed unphosphorylated P
protein was first phosphorylated with 0.05 milliunits of CKII in a
100-µl reaction mixture containing 0.5 mM unlabeled ATP.
The unlabeled P1 was then purified by DE52 column chromatography.
Approximately 1.0 µg of P1 protein was used in kinase reaction by
L. + indicates presence and - indicates absence of
corresponding agent.
Role of CKII-mediated Phosphorylation on the Template
Binding Activity of the P Protein
Since the physical and
functional interactions between the N-RNA template and the P protein
are required for the RNA synthetic process, we wanted to study whether
the phosphorylation by CKII facilitates binding of the P protein to the
N-RNA template in vitro. We used purified S-labeled unphosphorylated (P0 and P59/61) and
P-labeled phosphorylated P proteins (P1 and single serine
mutants) for this assay. N-RNA template (2 µg) was incubated with
increasing amounts of each of the P mutant proteins in a reaction
volume of 50 µl, and binding of P protein to the template was
quantified by the densitometric scanning of the autoradiogram as
detailed under ``Experimental Procedures.'' The results shown
in Fig. 10indicate that P0 and P59/61 bind to the template in a
manner which is sigmoidal in nature (K
approximately 2 
10M),
whereas P1, P4+61, or P4+59 (data not shown) bound in a
linear fashion, indicating stoichiometric binding (K
10M). This suggests that both P1 and
phosphorylated single P mutant proteins interact with the N-RNA
template with higher affinity than the unphosphorylated P protein.
Thus, it seems that change in conformation and the apparent
dimerization induced by CKII facilitate the template binding activity.
S-labeled
protein in the case of P0 and P59/61, whereas
P-labeled
protein was used in the case of P1 and P4+61.
,
unphosphorylated P0; , P59/61 mutant;
, P1; ,
P4+61 mutant.
Inhibition of Viral Transcription by Double Mutant P
Protein
Since the P mutant P59/61 is transcriptionally inactive
due to its inability to be phosphorylated by CKII, it was of interest
to examine whether this mutant protein can act as a transdominant
negative mutant. To test the above possibility, we carried out in
vitro transcription reconstitution assay by using increasing
amounts of P59/61 mutant protein to study its effect, if any, on
transcriptional activity in the presence of P0 protein, which is
phosphorylated by CKII associated with the N-RNA template. The results
shown in Fig. 11demonstrate that only 2-fold molar excess of
P59/61 mutant protein completely inhibited the transcriptional activity
of wild-type P protein. However, addition of 10-fold molar excess of
wild-type P protein in the same reaction did not show any inhibitory
effect (data not shown), indicating that P59/61 mutant indeed acts as a
transdominant negative inhibitor.
,
indicates reaction where both wild-type and mutant P proteins were
included.
-helical structure (48%) compared with unphosphorylated P0 which
is predicted to contain only 27% -helix, similar to that of
phosphorylation defective double mutant P59/61 (Fig. 1, Table 1). Presumably, the increased -helical structure
directly plays an important role for the tranactivation property of the
P protein. Consistent with this observation, many eucaryotic
transcription factors have also been shown to posses extensive
-helical structures that are implicated in binding to cognate
proteins or promoter sequences on DNA(30) . Moreover,
three-dimensional structural analyses have shown that phosphorylation
can affect protein activity by inducing allosteric conformational
changes, as well as by electrostatic repulsive effects, and these
mechanisms are both likely to be important in regulating the function
of transcription factors(20, 21) . This alteration of
the secondary structure possibly induces efficient dimerization of the
transcription factors for their functional activity. Similar phenomena
seem to be operative for the P protein as it relates to its function.
Based on the results obtained from gel filtration analyses, it seems
that the wild-type P protein, in its unphosphorylated form, undergoes
dimerization according to the reversible reaction: 2(P0)
(P0)
. Phosphorylation of the P protein at Ser-59 and Ser-61
facilitates the dimerization process by increasing the association
constant of the monomers such that the reaction 2(P1) (P1)
takes place at a low protein concentration. Alternatively, since
gel filtration measures a change in the Stokes radius of the protein,
it is possible that phosphorylation of the P0 protein simply changes
its shape and not its state of oligomerization. A direct measurement
like chemical cross-linking or sedimentation analysis need to be
performed to come to a definite conclusion. Thus, it seems that the
first step toward activation of P protein by CKII is most likely the
alteration of the secondary structure with apparent dimerization of the
protein. The P protein then binds with the L protein and the N-RNA
template to initiate the RNA synthetic process. How precisely the
latter process manifests still remains an enigma. While this work was
in the review process, Gao and Lenard (39) have reported that
the active P1 protein of VSV (Indiana serotype) exists as multimeric,
probably tetrameric, structure as determined by gel filtration and
cross-linking analyses. Whether this apparent discrepancy relates to
the different serotypes of the P protein used in these studies remains
to be determined.(9) . It is interesting to note that the
phosphorylation-defective P mutant (P59/61) is transcriptionally
inactive due to its inability to undergo the phosphorylation process
that controls the proper folded structure of the P protein. Consistent
with this result, the CD analysis also suggests that the secondary
structure of this double mutant is identical to that of
unphosphorylated P protein (P0). However, this double mutant acts as a
transdominant negative mutant in an in vitro transcription
reconstitution with wild-type P protein (Fig. 11). It remains to
be seen whether this mutant forms a heterodimer with wild-type P
protein that leads to the formation of an inactive L(P-P59/61) complex. Regardless of the mechanism of inhibition of viral
transcription by this double mutant, this mutant may be utilized for
generating a resistant cell line to VSV. (38) . It is noteworthy that in all the cases, phosphorylation
by either CKII or PKC takes place within the acidic domains of the P
proteins in spite of the difference in their relative sites within the
polypeptide. Thus, it will be interesting to see whether the
introduction of phosphate groups brings about similar conformational
changes (as found in VSV P) in the acidic domains of measles, RSV, or
HPIV-3 P proteins, which in turn lead to their transcriptional
activation. Involvement of different protein kinases in the regulation
of gene expression of these RNA viruses suggests that these viruses
might infect the host organ in a tissue-specific manner where the
required source of the essential protein kinase is available. Further
studies along these lines would certainly be important to design and
develop antiviral agents specifically directed to the cellular kinases
which have an intimate relationship with the virus's life cycle.
)
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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ABSTRACT
