J. gen. Virol. (1977), 37, 75-83
75
Printed in Great Britain
Phosphorylation of Polyoma and SV40 Virus Proteins
By B. A. J. P O N D E R , A. K. R O B B I N S AND
L. V. C R A W F O R D
Department of Molecular Virology, Imperial Cancer Research Fund,
London WC2A 3PX
(Accepted 27 April 1977)
SUMMARY
The polypeptides of polyoma and SV4o virions are phosphorylated. An estimate
of the amount of phosphorylation of the major virus capsid protein (VPI) has
been made using two-dimensional gel electrophoresis to resolve phosphorylated
from non-phosphorylated forms. The results suggest that in both polyoma and
SV4o virions about I2 % of VPI molecules are phosphorylated. In unassembled
VPI molecules immunoprecipitated from extracts of infected cells the proportion
is greater, about 33 %. The possibility that phosphorylated VPI may form the
penton proteins of the virus capsid is discussed.
INTRODUCTION
The structure of the virus particles of polyoma and simian virus 40 (SV4o) is now well
understood (see review by Finch & Crawford, I975). The particles have icosahedral symmetry. The surface is made up of 72 capsomeres, I2 at the vertices of the icosahedron
('pentons', having 5 subunits), and 60 on the faces ('hexons', having 6 subunits). The
structure thus requires a total of 420 subunits. However, it is not clear how the different
polypeptides of the virus particle fit into the structure. Most of the capsid must be made up
of VPI, which is the major polypeptide. The smallest polypeptides, VP4, 5, 6 and 7, are
histones which are closely associated with the virus DNA to form the internal core of the
particle. The role of the minor capsid polypeptides VP2 and VP3 is the least clear. The
structure of the virus particle raises the possibility that the pentons (which have five nearest
neighbours) and the hexons (which have six nearest neighbours) may be made up of different polypeptides. For example the pentons could be made up of VPz plus VP3, these
polypeptides being present in about the right amount in the particle. Alternatively hexons
could be made of VPI and the pentons of some modified form of VPI. In the case of SV4o,
Tan & Sokol (I972) showed that all the virus polypeptides including VPI were phosphorylated, The extent of VPI phosphorylation was not determined, although it was later shown
to be low, of the order of a few per cent (K. B. Tan, personal communication). Phosphorylated VPI could therefore be a possible candidate for the penton protein.
With this possibility in mind we investigated the phosphorylation of the polypeptides of
both polyoma and SV4o. Since both viruses have similar structures it might be expected
that similar amounts of phosphorylated VPI would be present in the two viruses if it had
an essential structural role in the particle.
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76
B. A. J. P O N D E R ~ A. K. R O B B I N S A N D
L. V. C R A W F O R D
METHODS
Preparation of virus. Initial stocks of polyoma large plaque strain A2 and SV4o strain 777
were grown at low multiplicity from single plaques. Secondary whole mouse embryo ceils
(polyoma) or CVI cells (SV4o) in 9o mm plastic dishes in Dulbecco's modification of
Eagle's medium with 5 % calf serum were infected with virus from these stocks at a multiplicity of Io p.f.u./cell. The cells were harvested 5 days after infection, and polyoma virus
was purified as described previously (Crawford, I969). SV4o was purified from the cell
lysates by sedimentation on to caesium chloride cushions in an SW27 rotor (Beckman) and
then by equilibrium gradient centrifugation. Purified virus was stored at 4 °C in caesium
chloride and dialysed into I o mM-tris, ImM-EDTA at 4 °C immediately before use, except for
iso-focusing experiments when it was dialysed into 80 mM-tris, pH 7"5, 0"05 % SDS. Inexperiments to examine the extent of contamination of our radioactively labelled virus with labelled
material from cells, the harvest from infected unlabelled dishes was mixed with that from
mock-infected labelled dishes before purification.
