Download RNA Tumour Virus Phosphoproteins

J. gen. Virol. (1977), 3 6 , ~
459-469
459
Printed in Great Britain
R N A Tumour Virus Phosphoproteins: Evidence for Virus
Specificity of Phosphorylation
By E D W A R D G. H A Y M A N , B I J A Y K. P A L
AND P R A D I P R O Y - B U R M A N
Departments of Pathology and Biochemistry, University of Southern California
School of Medicine, Los Angeles, California 9o033, U.S.A.
(Accepted 19 April I977)
SUMMARY
The purified I2OOO dalton (pI2) phosphoprotein of Rauscher (R) and wild
mouse (WM) strains of murine leukaemia virus (MuLV) was analysed for the
distribution patterns of its variously charged molecular species by urea-polyacrylamide gradient gel electrophoresis. The distribution patterns of the pI 2 of two
different field isolates of WM viruses, 292 and I5O4, and the mouse-tropic and
amphotropic clonal sub-populations of 15o4 field isolate were very similar but
different from that of MuLV-R. A unique characteristic of the pI2 of the WM
isolates is the presence of two major apparently non-phosphorylated species in
approximately constant proportions relative to the phosphorylated species.
Similar studies on the pI2 of the same virus (MuLV-R or WM viruses) grown in
different host cells showed that the patterns of phosphorylated and non-phosphorylated species are virus-specific and independent of the cell lines of propagation. These analyses and their comparison with urea-gel patterns of the
phosphoproteins of other mammalian type C viruses indicated that the number
and relative proportion of the variously phosphorylated and non-phosphorylated
species are predetermined for a virus. Therefore, the virus must have the genetic
information for the phosphoprotein as well as other necessary genetic information
which functions, perhaps in conjunction with appropriate cellular factors, in regulating the specific proportions of these multiple species. Possible biological significance
of the variously charged molecular species in the phosphoprotein of RNA tumour
viruses is discussed.
INTRODUCTION
Type C RNA tumour viruses contain phosphoproteins as their structural components
(Pal et al. 1975; Pal & Roy-Burman, 1975). The proteins that are phosphorylated in vivo
during virus synthesis belong to the major structural polypeptides. These phosphorylated
proteins have been identified in various rodent type C viruses, in the exogenous and
endogenous classes of feline type C viruses, and in type C viruses isolated from primates,
including simian sarcoma associated virus, gibbon ape lymphosarcoma virus and baboon
endogenous virus (Pal et al. 1975; Pal & Roy-Burman, 1975). In general, viruses originated
from the lower mammalian species contain a major phosphoprotein of 12000 daltons (pI2)
and the endogenous viruses of primate origin (baboon endogenous virus and the RD-II4
virus) contain a major phosphoprotein of 15ooo daltons (pI5). Besides the pI2 phosphoprotein, a second major phosphoprotein of IOOOO daltons (plo) is present only in viruses
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~1m46o
E . G . H A Y M A N , B. K. P A L A N D P. R O Y - B U R M A N
genetically related to the rat species. Recently it was shown that the major phosphorylated
protein of avian type C viruses is the pl 9 protein (Lai, 1976).
The phosphoprotein p15, pt2, or plo is present in the virion in several different phosphorylated states (Pal et al. 1975). These phosphorylated states and their relative proportions
in the pI2 of Rauscher murine leukaemia virus (MuLV-R), murine leukaemia virus field
isolates from wild mice (15o4, 292; Officer et al. 1973; Pal et al. 1973; Gardner et al. 1976),
and the cloned mouse-tr0pic and amphotropic components (Bryant & Klement, 1976;
Hartley & Rowe, 1976; Rasheed, Gardner & Chan, 1976) of the 15o4 isolate have been
investigated for comparison after propagation of the viruses in different mouse and human
cell lines. These results suggest that the phosphorylation patterns of the phosphoprotein
are virus-specific and independent of the host cell line of propagation. In addition, these
experiments reveal a unique characteristic of having two major non-phosphorylated species
of pi2 in all uncloned or cloned isolates of wild mouse type C viruses tested.
