VIROLOGY
74,
287-301
(1976)
Phosphoproteins
of Rous Sarcoma
MICHAEL Department
of Microbiology,
University Los Angeles, Accepted
Viruses
M. C. LA1 of Southern California May
California, 90033
School
of Medicine,
31,1976
:‘2P-labeled proteins from Rous sarcoma viruses were extracted with phenol and analyzed by SDS-polyacrylamide gel electrophoresis. p19 was found to be the major phosphorylated protein. ~12, a ribonucleoprotein, was also phosphorylated. Phosphorylation took place at both serine and threonine. The phosphorylation patterns of viral proteins were studied with respect to subgroup specificity, transforming ability, and maturation ability of the viruses. It was found that some of the viruses belonging to subgroup A, including RAV-3, RSV(RAV-11, and PR-B:RAV-3, contain a novel p23 phosphoprotein in addition to phosphorylated p19 and ~12. It could not be determined whether p23 phosphoprotein was cellular or viral in origin. The phosphorylation patterns did not vary with regard to other viral properties. Furthermore, the in viuo phosphorylated proteins were found to be different from the viral proteins phosphorylated in vitro by virion-associated protein kinases. It was suggested that the phosphorylation of viral proteins in uiuo is a specific process.
species (Pal and Roy-Burman, 1975; Pal et al ., 1975). The significance of the phosphorylation of the viral proteins is unclear. The present paper reports that two of the internal viral proteins, p19 and ~12, in Rous sarcoma viruses are also phosphorylated. Since phosphorylation of proteins is one of the mechanisms for regulating the functions of proteins (Taborsky, 19741, the state of phosphorylation of the viral proteins was examined with regard to the subgroup specificity, transforming ability, and infectivity of the viruses. It was found that the pattern of phosphorylation of the viral proteins varied with the viral strains. Some of the viruses belonging to subgroup A had a new phosphorylated component, ~23, in addition to phosphorylated p19 and ~12. On the other hand, phosphorylation of the proteins did not vary with regard to other viral properties. The specificity of the phosphorylation was studied.
INTRODUCTION
Rous sarcoma viruses contain at least seven structural proteins (Fleissner, 1971; August et al., 19741, two of which, gp85 and gp37, are glycosylated (Duesberg et al., 1970; Hung et al., 1971) and are the components of the viral envelope (Rifkin and Compans, 1972; Bolognesi et al., 1972). They are responsible for the type-specific antigenicity of the virus (Duesberg et al., 1970; Bolognesi et al., 1972). The rest of the viral proteins are nonglycosylated and are the internal components of the virion (Bolognesi et al., 1973). Among the internal proteins p27 and p15 are probably components of the viral core (Bolognesi et al., 1973; Stromberg et al., 19741, and ~12 is associated with the viral RNA (Bolognesi et al., 1973; Stromberg et al., 1974). On the other hand, the localizations and functions of p19 and p10 in the virion are poorly understood, except that p19 may contain viral type-specific antigens (Bolognesi et al., 1975; Stephenson et al., 1975). It has been reported that some of the structural proteins in mammalian RNA tumor viruses are phosphorylated, notably in ~12, ~15, or ~10, varying with the virus
MATERIALS
287 Copyright All rights
0 1976 by Academic Press, of reproduction in any form
Inc. reserved.
AND
METHODS
Viruses and cells. The growth of avian sarcoma and leukosis viruses and transformation-defective viruses in secondary
288
MICHAEL
M.