Radioactive precursors were added 24 h after infection except where indicated, a~PO4
(Ioo Ci/mg P), 35S-methionine (200 Ci/mmol), 3H-arginine (Io Ci/mmol) and aH-lysine
(20 Ci/mmol) were obtained from the Radiochemical Centre, Amersham. Labelling conditions were: asp, 250 #Ci per dish added to low (0"05 mM) phosphate medium and 2 % calf
serum; 35S-methionine, 75 #Ci per dish in low (6/zg/ml) methionine medium with 20%
normal serum and 5 % calf serum; 3H-arginine and 3H-lysine, 5o/zCi each per dish in low
(8 #g/ml) arginine-low (I 4 #g/ml) lysine medium with 5 % calf serum.
Separation of virus polypeptides. Virus suspensions were disrupted by heating at 80 °C for
3 min in sample buffer (IO % glycerol, 5 % mercaptoethanol, 2 % sodium dodecyl sulphate
[SDS], 0-05 M-tris, pH 6-8) and loaded on to I5 % polyacrylamide slab gels (Studier, I972),
run in the tris-glycine system described by Laemmli 0970). After electrophoresis the polypeptides were located by staining with Coomassie brilliant blue. z2p and 35S radioactivity
was located by autoradiography of the dried gels and 3H by fluorography of the gels after
impregnation with PPO in dimethyl sulphoxide (Bonner & Laskey, I974).
lsoelectric focusing separations were carried out by the method of O'Farrell 0975)- After
pre-electrophoresis samples were loaded on to the isoelectric focusing gels (I3O × 3 mm
diam.) containing pH 3 to to ampholines, run for I7 h at 400 V and then for I h at 800 V.
The frozen gels were then sliced into I mm sections and each slice suspended in I ml of
NCS ammonia scintillant (Ward, Wilson & Gilliam, I97O) and counted in a scintillation
spectrometer.
Newly synthesized VPr was isolated from polyoma infected mouse (3T6) cells at 70 h after
infection with 20 p.f.u, of large plaque virus/cell. Cells were transferred to methionine-free
medium and labelled with 50/zCi aSS-methionine per plate for 3 h at 37 °C. Extracts were
made by freezing and thawing Io 7 cells in 0"3 ml of extraction buffer (Io% glycerol,
80 mM-NaC1, 20 mM-EDTA, 20 mM-tris, pH 8-0, I mM-DTT, 200/zg/ml phenyl methyl
sulphonyl fluoride-Calbiochem). After spinning off cell debris (toooog for 30 min) the
supernatant was layered on to 0"4 ml 20% sucrose in o-I M-NaC1, o-oI M-tris, pH 8"0, and
centrifuged for 2 h at tooooog to remove cell debris and virus particles. The top 0.2 ml
was then collected and VPI immunoprecipitated with rabbit anti-virion serum followed by
sheep anti-rabbit IgG serum. The immunoprecipitate was then treated as described above
for isoelectric focusing.
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Polyoma and SV4o virus protein phosphorylation
(a)
SV40
(b)
(c)
(d)
Polyoma
(e)
77
(f)
(1)
Coomassie
VPI
VP1
VP3
VP2
VP3
} histones
histones (
(2)
Autoradiograph
m
v
Fig. I. Polyacrylamide gel electrophoresis of polyoma virus preparations. Coomassie stain (I) and
autoradiograph (2) of the same I5% polyacrylamide gel. SV4o full particles: (a) ~zP labelled;
(b) unlabelled; (c) unlabelled, purified from 32P-labelled cells. Polyoma full particles: (d) unlabelled,
purified from 3~P-labelled cells; (e) 3~S-methionine labelled; (f) 32P-labelled. The material at the
top of the gel in the autoradiograph of sample (a) and (f) is 32P-labelled DNA.
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78
B.A.J.