METHODS
Reagents, media, and sera. Reagent or analytical grade chemicals were used in all
experiments. All radiochemicals were obtained from New England Nuclear Corp., Boston,
Massachusetts. Ultrapure urea and sucrose were purchased from Schwarz/Mann, Orangeburg, New York; agarose, acrylamide, and bisacrylamide from Bio-Rad Lab., Richmond,
California; guanidine hydrochloride and Triton X-IOO from Sigma Chemical Co., St Louis,
Missouri; pancreatic ribonuclease from Worthington Biochemical Corp., Freehold, New
Jersey; Eagle's minimum essential medium (MEM) and foetal bovine serum from Flow Labs,
Inglewood, California; gentamicin from Microbiological Associates, Bethesda, Maryland;
and polybrene from Aldrich Chemical Co., San Leandro, California. Sodium dodecyl
sulphate (SDS) obtained from Matheson, Los Angeles, California, was recrystallized before
use.
Cells and viruses. The Rauscher strain of MuLV was grown in NIH Swiss mouse embryo
cells and in human rhabdomyosarcoma (RD) cells (McAllister et al. 1969). For infection of
the cell lines, cells were pre-incubated for 18 to 24 h with 2/zg of polybrene per ml before
exposure to the virus. Wild mouse (Mus musculus) virus field isolates, i5o4 and 292, were
grown in NIH Swiss and cloned wild mouse embryo cell line (SC-I ; Hartley & Rowe, 1975).
The mouse-tropic and amphotropic components of the t 5o4 virus were also grown separately
in SC-I cells. All virus-infected cell lines were cultured in MEM supplemented with lO %
foetal bovine serum, z mM-glutamine, and 8o #g of gentamicin per ml.
Radiolabelling of virus and fractionation of virion phosphoproteins. Virus-producing cells
were grown in 5o ~o phosphate-free MEM containing IO ~o dialysed foetal bovine serum in
the presence of 3H-labelled L-amino acid mixture (2/zCi/ml) and carrier-free 32P-phosphate
(8o #Ci/ml). Two changes of the medium were made at 24 h intervals, and the virus was
purified from the pooled culture fluids as described (Pal et al. I973). The pelleted virus was
then disrupted in 2o mM-tris-HC1 (pH 7"4) containing o.I °jo Nonidet P-4o at o °Cfor I h,
treated with pancreatic ribonuclease (5oo #g/ml) at 37 °C for z h, and adjusted to 8 Mguanidine hydrochloride and o'3 % fl-mercaptoethanol. After heating at 56 °C for 45 min,
the solution was applied on a Bio-Gel A-5m column for fractionation of virion proteins
(Nowinski et aL 1972). The fractions containing the phosphoprotein were pooled, dialysed,
and lyophilized (Pal et al. I975; Pal & Roy-Burman, I975).
SDS-polyacrylamide gel electrophoresis. The protein peaks containing both ZH
and 32p labels, isolated by the guanidine-agarose chromatography, were analysed by
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R N A tumour virus phosphoproteins
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Fig. I. Urea-polyacrylamide gel electrophoresis of ~H-amino acid (0--(3) and 3~P-phosphate
( • - - - • ) labelled p 12 of Rauscher MuLV grown in NIH Swiss cells. Migration_wasfrom cathode
(left) to anode (right). It should be noted that 8H was counted in a narrow channel to avoid 3~p
spillover, and the values were not corrected for counting efficiency.The background values in the
narrow 3H channel (8 ct/min) and 32p channel (20 ct/min) were deducted from the data shown.
SDS-polyacrylamide gel electrophoresis (Pal et al. I975; Pal & Roy-Burman, 2975) for
determination of the homogeneity of the labelled polypeptides.
Urea-polyacrylamide gradient gel electrophoresis. Further separation of the variously
charged species of polypeptides of similar size class was accomplished by electrophoresis in
4 to IO % gradient polyacrylamide gels with 3 % stacking gel in the presence of denaturing
5"25 M-urea. The procedure used was essentially the same as described previously (Pal et al.
1975). The electrophoresis was continued until the bromophenol blue marker reached the
end of the gel.
