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Transforming proteins of some feline and avian sarcoma viruses are related structurally and functionally
Cell. Vol. 24, 145-l
53, April 1981,
Copyright
0 1981
by MIT
Transforming Proteins of Some Feline and Avian Sarcoma Viruses Are Related Structurally and Functionally Karen Beemon Tumor Virology Laboratory The Salk institute P. 0. Box 85800 San Diego, California 92138
Summary Transformation of chicken cells by Fujinami sarcoma virus (FSV), PRC II or Y73 (three independently isolated avian sarcoma viruses that are replication-defective and lack the Rous sarcoma virus src gene) resulted in significant elevation (4-13 fold) of phosphotyrosine levels in cellular protein. The gag-related proteins encoded by these avian sarcoma viruses (ASVs) were all associated with tyrosine-specific protein kinase activity when assayed in immune complexes and were phosphorylated at both tyrosine and serine residues in vivo. Both the phosphotyrosine level in protein of FSVinfected cells and the protein kinase activity assayed in immune complexes containing the FSV protein P140 were temperature-sensitive. The presumed transforming proteins of these ASVs were compared with those of Rous sarcoma virus (RSV), Abelson murine leukemia virus and the Snyder-Theilen and Gardner-Arnstein strains of feline sarcoma virus (FeSV), which have previously been associated with tyrosine-specific protein kinase activity. FSV and PRC II proteins were shown to be structurally related to one another and to the FeSV proteins by tryptic peptide mapping and by immunological studies. No homology was observed, however, between the transforming proteins of RSV, Y73, Abelson murine leukemia virus and the FSV/PRC II/ FeSV class, suggesting there may be at least four classes of retroviruses whose transformation mechanisms involve aberrant phosphorylation of cellular protein at tyrosine residues. Introduction Fujinami sarcoma virus (FSV), PRC II and Y73 are three replication-defective avian sarcoma viruses (ASVs) that induce sarcomas in birds and transform fibroblasts in culture; however, they lack the Rous sarcoma virus (RSV) src gene. Each of these viruses was isolated independently from a spontaneous chicken fibrosarcoma (Fujinami and Inamoto, 1914; Carr and Campbell, 1958; itohara et al., 1978). The organization of the genomes of these ASVs is also different from that of RSV, but resembles that of avian acute leukemia viruses and certain mammalian RNA tumor viruses. The approximately 5 kilobase (kb) FSV genome shares sequences at its 5’ terminus (which include part of the viral gag gene coding for the internal structural proteins) and at its 3’ terminus (from
the noncoding “common” region), with its associated avian leukosis virus (ALV) helper. The internal 3 kb region contains transformation-specific sequences, probably derived from the cell (Lee et al., 1980; Hanafusa et al., 1980). PRC II and Y73 appear to have similar genomic organizations. Each virus encodes a large phosphoprotein containing sequences from the N terminus of the gag gene linked to transformation-specific sequences. The presumed transforming proteins encoded by these viruses are FSV P140 (Lee et al., 1980; Hanafusa et al., 1980), PRC II P105 (Neil et al., 1981) and Y73 P90 (Kawai et al., 1980). In contrast, RSV encodes a phosphorylated transforming protein, p60”“, which does not contain gag sequences (Brugge and Erikson, 1977; Sefton et al., 1978). The protein p60 “’ is associated with protein kinase activity as assayed both in immune complexes (Collett and Erikson, 1978; Levinson et al., 1978) and in partially purified preparations (Erikson et al., 1979). A tyrosine residue is phosphorylated by this protein kinase activity in vitro (Hunter and Sefton, 1980; Collett et al., 1980). Furthermore, transformation of cells with RSV increases the level of phosphotyrosine found in cellular protein 6-10 times, suggesting this protein kinase activity is involved in transformation by RSV (Sefton et al., 1980b). Cells