C. LA1
chicken embryo fibroblasts has been de- ethanol, and were used for electrophoretic and chromatographic analyses. scribed previously (Lai and Duesberg, Polyacrylamide gel electrophoresis. 1972). The cloned Prague strain Rous sarMost electrophoretic analyses of viral coma virus of subgroup C, PR-C, was origiphosphoproteins were performed in 7 cm of nally obtained from Dr. Peter Vogt. The 10% bisacrylamide-linked polyacrylamide recombinant viruses used in this study, PR-B:RAV-1 and PR-B:RAV-3, were also gels in the presence of 1% SDS as described previously (Lai and Duesberg, 1972). Viral kindly provided by Dr. Vogt and were isolated according to the published method proteins extracted from the phenol were electrophoresed at 50 V for 3 hr. Polyacryl(Vogt, 1971). These recombinant viruses carry the transforming ability of the paramide gel electrophoresis in the presence ent sarcoma virus, PR-B, and the host of urea at acid pH was carried out as described by Panyim and Chalkley (1969). In range (subgroup A) of the parent leukosis viral proteins were disviruses RAV-1 and RAV-3. The defective this procedure, solved in 6 A4 urea, 0.1 M dithiothreitol Bryan high titer strain of Rous sarcoma (DTT), and 1 M acetic acid, incubated at 37’ virus [BH RSV(-)I was fused into C/B for 60 min, and were then electrophoresed cells by Sendai virus and was a gift of Dr. in 7.5% bisacrylamide-linked polyacrylY.-C. Chen. LA334, a replication-defective temperature-sensitive avian sarcoma viamide gels containing 6 M urea. Electrophoresis was performed in 1 M acetic acid rus, was derived from the B77 strain avian sarcoma virus (Toyoshima and Vogt, at pH 3.2 and was run at 100 V for 210 min. 1969). This mutant produces noninfectious Under these conditions, the viral proteins virions at the nonpermissive temperature move from anode to cathode. After electro(41”) (Hunter et al., 1976). phoresis the gels were sliced into l-mm Radioactive labeling of virus. Virus-infractions, and each slice was incubated in 4 fected cells were labeled with carrier-free ml of toluene-based scintillation fluid con[“2P]orthophosphate (New England Nutaining 10% NCS (Nuclear Chicago, Inc.) clear) in phosphate-free medium suppleand 1% H,O at 55” overnight. Radioactivmented with 2% calf serum and 1% di- ity was measured in a Packard liquid scinmethyl sulfoxide (DMSO) at a concentratillation counter. tion of about 0.5 mCi/ml. The medium was Discontinuous SDS-polyacrylamide gel harvested at 12-hr intervals for 2 days. electrophoresis was performed in 2-mmAfter each harvest, new phosphate-free thick gel slabs essentially as described by medium was added to the cell culture and Laemmli (1970). After electrophoresis the the virus harvest was frozen at -70”. More gel was stained with Coomassie brilliant frequent harvests of the medium (every 3 blue (Gurr) by the techniques of Fairbanks hr) did not alter the pattern of phosphorylet al. (1971) and then autoradiographed ation. The labeling of the viruses with after it was dried. both [3ZP]orthophosphate (500 pCi/ml) and Gel filtration chromatography of viral [3H]amino acids (50 @i/ml, New England proteins. This was carried out essentially Nuclear) was carried out in medium de- as described by Fleissner (1971). Viral propleted of phosphate and 90% of amino teins precipitated from the phenol phase acids. The medium was harvested as were dissolved in 8 M guanidine HCl, 0.01 above. M EDTA, 0.05 M Tris-HCl (pH 8.1), and Purification of virus and viral proteins. 2% mercaptoethanol, and incubated at 56” Virus was purified from tissue culture me- for 2 hr. The mixture was then applied to a dia according to published methods (Lai column (1 x 85 cm) of agarose A-5m (200and Duesberg, 1972). The purified virus 400 mesh, Bio-Rad Lab) equilibrated with was disrupted with 1% sodium dodecyl sul- 6 M guanidine HCl in 0.02 M sodium phosphate (pH 6.5) and 0.01 M DTI’. The elufate (SDS) and 50 mM mercaptoethanol and extracted with phenol three times. tion was carried out in the same buffer at a Viral proteins were precipitated from the flow rate of 0.5 ml/hr with l-ml fractions phenol phase by addition of 5 volumes of being collected. Each fraction was diluted
PHOSPHOPROTEINS