P O N D E R ~ A. K. R O B B I N S A N D L. V. CRAWFORD
Table I. The extent of phosphorylation of polyoma and SV4o virus polypeptides
% phosphorylation*
' Polyoma
VPI
¥P2
VP3
Histones
I6'4 (r5-3-I8"o)
7"7 (4"5- I 1.2)
7"8 (5"7-Io'8)
7"5 (5'5-IO'3)
SV4o
4'9 (4'2-5"7)
Not determined
9"6 (6"o--I5"3)
I'8 (i.6-2.2)
* The percentage pbosphorylation of each polypeptide separated by SDS gel electrophoresis was determined relative to the amount of D N A a2p as described in the text. The range of values observed in three
determinations on independent virus preparations is given in parentheses after the mean value. A preparation
of polyoma virus in which sap was added 2 h after infection (as compared to 24 h after infection) gave a
somewhat lower value, I I-2 % phosphorylation. In the case of SV4o VP2 the amount of radioactivity was too
low to give a significant value.
RESULTS
The first experiment is based on the analysis of virus grown in the presence of asp_
inorganic phosphate. The virus was purified and the virus polypeptides sepalated by electrophoresis on polyacrylamide gels. An example of such a gel is shown in Fig. i. z2p has been
incorporated into polyoma and SV4o virions, and it migrates in the same position as Coomassie stained or 35S-methionine labelled polypeptides of the virus particle, suggesting that
the polypeptides are phosphorylated. The major phosphorylated species is VPI, but the
other polypeptides of both viruses are also phosphorylated to some extent, including the
histones. Most of the histone phosphorylation appears to correspond to one of the bands,
probably f2az. As a control against contamination of VP I with ~2P-labelled cellular material
during the purification of the virus, lysates of non-radioactive infected cells and asP-labelled
uninfected cells were mixed, and the virus was purified from them and analysed in parallel.
VPI from non-radioactive 'full' particles did not show any 3~p contamination (Fig. I,
tracks c and d). VPI from empty capsids, on the other hand, showed a small amount of
contamination. This complicates the study of VPI phosphorylation in empty particles.
It is possible to make a quantitative estimate of the extent of phosphorylation of VPI
by measuring the relative amounts of radioactivity in the virus DNA and in the VPI band
on gels such as that shownin Fig. I. The virus contains DNA of tool. wt. 3"4 x 106corresponding
to about 5250 base pairs of DNA (Io 500 phosphates), and there are 42o structural subunits
in the virus capsid corresponding to VPI molecules. Since the virus DNA does not enter the
15 % polyacrylamide running gel but remains in the stacking gel, the amounts of DNA phosphate and VPI phosphate can be estimated together on each sample. An estimate can thus be
made of the number of phosphorylated VPI molecules as a percentage of the total. This was
done for the polypeptides of both polyoma and SV4o, with the result shown in Table I. It is
important to realize that this method will only give a reliable estimate for the degree of
phosphorylation of VPI if the specific activities of the pools from which DNA phosphate
and protein phosphorylation are drawn are equal. This might seem to be a reasonable
assumption, since the amount of phosphate present during virus growth was not limiting,
nor was the amount of radioactivity in the medium depleted substantially by incorporation
into the infected cells. However, it is probably not valid: for example, the time of addition
of phosphate to infected cells appeared to influence the values obtained (Table I). The
amount of VPI phosphorylation, as estimated by this method, was reasonably consistent
for each virus but significantly different for polyoma and SV4o. Since the two viruses were
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Polyoma and SV4 o virus protein phosphorylation
I
I
79
!
!
?
X
~
4
8
"6
#
~
×
._=
2
0~--
4~
-
~
I
60
65
I
70
Fraction number (mm)
---
0
I
I
75
80
Fig. 2. Separation of VPI and phosphorylated VPI by isoelectric focusing (O'Farrell, I975).