infected with a large number of other transforming viruses, including Moloney and Kirsten murine sarcoma viruses, avian myelocytomatosis virus MC29, SV40 and polyoma virus, as well as cells transformed by some chemical carcinogens, have been screened for increased levels of phosphotyrosine in cellular proteins. None of these transformed cells showed more phosphotyrosine in protein than the corresponding nontransformed cells (Sefton et al., 1980b). Both Abelson murine leukemia virus (Ab-MuLV) and Snyder-Theilen feline sarcoma virus (ST-FeSV), however, induce an elevation of 5-10 times in the level of phosphotyrosine in cellular protein (B. M. Sefton, T. Hunter and W. Raschke, manuscript submitted for publication; Barbacid et al., 1980a). Furthermore, the gag-related proteins encoded by these viruses, AbMuLV P120 and ST-FeSV P85, are associated with tyrosine-specific protein kinase activity when assayed in immune complexes (Witte et al., 1979a; Barbacid et al., 1980a). The Gardner-Arnstein strain of FeSV (GA-FeSV) encodes a protein, P95, structurally related to P85 (Barbacid et al., 1980b), which also has associated kinase activity (Van de Ven et al., 1980). This study examines the structure and function of the gag-related proteins encoded by PRC II, FSV and Y73. Although none of these viruses contains RSV src gene sequences, the pathogenic similarity of these viruses to RSV suggests they might transform cells by a mechanism similar to that of RSV. Furthermore, all gag-related ASV proteins are phosphorylated, as are RSV p60src, Ab-MuLV P120 and ST-FeSV P85. In
Cell 146
addition, tyrosine-specific protein kinase activity has been detected in immunoprecipitates containing FSV P140 (Feldman et al., 1980; Pawson et al., 1980) and Y73 P90 (Kawai et al., 1980). I have extended the comparison between RSV and the other ASVs by asking whether these viruses also induce elevated phosphorylation of tyrosine residues in cellular protein. In addition, I have investigated possible structural relationships between the presumptive transforming proteins of these ASVs and those of other retroviruses. Results Cells Infected with PRC II, FSV and Y73 Have Elevated Levels of Phosphotyrosine To analyze the levels of tyrosine phosphorylation in cellular protein after infection with PRC II, FSV or Y73, well transformed cultures were labeled with 32P-orthophosphate for 18 hr; cellular protein was extracted and partially hydrolyzed with HCI, and the acid-stable phosphoamino acids were resolved by two-dimensional electrophoresis. Table 1 indicates the relative amount of phosphotyrosine recovered in each case. The relative levels of the major phosphoamino acids, phosphothreonine and phosphoserine, were essentially unchanged by transformation by these viruses. Cells infected with PRC II showed a dramatic increase in phosphotyrosine to a level 13 times that seen in protein of uninfected chicken embryo fibroblasts (Table 1). Y73-infected cells showed a ninefold increase in relative phosphotyrosine levels (Table l), similar to that previously reported for cells transformed by RSV. Infection only with ALVs, similar to the helper viruses present here, has been demonTable 1. Effect Cellular Protein Infecting
Virus
of ASV Infection
Growth ture
on the Level of Phosphotyrosine
Tempera-
in
Relative Abundance of Phosphotyrosinea
None
38°C
0.021
PRC II
38°C
0.282
None
37°C
0.030
Y73
37oc
0.266
None
41 “C
0.068
FSV
41 “C
0.079
FSV
36°C
0.303
Chicken cells were labeled with 32P-orthophosphate for 18 hr at the temperatures shown, and proteins were extracted and partially hydrolyzed with acid. Approximately 2 X 1 O6 cpm of each hydrolysate plus 0.5 pg of each phosphoamino acid marker were analyzed by two-dimensional electrophoresis as in Fig. 2. a The radioactivity in phosphotyrosine is expressed as a percentage of the total radioactivity recovered in phosphoserine, phosphothreonine and phosphotyrosine. Phosphothreonine represented approximately 6%. and phosphoserine approximately 94%, of the recovered phosphoamino acids in all cases.