with H,O and counted in a Triton X-lOOtoluene scintillation fluid (Patterson and Greene, 1965). Determination of phosphoamino acid linkage. 32P-labeled viral proteins separated by SDS-polyacrylamide gel electrophoresis were identified by autoradiography of the gel. The 3”P-labeled bands were cut out of the gel and eluted with a buffer containing 0.1 M ammonium bicarbonate and 0.1% SDS at room temperature for 24 hr. Viral proteins were then precipitated with 5 volumes of ethanol after adding 50 pg of bovine serum albumin. The precipitated phosphoproteins were dissolved in 0.1 ml of 2 N HCl and hydrolyzed in a sealed ampule at 110” for 15 hr. The hydrolysate was dried and dissolved in 5 ~1 of H,O and spotted on Whatman 3MM paper. It was subjected to electrophoresis in formic acid-acetic acid-water (30:90:880, pH 1.9) at 1000 V for 2 hr (Takeda and Ohga, 1971). Twenty micrograms each of phosphoserine and phosphothreonine (Sigma Co.) were included as markers and were located by ninhydrin. Under the conditions used for acid hydrolysis, about 60-80% of 32P-counts associated with the viral proteins were dissociated and migrated as inorganic phosphates which migrated faster than phosphoserine and phosphothreonine in paper electrophoresis. In order to increase the resolution between these two phosphoamino acids, the electrophoresis of the acid hydrolysate was run until phosphoserine was close to the anode. The paper was cut into 5-mm strips and counted in toluenebased scintillation fluid. Protein kinase assay. The assay for protein kinase contained in the virion was a modification of the procedures by Hatanakaet al. (1972). The reaction mixture in a final volume of 40 ~1 contains 1 pmol TrisHCl (pH 8.01, 0.05 pmol MgC12, 0.05 Fmol DTT, 0.5 nmol [y-““PIATP (16 Ci/mmol, Amersham-Searle), and 0.02% Nonidet-P 40. The viral proteins serve as endogenous phosphate acceptors. The reaction was allowed to proceed at room temperature for 30 min and was stopped by addition of 5 ml of 5% cold trichloroacetic acid (TCA). The TCA-insoluble counts were determined in
OF
289
RSV
a Packard liquid scintillation counter. For the identification of in u&o-labeled viral phosphoproteins, the reaction mixture from the protein kinase assay was extracted with phenol three times followed by the precipitation of the viral proteins from the phenol phase. RESULTS
Electrophoresis and Chromatography of “2P-Labeled Viral Proteins of Prague Strain of Rous Sarcoma Virus :j’P-labeled Prague strain of Rous sarcoma virus of subgroup C, PR-C, is expected to contain “?P-labeled RNA and 02Plabeled phospholipid (Quigley et al., 1972). In order to determine whether viral proteins were phosphorylated, the viral proteins were separated from viral RNA by extraction with phenol and were further separated from lipid by precipitation of the viral proteins from phenol phase with 5 volumes of ethanol as described in Materials and Methods. The “‘P-labeled proteins were then mixed with [:‘Hlamino acid-labeled virus previously disrupted with SDS, and coelectrophoresed in polyacrylamide gels in the presence of SDS. As shown in Fig. 1, the %P-labeled materials from the phenol phase were separated into three peaks. The majority of “‘P-counts comigrated with the [:‘H]amino acid-labeled p19, suggesting that p19 is phosphorylated. However, occasionally :‘?P-labeled proteins could be seen to migrate slightly slower than the majority of p19 (see Fig. 8), suggesting that possibly only a portion of the p19 molecules are phosphorylated (see Discussion). The second ““P-labeled peak migrated at the trailing half of the p12-~15~10 complex which could not be completely resolved in this system. Since p12 migrates slower than both ~15 and ~10 (Bolognesi et al., 19731, the data suggest that the second phosphorylated protein peak migrates together with ~12. However, phosphorylation of ~15 or p10 could not be ruled out. The third :l’P-labeled peak migrated faster than all of the viral proteins. The amount of :?“P-labeled material in this position varied considerably from preparation to preparation. It could
290
MICHAEL
0
IO
20
30 olrtonce
M. C. LA1
40 Movad.
50
60
70
mm
FIG. 1. SDS-polyacrylamide gel electrophoresis of [“‘PI- and [3H]amino acid-labeled PR-C. The anPlabeled PR-C was extracted with phenol and the 3ZP-labeled materials were precipitated from the phenol phase. The [3H]amino acid-labeled PR-C was disrupted by incubation for 30 min at 3’7” in 0.1 Tris-HCl, pH 7.4, 1% SDS, 2 mM EDTA, and 5 m&f DTT. Both the [32P]- and [“HIamino acid-labeled viral proteins were dissolved in 40 ~1 of a solution containing 0.01 Tris-HCl, pH 8.1, 1% SDS, 2 m&f EDTA, 0.05 mercaptoethanol, 5 m&f DTT, and 10% glycerol. Electrophoresis was performed on a 10% SDS-polyacrylamide gel as described in Materials and Methods.
