(Received for publication, May 25, 1995; and in revised form, August 30, 1995)
From the
Phosphorylation of the polyomavirus major capsid protein VP1
plays a role in virus assembly and may function in virus-cell
recognition. Previous mapping of the in vivo phosphorylation
sites on VP1 identified phosphorylation of threonine residues Thr-63
and Thr-156 (Li, M., and Garcea, R. L.(1994) J. Virol. 68,
320-327). Phosphoserine was detected in a tryptic phosphopeptide
encompassing residues 58-78. Because of consensus casein kinase
II (CK II) sites in this peptide, we examined the in vitro phosphorylation of the purified recombinant VP1 protein by CK II.
CK II phosphorylated VP1 on serine, and the resulting tryptic
phosphopeptide eluted in a 30-31 min high performance liquid
chromatography fraction corresponding to residues 58-78. The VP1
tryptic phosphopeptide also co-migrated in two-dimensional peptide
analysis with one of the tryptic peptides obtained from VP1 isolated
after in vivoP labeling of virus-infected cells.
A site-directed mutant VP1 protein, Ser-66 to Ala, was phosphorylated
poorly by CK II in vitro. As determined by electron
microscopy, all of the mutant proteins were isolated in pentameric form
similar to the wild-type protein, although the Ala-66 pentamers had a
tendency to self-assemble in vitro into tubular as well as
capsid-like structures. These findings identify Ser-66 as a site of VP1
phosphorylation in vitro, and suggest that VP1 may serve as a
substrate for CK II in vivo.
The polyomavirus capsid is composed of 72 pentamers of the major coat protein VP1(1) . The minor capsid proteins VP2 and VP3 are present in approximately one-tenth the abundance of VP1 in the virion and play an unknown structural role(2) . VP1 is synthesized late in the viral lytic cycle and transported to the nucleus of the infected cells where encapsidation of the viral minichromosome occurs. Studies have suggested that post-translational modifications of VP1 play an important role in virus assembly and cell infection(3, 4, 5, 6) . VP1 is phosphorylated in serine and threonine residues(7, 8) . Recently, we mapped the phosphorylation sites of VP1 isolated from virus-infected mouse cells(9) . Threonine phosphorylation of VP1 was identified on residues Thr-63 and Thr-156 in the BC and DE loops, respectively, which are exposed on the exterior viral surface(10) . A defect in virus assembly was associated with a mutation at threonine 156(9) . Serine sites, although present in the same tryptic phosphopeptides as the threonine sites, could not be assigned because viruses reconstructed with mutations at these serine residues were nonviable.
Polyoma host-range nontransforming mutant viruses have
genetic alterations in both middle and small tumor (T)-antigens, and
are defective in cell transformation in vitro and tumor
induction in vivo(11) . The host-range nontransforming
mutant viruses are blocked in virus assembly when grown on
nonpermissive cells(3) . The assembly defect of host-range
nontransforming mutant viruses is associated with underphosphorylation
of VP1 on threonine(8) . In vivo phosphate labeling of
VP1 during host-range nontransforming mutant virus infections showed
that phosphorylation of VP1 on both residues Thr-63 and Thr-156 was
defective(9) . This regulation could be controlled by
activation of a cellular kinase, e.g. pp60, or inactivation of a specific
phosphatase, e.g. phosphatase 2A, both activities known to be
associated with middle
T-antigen(12, 13, 14) . VP1 serine
phosphorylation appears constitutive, however, at least in the presence
of an intact large T-antigen(8) .
Casein kinase II (CK II) ()is a ubiquitous cyclic nucleotide independent
serine-threonine kinase present in the cell nucleus and
cytoplasm(15, 16) . CK II is activated rapidly when
cells are treated with certain growth factors such as serum, epidermal
growth factor, and insulin-like growth factor (17, 18, 19) . These findings raise the
possibility that CK II plays an important role in cellular activities
related to cell growth and proliferation(20, 21) .