Polyoma virus samples labelled with 32p_ and with 3H-lysine plus arginine were mixed, lyophilized
and resuspended in sample buffer. After heating at 80 °C for 3 rain and cooling, 20 mg of urea was
added to each 20 #1 sample followed by 40 #1 of lysis buffer. The samples were then run as described
in Methods. After running the gels were removed from the tubes, frozen, sliced and the 82p and 3H
radioactivity determined in a scintillation spectrometer. The fractions are numbered in m m from
the top of the gel. •
• , 3H; (2- - -D, 32p.
grown in cells from different host species the different results may simply reflect differences
in the metabolism of the cells during infection and of the specific activities of their phosphate pools.
Because of these difficulties we used an alternative method, based on separation of the
phosphorylated form of VPI by isoelectric focusing. The advantage of obtaining a physical
separation of the phosphorylated and non-phosphorylated forms of VPI is that an estimate
of the relative amounts of protein in each can be made from measurement of incorporated
amino-acids alone. The method is therefore independent of any uncertainty about the pool
specific activity of phosphate precursors.
Resolution of VPI into a major and a heterogeneous minor component on isoelectric
focusing gels has been reported for SV4o by O'Farrell & Goodman (I976), and for polyoma
by Hewick 0976). Although it has not been possible to obtain sufficient of the minor component to show directly by peptide analysis that it is a derivative of VPI, the indirect evidence
is strong. The minor component is consistently obtained from purified virions, and it has
the same mobility as VPI on SDS gels. A minor component with theI same properties
is immunoprecipitated from infected cells by anti-VPI serum (see Fig. 3). No material with
similar properties is found in immunoprecipitates of uninfected cells, so it is unlikely that
the minor component is a cellular protein which becomes incorporated into the virus
particle. In conjunction with our finding that a minority of VPt from polyoma and SV4o was
6
VIR 37
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8o
B. A. J. P O N D E R , A. K. R O B B I N S A N D
VPI
L. V. C R A W F O R D
Phosphorylated
VPI'
÷
(a)
(b)
(c)
......
w
0
=
(d)
f
o
(e)
Fig. 3. Two-dimensional separation of VPI and phosphorylated VPI. Samples of a2p. and 3H-lysine
plus arginine-labelled virus were subjected to two-dimensional electrophoersis as described by
O'Farrell (1975). The 8H and 3zp were detected separately on the same gel as described in the text. The
VPI regions of the fluorographic and autoradiographic films have been cut out and aligned with each
other so that corresponding areas are aligned vertically. 3,.p is less well resolved than ~H here because
of the range of the fl particle emitted and the interposition of a plastic sheet between gel and film.
The tracks are as follows: (a) polyoma virions 3H; (b) polyoma virions z~P; (c) polyoma empty
particles 3H; (d) polyoma empty particles 32p. Polyoma VPI labelled with 85S-methionine in a 3 h
pulse, immunoprecipitated as described and then treated as for the samples above is shown in track
(e). Immunoprecipitates from mock infected cells showed no labelled protein in this region.
phosphorylated, these observations suggested that the minor component was phosphorylated VPI. Analysis of a mixture of asp. and 3H-lysine plus arginine-labelled virus by isoelectric focusing, shown in Fig. 2, supports this view.
The one-dimensional separation of phosphorylated and non-phosphorylated VPI by
isoelectric focusing shown in Fig. 2 is unsuitable for quantification of the phosphorylated
component, because some of both the 3,p and the aH running near VPI may be derived
from unrelated polypeptides having isoelectric points close to that of VPI. For this reason
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Polyoma and SV4o virus protein phosphorylation
81
Table 2. Determination of phosphorylation by two-dimensionalgel separation*
% of 8H in phosphorylated VPI (2 determinations)
Polyoma full particles
I2-o, 12"I
Polyoma empty particles I7"o, 16.8
SV4o full particles
13-o, t2-8
Immunoprecipitation of 8~S-labelledpolyoma VP~ from cell extract
% of 35S in phosphorylated VPI 33"0,35"0
* Radioactively labelled virus polypeptides were separated by two-dimensional electrophoresis, as shown
in Fig. 3. Phosphorylated and non-phosphorylated components of VPI were located by fluorography
(3H), or by autoradiography (35S),excised, and the radioactivity determined as described in Methods.