M
M
be reduced but not completely removed by treatment with pancreatic RNase (20 pgl ml at 37” for 30 min) (see Fig. 5b and c). Also, it could not be removed by further extraction with methanol-chloroform (1:l) mixture (see below). However, it could be separated entirely from the viral proteins (see Fig. 7). It suggests that the fast-moving “2P-labeled material might represent RNA fragments not completely removed by phenol extraction. To separate viral proteins further, %Plabeled proteins of PR-C were electrophoresed in polyacrylamide gels in the presence of 6 M urea at pH 3.2. The basic protein in the virion, p12 (Bolognesi et al., 1973) was separated from the rest of the viral proteins under these conditions. As can be seen in Fig. 2, the [3Hlamino acidlabeled p19 moved slightly heterogeneously; the =P-labeled protein corresponding to p19 was also found to be slightly heterogeneous. Whether this heterogeneity represents multiple components of p19 or degradation of p19 was not determined. Some “‘P-radioactivity was associated with ~12. However, the ““P-la-
M
beled protein peak moved slightly slower than the [3Hlamino acid-labeled ~12, again suggesting that only part of p12 is phosphorylated (see Discussion). No 32P radioactivity was found to be associated with ~27, ~15, or ~10. Thus, from the polyacrylamide gel electrophoresis in the presence of SDS and in the presence of urea at acid pH, it is suggested that p19 is the major phosphorylated protein in the virion. A small fraction of p12 is also phosphorylated. To rule out the possible nonspecific association between 32P-labeled material and viral proteins, the [32Pl- and 13Hlamino acid-labeled viral proteins were further separated by gel filtration chromatography in 6 M guanidine HCl (Fleissner, 1971). As can be seen in Fig. 3, all of the r3H]amino acid-labeled viral proteins were separated. The major phosphorylated protein is p19. A small amount of 32P-counts was also found to be associated with ~12, in agreement with the finding by polyacrylamide gel electrophoresis (Figs. 1 and 2). Very little 32P was associated with p15 or ~10. However, occasionally ~15 or p10
PHOSPHOPROTEINS I
I
1 P27
0
20
IO
30
40 Dlntonce
OF
PI5 PI0
SO Moved,
291
RSV I
I
I
I
60
70
60
90
mm
FIG. 2. Acid urea polyacrylamide gel electrophoresis of :“P-labeled PR-C. [“‘PI- and l:
74,
287-301
(1976)
Phosphoproteins
of Rous Sarcoma
MICHAEL Department
of Microbiology,
University Los Angeles, Accepted
Viruses
M. C. LA1 of Southern California May
California, 90033
School
of Medicine,
31,1976
:‘2P-labeled proteins from Rous sarcoma viruses were extracted with phenol and analyzed by SDS-polyacrylamide gel electrophoresis. p19 was found to be the major phosphorylated protein. ~12, a ribonucleoprotein, was also phosphorylated. Phosphorylation took place at both serine and threonine. The phosphorylation patterns of viral proteins were studied with respect to subgroup specificity, transforming ability, and maturation ability of the viruses. It was found that some of the viruses belonging to subgroup A, including RAV-3, RSV(RAV-11, and PR-B:RAV-3, contain a novel p23 phosphoprotein in addition to phosphorylated p19 and ~12. It could not be determined whether p23 phosphoprotein was cellular or viral in origin. The phosphorylation patterns did not vary with regard to other viral properties. Furthermore, the in viuo phosphorylated proteins were found to be different from the viral proteins phosphorylated in vitro by virion-associated protein kinases. It was suggested that the phosphorylation of viral proteins in uiuo is a specific process.
species (Pal and Roy-Burman, 1975; Pal et al ., 1975). The significance of the phosphorylation of the viral proteins is unclear. The present paper reports that two of the internal viral proteins, p19 and ~12, in Rous sarcoma viruses are also phosphorylated. Since phosphorylation of proteins is one of the mechanisms for regulating the functions of proteins (Taborsky, 19741, the state of phosphorylation of the viral proteins was examined with regard to the subgroup specificity, transforming ability, and infectivity of the viruses. It was found that the pattern of phosphorylation of the viral proteins varied with the viral strains. Some of the viruses belonging to subgroup A had a new phosphorylated component, ~23, in addition to phosphorylated p19 and ~12. On the other hand, phosphorylation of the proteins did not vary with regard to other viral properties. The specificity of the phosphorylation was studied.