This enzyme has a number of substrates including MYC and
p53(22, 23) . In addition, CK II activity is
stimulated by many of the agents that activate c-fos transcription(18, 24, 25) . These
substrates suggest that CK II may link signal transduction pathways and
nuclear proteins that control cell
proliferation(26, 27) . CK II has also been found to
phosphorylate structural and nonstructural viral proteins such as SV40
large T-antigen and varicella-zoster virus
glycoprotein(28, 29) . The data presented in this
report show that the polyomavirus major capsid protein VP1 is also
phosphorylated by CK II.
The peptides
GQPPTPESLTEGGQYYGWSRGINC and DVHGFNKPTDTVNTKGISTPVEGC, corresponding to
residues 59-81 and 138-160 of VP1, were reacted with CK II
in kinase buffer. The P-labeled peptide was spotted on
Immobilon-P membrane and free [
-
P]ATP
eluted with water. The peptide was then subjected to phosphoamino acid
analysis.
Figure 1:
In vitro phosphorylation of VP1 by CK II. Purified recombinant VP1 protein
was incubated with CK II and [-
P]ATP, and
the products analyzed by SDS-PAGE and autoradiography. Panel
A, Coomassie Blue stained; and panel B, autoradiogram.
Full-length VP1 (lanes 1 and 2) and
NCOVP1 (lanes 3 and 4) were incubated with (lanes 2 and 4) or without (lanes 1 and 3) CK
II. Panel C, phosphoamino acid analysis of the
P-labeled VP1 (full-length, lane 1;
NCOVP1, lane 2).
Immunoprecipitates of VP1 from
virus-infected cells were also tested for associated kinase activity by
direct incubation with [-
P]ATP (data not
shown). No significant VP1 ``autophosphorylation'' was
detected and the addition of CK II to the immunoprecipitates did not
lead to increased VP1 phosphorylation. Mitogen-associated protein
(p44), cdc2 (p34), and cdk2 kinases were also tested for their ability
to phosphorylate VP1 in vitro. (
)Mitogen-associated
protein kinase did not phosphorylate VP1. cdc2 and cdk2 kinases did
phosphorylate VP1 in vitro, but the tryptic phosphopeptides
resulting from these reactions did not correspond with those seen in vivo (see below) and these enzymes were therefore not
further characterized.
Figure 2:
HPLC analysis of VP1 tryptic
phosphopeptides. VP1 was either labeled in vivo with
[P]orthophosphate or in vitro by CK II. Panel A, the tryptic peptides from in vivo (
)
and in vitro (
)
P-labeled VP1 were
fractionated by C18 reverse-phase HPLC. Panel B,
phosphopeptides (30-31 min fraction) from in vivo (lane 1) and in vitro (lane 2)
P labeling were subjected to phosphoamino acid
analysis.
Phosphoamino acid analysis of VP1 isolated
from polyomavirus-infected cells showed that the phosphopeptide in the
36-37 min fraction is phosphorylated only on threonine residues (9) and the phosphopeptide in the 30-31 min fraction is
phosphorylated on both threonine and serine residues (Fig. 2B, lane 1). The phosphopeptide in the
30-31 min fraction of in vitro CK II-kinased recombinant
VP1 contained only phosphoserine (Fig. 2B, lane 2).
These results suggest that phosphorylation of VP1 by CK II was
primarily on serine sites between residues 58 and 78. In order to
verify that the in vivo and in vitro phosphopeptides
were identical, they were further analyzed by two-dimensional
phosphopeptide mapping (Fig. 3). The two-dimensional mapping
showed that VP1 isolated from polyomavirus-infected mouse cells has
three phosphopeptide spots (Fig. 3A)(9) .
Phosphopeptide spots 1 and 2 contained phosphothreonine, and spot 3
phosphoserine (9) (data not shown). Recombinant VP1 protein
phosphorylated by CK II showed a major phosphopeptide spot containing
phosphoserine (Fig. 3B; data not shown). When P-labeled VP1 from polyomavirus-infected cells was mixed
with the recombinant VP1 phosphorylated by CK II, spot 3 from the in vivo labeled VP1 co-migrated with the phosphopeptide
generated by CK II (Fig. 3C). HPLC analysis showed that
both spots 2 and 3 eluted in the 30-31 min fraction (9) (data not shown). This result may be due to two different
peptides eluting coincidently from the 30-31 min fraction, or the
same peptide which has been differentially modified. The
two-dimensional chromatography analysis supports the latter, suggesting
that the peptide from residues 58 to 78 is phosphorylated on either
threonine or serine residues, but not both, and each of these
modifications yields a distinctive species in the two-dimensional
chromatogram (spot 2 or 3).