High voltage paper electrophoresis of virus phosphoprotein hydrolysates. The phosphoprotein (pI2) of WM-29z virus labelled in vivo with 32P-phosphate was hydrolysed in
6 N-HCI at I IO °C for 5 h and the hydrolysate was subjected to high voltage paper electrophoresis as described (Pal et al. 1975) in presence of o-phosphoserine and o-phosphothreonine
markers.
RESULTS
Urea-gel electrophoresis patterns of the p 12 of MuL V-R
The 32P-phosphate and 3H-amino acid labelled phosphoprotein of MuLV-R grown in
N I H Swiss cells isolated by the guanidine agarose chromatography technique (Pal & RoyBurman, 1975) was analysed by SDS- and urea-gel electrophoresis. While a single homogeneous radioactive peak was obtained in guanidine-agarose gel filtration and in SDS-gels
(Pal et al. I973; Pal & Roy-Burman, I975), resolution into 3 to 4 major components was
accomplished in gradient gel electrophoresis in the presence of denaturing urea (Fig. I). As
previously observed with the phosphoproteins of the RD-I I4, gibbon ape lymphosarcoma
and feline leukaemia viruses (Pal et aL 1975), these multiple components apparently represent
varying degrees of phosphorylation of the pI2 protein of MuLV-R. It was evident that all
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462
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Fig. 2. Comparison of the urea-polyacrylamide gel electrophoresis patterns of 3~P-phosphate
labelled p i z of Rauscher MuLV grown in h u m a n rhabdomyosarcoma (RD) cells (a) and in N I H
Swiss embryo cells (b). Migration was from left to right.
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RNA tumour virus phosphoproteins
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Fig. 3. Comparison of the urea-polyacrylamide gel electrophoresis patterns of 3H-amino acid
((3--(3) and zzP-phosphate ( O - - - Q ) labelled pi2 of a wild mouse virus field isolate, strain 292,
propagated in NIH Swiss cells (a) and in wild mouse cloned (SC-x) embryo cells (b). Migration was
from left to right.
major components were phosphorylated since all of them contained superimposable *~P
and ZH counts.
To determine whether the phosphorylation patterns exhibited in the urea-gel electrophoresis are virus- or host cell-specific, MuLV-R was propagated in a heterologous human
cell line (RD) for isolation and analysis of the pI2. I f phosphorylation characteristics are
virus-specific, then the urea-gel patterns of the virus p I z should be independent of the cell
line in which it was propagated. The results (Fig. z) show that the phosphorylation patterns
o f the p I z of MuLV-R grown in N I H Swiss cells (Fig. zb) were very similar to that o f
M u L V - R grown in R D cells (Fig. 2a). Three to four major peaks were obtained in both
cases and the relative proportion of these peaks appeared to be almost identical. These
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Fig, 4. Urea polyacrylamide gel electrophoresis patterns of 8H-amino acid ( 0 - - 0 ) and z2P-phosphate (O---O),~a~oefled pI2 protein of the (a) cloned mouse-tropic component of wild mouse
virus, strain 15o4, grown in SC-I cells; (b) cloned amphotropic component of the I5O4virus grown
in SC-I cells; and (c) uncloned I5o4 field isolate grown in NIH Swiss cells. Migration was from
left to right.
experiments were repeated and identical patterns were derived, suggesting that the number
and amount of the various phosphorylated species are intrinsic characteristics of the virus.
Urea-gel electrophoresis patterns of the pI 2 of wild mouse type C viruses
Virus 292 field isolate was propagated in two different mouse cell lines, namely NIH Swiss
embryo cells and SC-I cells. The pI2 phosphoprotein was isolated from these two separate
virus preparations and was analysed by urea-gel electrophoresis. The data (Fig. 3) show that
peak distribution patterns obtained in the two runs were very similar. Three phosphorylated
species were recognized in each case. However, in contrast to the MuLV-R pI2 patterns,
292 pI 2 showed the presence of two major species which were not detectably phosphorylated.
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465 ul~
RNA tumour virus phosphoproteins
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20
30
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Fig. 5- Urea polyacrylamide gel electrophoresis patterns of ~H-amino acid labelled p3o (a), p15
(b), and pxo (e) of W M q 5 o 4 virus grown in N I H Swiss cells. Migration was from left to right.