INTRODUCTION
Rous sarcoma viruses contain at least seven structural proteins (Fleissner, 1971; August et al., 19741, two of which, gp85 and gp37, are glycosylated (Duesberg et al., 1970; Hung et al., 1971) and are the components of the viral envelope (Rifkin and Compans, 1972; Bolognesi et al., 1972). They are responsible for the type-specific antigenicity of the virus (Duesberg et al., 1970; Bolognesi et al., 1972). The rest of the viral proteins are nonglycosylated and are the internal components of the virion (Bolognesi et al., 1973). Among the internal proteins p27 and p15 are probably components of the viral core (Bolognesi et al., 1973; Stromberg et al., 19741, and ~12 is associated with the viral RNA (Bolognesi et al., 1973; Stromberg et al., 1974). On the other hand, the localizations and functions of p19 and p10 in the virion are poorly understood, except that p19 may contain viral type-specific antigens (Bolognesi et al., 1975; Stephenson et al., 1975). It has been reported that some of the structural proteins in mammalian RNA tumor viruses are phosphorylated, notably in ~12, ~15, or ~10, varying with the virus
MATERIALS
287 Copyright All rights
0 1976 by Academic Press, of reproduction in any form
Inc. reserved.
AND
METHODS
Viruses and cells. The growth of avian sarcoma and leukosis viruses and transformation-defective viruses in secondary
288
MICHAEL
M.
C. LA1
chicken embryo fibroblasts has been de- ethanol, and were used for electrophoretic and chromatographic analyses. scribed previously (Lai and Duesberg, Polyacrylamide gel electrophoresis. 1972). The cloned Prague strain Rous sarMost electrophoretic analyses of viral coma virus of subgroup C, PR-C, was origiphosphoproteins were performed in 7 cm of nally obtained from Dr. Peter Vogt. The 10% bisacrylamide-linked polyacrylamide recombinant viruses used in this study, PR-B:RAV-1 and PR-B:RAV-3, were also gels in the presence of 1% SDS as described previously (Lai and Duesberg, 1972). Viral kindly provided by Dr. Vogt and were isolated according to the published method proteins extracted from the phenol were electrophoresed at 50 V for 3 hr. Polyacryl(Vogt, 1971). These recombinant viruses carry the transforming ability of the paramide gel electrophoresis in the presence ent sarcoma virus, PR-B, and the host of urea at acid pH was carried out as described by Panyim and Chalkley (1969). In range (subgroup A) of the parent leukosis viral proteins were disviruses RAV-1 and RAV-3. The defective this procedure, solved in 6 A4 urea, 0.1 M dithiothreitol Bryan high titer strain of Rous sarcoma (DTT), and 1 M acetic acid, incubated at 37’ virus [BH RSV(-)I was fused into C/B for 60 min, and were then electrophoresed cells by Sendai virus and was a gift of Dr. in 7.5% bisacrylamide-linked polyacrylY.-C. Chen. LA334, a replication-defective temperature-sensitive avian sarcoma viamide gels containing 6 M urea. Electrophoresis was performed in 1 M acetic acid rus, was derived from the B77 strain avian sarcoma virus (Toyoshima and Vogt, at pH 3.2 and was run at 100 V for 210 min. 1969). This mutant produces noninfectious Under these conditions, the viral proteins virions at the nonpermissive temperature move from anode to cathode. After electro(41”) (Hunter et al., 1976). phoresis the gels were sliced into l-mm Radioactive labeling of virus. Virus-infractions, and each slice was incubated in 4 fected cells were labeled with carrier-free ml of toluene-based scintillation fluid con[“2P]orthophosphate (New England Nutaining 10% NCS (Nuclear Chicago, Inc.) clear) in phosphate-free medium suppleand 1% H,O at 55” overnight. Radioactivmented with 2% calf serum and 1% di- ity was measured in a Packard liquid scinmethyl sulfoxide (DMSO) at a concentratillation counter. tion of about 0.5 mCi/ml. The medium was Discontinuous SDS-polyacrylamide gel harvested at 12-hr intervals for 2 days. electrophoresis was performed in 2-mmAfter each harvest, new phosphate-free thick gel slabs essentially as described by medium was added to the cell culture and Laemmli (1970). After electrophoresis the the virus harvest was frozen at -70”. More gel was stained with Coomassie brilliant frequent harvests of the medium (every 3 blue (Gurr) by the techniques of Fairbanks hr) did not alter the pattern of phosphorylet al. (1971) and then autoradiographed ation. The labeling of the viruses with after it was dried. both [3ZP]orthophosphate (500 pCi/ml) and Gel filtration chromatography of viral [3H]amino acids (50 @i/ml, New England proteins. This was carried out essentially Nuclear) was carried out in medium de- as described by Fleissner (1971). Viral propleted of phosphate and 90% of amino teins precipitated from the phenol phase acids. The medium was harvested as were dissolved in 8 M guanidine HCl, 0.01 above. M EDTA, 0.05 M Tris-HCl (pH 8.1), and Purification of virus and viral proteins. 