Figure 3:
Two-dimensional mapping of VP1 tryptic
phosphopeptides. The in vivoP-labeled VP1 and
VP1 phosphorylated by CK II in vitro were digested with
trypsin, and analyzed by two-dimensional phosphopeptide mapping. A,
P-labeled VP1 from polyomavirusinfected cells; B, recombinant VP1 protein phosphorylated in vitro by
CK II; C, a mixture of samples in panels A and B.
To confirm that VP1 residues 58-78 contained a serine that was a substrate for CK II, a peptide of VP1 corresponding to residues 59-81 was synthesized. This peptide was incubated with CK II in vitro, and phosphoamino acid analysis of the reaction product demonstrated phosphoserine (Fig. 4, lane 1). In addition, a peptide corresponding to residues 138-160 (the DE loop of VP1) was also phosphorylated by CK II (Fig. 4, lane 2). This peptide contains a potential consensus CK II site (residue Thr-156), a site phosphorylated in vivo. In vitro the intact protein is not phosphorylated in this region, but the synthetic peptide is phosphorylated on both serine and threonine. These results suggest that in the intact VP1 pentamer, potential CK II sites are conformationally distinct for kinase recognition.
Figure 4: BC and DE loop peptides of VP1 are phosphorylated by CK II in vitro. BC (lane 1) and DE (lane 2) loop peptides of VP1 were phosphorylated by CK II, and the phosphorylated peptides were analyzed for phosphoamino acids.
Figure 5:
In vitro phosphorylation of
mutant VP1 proteins by CK II. Wild-type and mutant VP1 proteins were
incubated with CK II, and the phosphorylated proteins analyzed by
SDS-PAGE and autoradiography. Lanes 1 and 6,
wild-type VP1; lanes 2 and 7, Ala-66 VP1; lane
3, Ala-77 VP1; lanes 4 and 8, Gly-63 VP1; lanes 5 and 9, Ala-156 VP1. Lanes 1-5,
full-length; lanes 6-9, NCOVP1 variants of these
proteins. A, Coomassie Blue stain; B,
autoradiogram.
Figure 6: In vitro assembly of mutant VP1 proteins. The Ala-77 (A) and Ala-66 (B) mutant VP1 proteins were incubated under assembly conditions and examined by electron microscopy (see ``Materials and Methods''). The Ala-77 protein formed typical capsid-like aggregates similar to the wild-type protein, whereas the Ala-66 protein formed both tubular and capsid-like structures. Bar equals 100 nm.
These data demonstrate that the polyomavirus major capsid protein VP1 is an in vitro substrate for phosphorylation by CK II. VP1, purified after expression in E. coli, was phosphorylated in vitro by CK II, and phosphoamino acid analysis demonstrated only phosphoserine residues. The major phosphorylated serine site modified by casein kinase II was located in the tryptic peptide encompassing residues 58 to 78. A site-directed mutant VP1 protein, with a serine-to-alanine change at residue 66, was defective in CK II phosphorylation. Therefore, Ser-66 is the phosphorylation site modified by CK II in vitro, and the data suggest that the in vivo phosphopeptide representing residues 58-78 is also phosphorylated on Ser-66.
Recently the VP1
threonine phosphorylation sites were mapped(9) . Two major
phosphopeptides, in residues 58-78 and residues 153-173,
were identified from in vivoP labeling
polyomavirus-infected cells. Viruses with site-directed mutations
confirmed that VP1 was phosphorylated on Thr-63 and Thr-156. However,
the serine phosphorylation site(s) was unidentified because mutant
viruses with substitutions at possible serine residues (Ser-66 and
Ser-77) were non-viable. We conclude from this previous result that
Ser-66 is essential for virus growth. HPLC analysis from in vivo
P labeling polyomavirus-infected cells showed that
the peptides eluted in the 30-31 min fraction were phosphorylated
on both threonine and serine residues. Two-dimensional peptide mapping
showed that the peptide fragments eluted from this fraction migrated in
different locations (Fig. 2). However, the fragments in the
30-31 min fraction contained only one peptide sequence from
residue 58 to 78 determined by NH
-terminal sequencing and
mass spectrometry(9) . Therefore, we conclude that
phosphorylation of Thr-63 and Ser-66 occur in different VP1 monomers.