we ran two-dimensional gels, isoelectric focusing in the first dimension followed by polyacrylamide gel electrophoresis in the second dimension (O'Farrell, I975). 3H was detected
by fluorography (Bonner & Laskey, 1974) and the a~P was present at a sufficiently low level
to have a negligible effect here. Radioautography of the same gel with an opaque plastic
sheet between the gel and the film gave a record of the a2p alone. The relevant areas of
several such two-dimensional separations are shown in Fig. 3. This demonstrates that the
minor spot of VPI is more negatively charged than VPI, the increment being that expected
for a singly phosphorylated derivative. In the SDS dimension VPI and phosphorylated
VPI have the same mobility. In some gels both the major and minor spots of VPI appeared
to be double in the isofocusing dimension, probably reflecting differences caused by variation
in the number of other charged groups present. The relative amounts of 3H (from lysine and
arginine) and ~P appear to be the same across the phosphorylated VP1 doublet, so that
there is no indication of multiple phosphorylation of the most negatively charged species.
The values obtained for the amounts of aH in VPI and phosphorylated VPI from twodimensional separations, including those shown in Fig. 3, are given in Table 2.
DISCUSSION
Analysis on SDS-polyacrylamide gels shows that the proteins of both polyoma virus
and SV4 o are phosphorylated. An estimate of the amount of phosphorylation of the major
capsid protein, VPI, can be made from the two-dimensional gel separation of the phosphorylated and non-phosphorylated forms, using isoelectric focusing in the first dimension. SV4o
and polyoma give similar values of about I2 ~o for the full particles. A slightly higher value
of I7 % for empty particles may be due to the contamination with cellular material shown in
control experiments (Fig. 1). In a recent paper O'Farrell & Goodman (1976) showed that
SV4o VP1 could be separated into six species on two-dimensional gels. Two of the minor
species were phosphorylated and 1o % of the total protein was contributed by the minor
species. This is somewhat lower than the figure we have obtained.
Our value of I2 ~o for the phosphorylation of VP~ in full particles may be compared with
the calculated contribution of penton protein to the total capsid protein, 60 out of 420
subunits - I4-3 %. In view of the difficulties inherent in making a precise estimate of a minor
component of the virus structure, the value obtained is in good agreement with the expectation if each penton polypeptide carried one phosphate. We conclude that the amount of
VPI phosphorylation observed here is consistent with the idea that the pentons of the virus
particle are made up of VPI molecules each carrying a single phosphate residue. Further
work will be necessary to show where the phosphate is attached to VPI and whether this
attachment is specific, as would be predicted if it had structural significance. We found that
6-2
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82
B.A.J.
P O N D E R , A. K. ROBBINS AND L. V. CRAWFORD
the linkage of phosphate to VPt was unusually acid labile, suggesting that some bond
other than the usual ester linkage to serine or threonine was involved.
The high value we obtained for the phosphorylation of free VPI inside the cell is intriguing.
Several possible explanations were considered for this finding. It is unlikely to be the result
of other cellular proteins running with the phosphorylated VPI since these would not be
immunoprecipitated and no spots were observed in the VPI region of gels from extracts of
uninfected cells. Possibly a degradation product of VPI coincides exactly with phosphorylated V P L but this also seems unlikely on a two-dimensional separation. Only one major
phosphorylated species was seen in the immunoprecipitated free VP~ and this corresponded
to the species from virus particles. It may be that phosphate is removed f r o m VPI during
virus assembly, as an integral part of the process. Since the amount of phosphorylation of
free VPt appears to be higher than that of virion VPI the level of virion VPI phosphorylation cannot be limited by that of the available pool of free VPI and therefore assumes
greater significance.