2% mercaptoethanol, and incubated at 56” Virus was purified from tissue culture me- for 2 hr. The mixture was then applied to a dia according to published methods (Lai column (1 x 85 cm) of agarose A-5m (200and Duesberg, 1972). The purified virus 400 mesh, Bio-Rad Lab) equilibrated with was disrupted with 1% sodium dodecyl sul- 6 M guanidine HCl in 0.02 M sodium phosphate (pH 6.5) and 0.01 M DTI’. The elufate (SDS) and 50 mM mercaptoethanol and extracted with phenol three times. tion was carried out in the same buffer at a Viral proteins were precipitated from the flow rate of 0.5 ml/hr with l-ml fractions phenol phase by addition of 5 volumes of being collected. Each fraction was diluted
PHOSPHOPROTEINS
with H,O and counted in a Triton X-lOOtoluene scintillation fluid (Patterson and Greene, 1965). Determination of phosphoamino acid linkage. 32P-labeled viral proteins separated by SDS-polyacrylamide gel electrophoresis were identified by autoradiography of the gel. The 3”P-labeled bands were cut out of the gel and eluted with a buffer containing 0.1 M ammonium bicarbonate and 0.1% SDS at room temperature for 24 hr. Viral proteins were then precipitated with 5 volumes of ethanol after adding 50 pg of bovine serum albumin. The precipitated phosphoproteins were dissolved in 0.1 ml of 2 N HCl and hydrolyzed in a sealed ampule at 110” for 15 hr. The hydrolysate was dried and dissolved in 5 ~1 of H,O and spotted on Whatman 3MM paper. It was subjected to electrophoresis in formic acid-acetic acid-water (30:90:880, pH 1.9) at 1000 V for 2 hr (Takeda and Ohga, 1971). Twenty micrograms each of phosphoserine and phosphothreonine (Sigma Co.) were included as markers and were located by ninhydrin. Under the conditions used for acid hydrolysis, about 60-80% of 32P-counts associated with the viral proteins were dissociated and migrated as inorganic phosphates which migrated faster than phosphoserine and phosphothreonine in paper electrophoresis. In order to increase the resolution between these two phosphoamino acids, the electrophoresis of the acid hydrolysate was run until phosphoserine was close to the anode. The paper was cut into 5-mm strips and counted in toluenebased scintillation fluid. Protein kinase assay. The assay for protein kinase contained in the virion was a modification of the procedures by Hatanakaet al. (1972). The reaction mixture in a final volume of 40 ~1 contains 1 pmol TrisHCl (pH 8.01, 0.05 pmol MgC12, 0.05 Fmol DTT, 0.5 nmol [y-““PIATP (16 Ci/mmol, Amersham-Searle), and 0.02% Nonidet-P 40. The viral proteins serve as endogenous phosphate acceptors. The reaction was allowed to proceed at room temperature for 30 min and was stopped by addition of 5 ml of 5% cold trichloroacetic acid (TCA). The TCA-insoluble counts were determined in
OF
289
RSV
a Packard liquid scintillation counter. For the identification of in u&o-labeled viral phosphoproteins, the reaction mixture from the protein kinase assay was extracted with phenol three times followed by the precipitation of the viral proteins from the phenol phase. RESULTS
Electrophoresis and Chromatography of “2P-Labeled Viral Proteins of Prague Strain of Rous Sarcoma Virus :j’P-labeled Prague strain of Rous sarcoma virus of subgroup C, PR-C, is expected to contain “?P-labeled RNA and 02Plabeled phospholipid (Quigley et al., 1972). In order to determine whether viral proteins were phosphorylated, the viral proteins were separated from viral RNA by extraction with phenol and were further separated from lipid by precipitation of the viral proteins from phenol phase with 5 volumes of ethanol as described in Materials and Methods. The “‘P-labeled proteins were then mixed with [:‘Hlamino acid-labeled virus previously disrupted with SDS, and coelectrophoresed in polyacrylamide gels in the presence of SDS. As shown in Fig. 1, the %P-labeled materials from the phenol phase were separated into three peaks. The majority of “‘P-counts comigrated with the [:‘H]amino acid-labeled p19, suggesting that p19 is phosphorylated. However, occasionally :‘?P-labeled proteins could be seen to migrate slightly slower than the majority of p19 (see Fig. 8), suggesting that possibly only a portion of the p19 molecules are phosphorylated (see Discussion). The second ““P-labeled peak migrated at the trailing half of the p12-~15~10 complex which could not be completely resolved in this system. Since p12 migrates slower than both ~15 and ~10 (Bolognesi et al., 19731, the data suggest that the second phosphorylated protein peak migrates together with ~12. However, phosphorylation of ~15 or p10 could not be ruled out. The third :l’P-labeled peak migrated faster than all of the viral proteins. The amount of :?“P-labeled material in this position varied considerably from preparation to preparation. It could