30
VIR 36
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i(J466
E.G. HAYMAN, B. K. PAL AND P. ROY-BURMAN
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p12 WM-1504
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Fig. 6. Diagrammatic representation of the variously charged populations of molecules in phosphoproteins of mammalian type C RNA tumour viruses. Separation was accomplished by electrophoresis in urea-polyacrylamide gels. Migration was from left to right. Proteins were labelled with
~H-amino acids and ~zP-phosphateand the heights of the peaks represent 3H-radioactivity. Species
of molecules also containing 3~P-radioactivity were shaded black (J) for distinguishing from those
not detectably phosphorylated ([]). The data for the pI5 of RD-Ir4 al~d pI2 of GaLV were
previously reported by Pal et al. 0975).
Similar experiments were done with another wild mouse virus isolate, t5o 4. The field
isolate of I5o4 was grown in N I H Swiss cells, and its cloned mouse-tropic and amphotropic
components were grown separately in SC-r cells. The isolated p I z proteins were run in
urea-gels for comparison (Fig. 4). Two major non-phosphorylated species were present in
each case and the distribution of the phosphorylated species, although not well resolved,
showed good similarity to each other. Again, these experiments were repeated and the
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R N A tumour virus phosphoproteins
467
patterns were found to be reproducible. These data supported the observation with MuLV-R
that the phosphorylation patterns are virus-specific and are not grossly altered by propagation of the virus in different host cells. In addition, the data revealed a unique characteristic
of the pi2 of wild mouse derived viruses, whether uncloned or cloned, in having two major
non-phosphorylated species whose relative concentration appeared to be constant. It should
be noted that the charge heterogeneity detected in polyacrylamide gel electrophoresis under
urea denaturing conditions was restricted to the virion phosphoproteins. The other major
structural proteins showed primarily single species distribution under analogous conditions.
This is illustrated in Fig. 5 showing the electrophoretic pattern of p3o, p I 5 and p I o proteins
of the 15o4 virus in gradient gels in the presence of urea. Proteins p3o and plo of feline
leukaemia virus (Pal et al. 1975) and p3o of mouse sarcoma virus (Kirsten) and avian
sarcoma viruses also showed similar charge homogeneity in urea-gels (data not shown).
Phosphoamino acids of the phosphoprotein of wiM mouse type C virus
For identification of major phosphoamino acids, the ~2P-labelled p12 of WM-292 virus
was hydrolysed in acid and subjected to high voltage paper electrophoresis with o-phosphoserine and o-phosphothreonine as markers (Pal et al. I975). Consistent with the previous
observation on mouse type C viruses (Pal et al. 1975; Pal & Roy-Burman, I975), pI2 of
292 virus contained phosphoserine as the major phosphoamino acid. A small amount of
phosphothreonine was also detected.
DISCUSSION
Consistent with our previous observation (Pal et al. i975) on the presence of several
different phosphorylated species within the phosphoproteins of feline leukaemia virus
(FeLV), gibbon ape lymphosarcoma virus (GaLV), and the endogenous cat type C virus
(RD-II4), we report here the occurrence of multiple phosphorylated species in the pI2
phosphoproteins of the Rauscher (R) and wild mouse (WM) strains of murine leukaemia
virus (MuLV). The number and relative proportion of the variously phosphorylated species
are found to be a characteristic of the origin of the virus. This is diagrammatically illustrated
in Fig. 6. The profiles of variously charged species reflect individual characteristics of the
phosphoproteins of FeLV, GaLV, RD-114, MuLV-R, and WM viruses. A unique characteristic of the phosphoprotein of the wild mouse virus isolates, whether uncloned or cloned,
for the mouse-tropic and amphotropic components is the occurrence of two distinct
non-phosphorylated species, apparently in constant amounts relative to the phosphorylated
species. These two non-phosphorylated species as well as the phosphorylated ones should
be considered as pI2 because they all co-migrate as a single peak in both guanidine-agarose
gel filtration and SDS-gel electrophoresis. A preparation of 3H-amino acid labelled pI2 of
WM-292 virus isolated from the guanidine-agarose column was iodinated in vitro by 125I
and run in SDS-gels. Again, a single major 12~I-labclledprotein peak ( > 95 % homogeneous)
was obtained, corresponding to a molecular size of approx. 12ooo daltons (data not shown).