VP1 isolated from polyomavirus-infected cells contains at least four
isoelectric subspecies identified by two-dimensional protein gel
analysis(3, 4) . Several of these subspecies may
represent distinct monophosphorylated molecules rather than multiply
modified forms.
In two other well characterized examples of the consequences of CK II phosphorylation, skeletal muscle glycogen synthase and acetyl-CoA carboxylase(41, 42) , the initial phosphorylation does not itself alter the activity of the substrate but is necessary for subsequent regulatory phosphorylation or dephosphorylation events. Serine phosphorylation of VP1 appears constitutive relative to the activity of middle T-antigen which appears to regulate VP1 threonine phophorylation (8, 30) . Because only a fraction, estimated by two-dimensional protein gel electrophoresis as 50% or less, of the VP1 monomers are modified it is possible that serine modification by a cellular CK II enzyme may affect the subsequent choice of VP1 molecules for threonine modification. This choice may dictate that a particular VP1 within a pentameric capsomere is phosphorylated either on serine or threonine but not both.
The in vitro assembly properties of the Ala-66 mutant VP1 protein demonstrated that this protein was structurally intact, although this mutant protein had a tendency to self-assemble into not only capsid-like but also tubular structures. Because inter-capsomere bonds are formed using the carboxyl termini of VP1 molecules between pentamers(38, 43) , this result suggests that changes in Ser-66, located on the surface of the VP1 pentamer(43) , may influence interactions between VP1 carboxyl termini. This structural interaction may be relevant to wild-type VP1 molecules phosphorylated at Ser-66, in that such a modification may facilitate formation of alternative carboxyl-terminal bonding interactions in the final capsid(43) . In addition, a perturbation of proper inter-capsomeric bonding may explain why a virus with the Ala-66 mutation could not be isolated(9) . However, additional studies of the Ala-66 mutant protein under a variety of assembly conditions (40) will be necessary before the significance of these structural interactions can be further assessed.
For the related
SV40 virus, the large T-antigenp53 complex isolated from
virus-infected cells is associated (in immunoprecipitates) with a
serine-specific kinase activity(44, 45) . This
activity autophosphorylates T-antigen on many of the same sites seen
for in vitro phosphorylation(29) . The two-dimensional
phosphopeptide map of SV40 large T-antigen was nearly reproduced in
vitro by the combination of CK I and CK II, and thus some of the
associated kinase activity may be related to their association with the
T-antigen
p53 complex(29, 46) . p53 is also a
substrate for CK II phosphorylation in vitro(23) ,
consistent with the findings from the in vivo T-antigen
associated kinase results. p53 is phosphorylated to a higher level
(10-fold) in SV40-infected cells, suggesting that its growth
suppressive activity may be inhibited by its
phosphorylation(46) .
CK II phosphorylation has been associated with other virus infections. CK II levels are stimulated upon adenovirus infection (12-fold within 15 min) of baby rat kidney cells(47) . This activation paralleled that seen for CK II in serum stimulation of WI38 cells, and correlates with the serum response early gene induction of myc, fos, and jun. Polyomavirus infection induces the serum response (platelet-derived growth factor-inducible) genes in a biphasic manner, with accumulation of these mRNAs 1 to 2 h post-infection associated with capsid protein addition, and a second phase associated with middle T-antigen expression(48, 49) . It is possible that CK II may also be induced during polyomavirus infection so that capsid protein phosphorylation would be facilitated. Although the biological functions of VP1 phosphorylation remain undetermined, the position of these sites on the exterior virion surface suggest a role in cell attachment and entry. To accomplish such an important function the virus may require recruitment of specific cellular kinases such as CK II.