The structure of polyoma and SV4o virus particles is such that the only other possible
candidates for the role of penton protein(s) are VP2 and VP3. Again they are present in
about the right amount to make up the pentons, but at present the evidence seems to indicate that they are not part of the outer shell of the particle. Disruption of the virus with SDS
alone in the absence of reducing agents releases all the minor proteins, leaving a shell consisting of VP~ molecules held together by disulphide bridges (Waiter & Deppert, I974).
Examination of polyoma virus particles treated with just sufficient SDS to release all the
VP2 and 3 showed that there was no indication of regular loss of pentons (J. T. Finch,
B. A. J. Ponder & L. V. Crawford, unpublished results) although in many of the particles
the regular arrangement of capsomeres had been disrupted to some extent. There is also
some immunological evidence which tends to the same conclusion. Antisera to intact
polyoma virions does not immunoprecipitate VP2 made in vitro, whereas antiserum made
against VP2 from disrupted virions does immunoprecipitate VP2 and probably VP 3 (Wheeler
et al. I976). This would indicate that VP2 is not exposed on the surface of the virion in an
antigenically active form.
The most likely explanation of all these findings is therefore that the virus capsid of
polyoma virus and SV4o consists entirely of VPI making up both pentons and hexons.
Some of the VPI molecules are phosphorylated, and this may be related to their role in penton
structure. The other virus particle proteins are probably all internal and may be associated
with the virus D N A .
This work was performed during the tenure by B.A.J.P. of a Clinical Research Fellowship of the Imperial Cancer Research Fund. We thank R. Hewick for instructing us in
isofocusing techniques and for helpful discussions, and Patricia O'Farrell for her comments.
REFERENCES
BONNER,W. M. &LASKEY,R. A. (I974). A film detection method for tritium-labelled proteins and nucleic acids
in polyacrylamide gels. European Journal of Biochemistry 46, 83-88.
CRAWFORD,L. V. 0969). Purification of polyoma virus. In Fundamental Techniques in Virology, pp. 75-8I.
Edited by K. Habel. New York: Academic Press.
FINCH,J. T. & CRAWFORD,L. V. (I975). Structure of small DNA-containing animal viruses. In Comprehensive
Virology, vol. 5, PP. II9-I54. Edited by Iff. Fraenkel-Conrat and R. R. Wagner. New York: Plenum
Publishing Corporation.
LAEMMLT,U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature, London z27, 68o-685.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 15:41:01
Polyoma and SV4 o virus protein phosphorylation
83
rmWICK, R. (1976). Ph.D. thesis, University of London.
O'FARgELL, P. (I975). High resolution two-dimensional electrophoresis of proteins. Journal of Biological
Chemistry z5o, 4oo7-4o2I.
O'rARRELL, P. Z. & GOODMAN,rL M. (I976). Resolution of simian virus 40 proteins in whole cell extracts by
two-dimensional electrophoresis: heterogeneity of the major capsid protein. Cell 9, 289-298.
STUDIER, V. W. (I972)- Bacteriophage T7. Science x76, 367-376.
TAN, ~. B. & SOKOL, r. (I972). Structural proteins of simian virus 40: phosphoproteins. Journal o f Virology
xo, 985-994.
WALTER, G. & DEPPERT, W. (I974). Intermolecular disulphide bonds: an important structural feature of the
polyoma virus capsid. CoM Spring Harbor Symposium on Quantitative Biology 39, 255-257.
WARt), S., WILSON, D. L. ~, 6ILLIAM, S. J. (I970). Methods for fractionation and scintillation counting of radioisotope-labelled polyacrylamide gels. Analytical Biochemistry 38, 90-97.
WHEELER, T., BAYLEY,S. T., HARVEY,R., CRAWEORD, L. V. & SMITH, A. E. (I976). Cell-free synthesis of polyoma
virus capsid proteins VPI and VP2. Journal o f Virology (in the press).
(Received 3 February I977)
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