290
MICHAEL
0
IO
20
30 olrtonce
M. C. LA1
40 Movad.
50
60
70
mm
FIG. 1. SDS-polyacrylamide gel electrophoresis of [“‘PI- and [3H]amino acid-labeled PR-C. The anPlabeled PR-C was extracted with phenol and the 3ZP-labeled materials were precipitated from the phenol phase. The [3H]amino acid-labeled PR-C was disrupted by incubation for 30 min at 3’7” in 0.1 Tris-HCl, pH 7.4, 1% SDS, 2 mM EDTA, and 5 m&f DTT. Both the [32P]- and [“HIamino acid-labeled viral proteins were dissolved in 40 ~1 of a solution containing 0.01 Tris-HCl, pH 8.1, 1% SDS, 2 m&f EDTA, 0.05 mercaptoethanol, 5 m&f DTT, and 10% glycerol. Electrophoresis was performed on a 10% SDS-polyacrylamide gel as described in Materials and Methods.
M
M
be reduced but not completely removed by treatment with pancreatic RNase (20 pgl ml at 37” for 30 min) (see Fig. 5b and c). Also, it could not be removed by further extraction with methanol-chloroform (1:l) mixture (see below). However, it could be separated entirely from the viral proteins (see Fig. 7). It suggests that the fast-moving “2P-labeled material might represent RNA fragments not completely removed by phenol extraction. To separate viral proteins further, %Plabeled proteins of PR-C were electrophoresed in polyacrylamide gels in the presence of 6 M urea at pH 3.2. The basic protein in the virion, p12 (Bolognesi et al., 1973) was separated from the rest of the viral proteins under these conditions. As can be seen in Fig. 2, the [3Hlamino acidlabeled p19 moved slightly heterogeneously; the =P-labeled protein corresponding to p19 was also found to be slightly heterogeneous. Whether this heterogeneity represents multiple components of p19 or degradation of p19 was not determined. Some “‘P-radioactivity was associated with ~12. However, the ““P-la-
M
beled protein peak moved slightly slower than the [3Hlamino acid-labeled ~12, again suggesting that only part of p12 is phosphorylated (see Discussion). No 32P radioactivity was found to be associated with ~27, ~15, or ~10. Thus, from the polyacrylamide gel electrophoresis in the presence of SDS and in the presence of urea at acid pH, it is suggested that p19 is the major phosphorylated protein in the virion. A small fraction of p12 is also phosphorylated. To rule out the possible nonspecific association between 32P-labeled material and viral proteins, the [32Pl- and 13Hlamino acid-labeled viral proteins were further separated by gel filtration chromatography in 6 M guanidine HCl (Fleissner, 1971). As can be seen in Fig. 3, all of the r3H]amino acid-labeled viral proteins were separated. The major phosphorylated protein is p19. A small amount of 32P-counts was also found to be associated with ~12, in agreement with the finding by polyacrylamide gel electrophoresis (Figs. 1 and 2). Very little 32P was associated with p15 or ~10. However, occasionally ~15 or p10
PHOSPHOPROTEINS I
I
1 P27
0
20
IO
30
40 Dlntonce
OF
PI5 PI0
SO Moved,
291
RSV I
I
I
I
60
70
60
90
mm
FIG. 2. Acid urea polyacrylamide gel electrophoresis of :“P-labeled PR-C. [“‘PI- and l:
