(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
-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.
The RNA-dependent RNA polymerase of vesicular stomatitis virus
(VSV) ()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) .
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 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.
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 -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.
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
[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).
Next, the P0 protein was phosphorylated by CKII to form P1
in the presence of [-
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.
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 () and
P counting (
), whereas the bottom panel shows the result for P0, for
comparison.
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 -helical structure as the wild-type P1.
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
[-
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.
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 (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.
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 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.
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. ,
indicates reaction where both wild-type and mutant P proteins were
included.
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
-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.
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)(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.
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 (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.