Thus, all available information suggests that the non-phosphorylated species belong to the
same size class as the phosphorylated species of the wild mouse viruses. They probably do
not represent cellular proteins as their occurrence and proportion relative to the phosphorylated species do not change when the virus is propagated in widely different host cells
like mouse and human. The possibility that the non-phosphorylated species may represent
degraded larger virion proteins was also considered. However, their presence in constant
proportion in the p12 protein fractions of all wild mouse isolates, cloned or uncloned,
suggests that it is not very likely. Further, the method used for the isolation of the pI2 rules
30-2
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468
E.G. HAYMAN, B. K. PAL AND P. ROY-BURMAN
out the presence of at least some precursor degradation products like p~5E or pI2E
(Buchhagen, Stutman & Fleissner, 1975; Naso et al. I976) which elute in the void volume of
the guanidine-agarose column. The presence or absence of homology between the nonphosphorylated pI2 species and the phosphorylated pI2 species of the wild mouse type C
viruses remains unknown. If the different species were to show overall similarity in peptide
patterns, protein modifications other than phosphorylation have to be invoked to explain
the charge differences between the two non-phosphorylated species. This possibility should
be considered as proteins are known to be modified in nature by acetylation, sulphuration,
carboxylation, and various other covalently linked charged substitutions.
When the overall distribution patterns of the variously charged molecular species of the
pI2 of wild mouse type C viruses are compared with that of MuLV-R, it appears that the
p~2 of MuLV-R has lost the two non-phosphorylated species that are present on the pI2 of
wild mouse viruses. The phosphorylated species of these wild mouse and laboratory MuLV
strains, however, still maintain a good degree of similarity. These findings raise the following
interesting questions. Is the absence of the major non-phosphorylated species in MuLV-R
p12 a function of genetic selection during long term laboratory propagation? Are the
additional non-phosphorylated pI2 species related to the neurotropic property (Gardner
et al. I973, 1976; Officer et al. 1973) of the wild mouse virus isolates?
Our results on the studies of the phosphoproteins of the same virus (MuLV-R or WM
isolates) grown in different cell lines suggest that the patterns of phosphorylated and nonphosphorylated species are virus-specific and apparently independent of the cell lines of
propagation, although it is recognized that the cell offers the setting for phosphorylation,
dephosphorylation or other protein modifications. These results indirectly suggest that the
information for the phosphoprotein resides in the virus genome, a concept that has already
been established by analysis of the translation products of the gag gene of mammalian type
C RNA tumour virus (Barbacid, Stephenson & Aaronson, 1976, and the references cited
therein).
Although the structural and biological significance of the occurrence of multiple species
of phosphorylated polypeptides within RNA tumour virions is largely unknown, some
recent studies strongly suggest that these phosphorylated polypeptides have a regulatory
role in vivo. It has been shown that the pIz phosphoprotein of mammalian type C viruses
binds specifically to the homologous virus RNA (Sen, Sherr & Todaro, 2976; Sen & Todaro,
1976) and the avian P~9 phosphoprotein to its RNA (Sen & Todaro, 1977). These binding
studies also indicate that there are at least two functionally distinct populations of molecules
in the pie protein preparations and one of these may be responsible for specific interaction
with the virus RNA (Sen & Todaro, ~976). It would appear very likely that this population
represents one or more of the several different species of p~z as described earlier (Pal et al.
I975) and presented in this report. Recent data on the binding studies, in fact, show that, of
the various phosphorylated forms of pIz protein of the Rauscher MuLV, only a species
containing relatively little phosphate can bind in vitro to purified homologous virus RNA
(A. Sen, C. J. Sherr & G. J. Todaro, personal communication).
We thankV. Klement and M. Bryant for supplying mouse-tropic and amphotropic clones
of WM 15o4 virus, M. B. Gardner for reading the manuscript, M. Akhavi for expert
technical assistance, and A. Dawson for her assistance in preparing the manuscript. This
investigation was supported by contract number NoI CP 535oo with the National Cancer
Institute.
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RNA tumour virus phosphoproteins
469
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