strated to have no effect on phosphotyrosine levels in cellular protein (Hunter and Sefton, 1980). When FSV-infected ceils were grown at 36°C the phosphotyrosine level in cellular protein increased fourfold, while there was essentially no increase over the level in uninfected cells when these cells were grown at 41 “C (Table 1). FSV-induced transformation also appeared to be temperature-sensitive by the criteria of cellular morphology and of focus formation in agar (data not shown). The rate of 3H-labeled 2-deoxyglucose uptake by cells infected with either FSV or PRC II also was measured to provide a more quantitative assay of transformation. FSV-infected cells grown at 36°C had an uptake rate three times that of FSV-infected cells grown at 41 “C, while cells infected with PRC II had an elevated rate of uptake at both temperatures (T. Ryden and K. Beemon, unpublished results). Furthermore, a striking difference was observed in the amount of protein kinase activity assayed in immunoprecipitates containing FSV P140 derived from cells grown at different temperatures (see below). In Vitro Protein Kinase Activity Tyrosine-specific protein kinase activity has recently been demonstrated in immunoprecipitates containing Y73 P90 and FSV Pi 40 (Kawai et al., 1980; Feldman et al., 1980; Pawson et al., 1980). In this report, immunoprecipitates containing PRC II P105 were also assayed for protein kinase activity. In addition, the protein kinase activities associated with different ASV proteins were compared with regard to phosphate acceptors. lmmunoprecipitates containing PRC II P105, FSV P140 or Y73 P90 all exhibited protein kinase activity, as demonstrated in Figure 1. The substrate that was phosphorylated, however, depended on the antiserum used for immunoprecipitation. When antiserum against the N-terminal viral gag protein pl9 was used, phosphorylation of a protein comigrating with the respective ASV-coded protein was observed (Fig. 1 B). When sera from rabbits bearing RSV-induced tumors (TBR sera) that contain antibodies to the viral structural proteins as well as to ~60’” (Brugge et al., 1978; Sefton et al., 1978) were used, phosphorylation of the immunoglobulin heavy chain was observed, as well as of the ASV proteins (Fig. 1A). No kinase activity was apparent in immunoprecipitates made from infected cells with sera from nonimmunized animals (data not shown). Analysis of in vitro phosphorylation products in immunoprecipitates of P105 revealed that all of the phosphate incorporated into the immunoglobulin heavy chain (Figure 2A) and into Pi05 (data not shown) was at tyrosine residues. Phosphorylation of the immunoglobulin heavy chain in this assay probably is not due to the activity of cellular p60”““, which is also recognized by the broadly cross-reactive TBR serum used, because the amount of phosphorylation observed was vastly
Retroviruses 147
and Tyrosine
Phosphorylation
2345
1234567123456712341
HC-
Figure
1. Assay
of Protein
Kinase
Activity
and lmmunoprecipitation
of ASV Proteins
Protein kinase activity was assayed in immune complexes made with (A) TBR serum or (6) anti-p1 9 serum from cells that were (lane 1) uninfected or infected with (lane 2) Y73. (lane 3) PRC II, (lane 4) FSV at 36”C, (lane 5) FSV at 41 “C, (lane 6) FSV shifted from 36’C to 41 ‘C for 1 hr before lysis or (lane 7) FSV shifted from 41 “C to 36’C for 1 hr. Bands comigrating with P105 and P140 were visible in lanes 3 and 4 of A when the autoradiogram was exposed for a longer time. for 2 hr. and lysates were immunoprecipitated with (lane 1) (C) PRC II-infected or (D) FSV-infected cells were labeled with “S-methionine nonimmune serum, (lane 2) anti-GA-FeSV, (lane 3) anti-ST-FeSV, (lanes 4 and 5) anb-pl9. FSV-infected cells in lanes 1, 2, 3 and 4 of D were grown at 36’C and in lane 5 of D at 41 “C. Samples were subjected to electrophoresis on a 12.5% acrylamide gel, which was fluorographed. Numbers on the sides represent molecular weight (x 1 O-? of viral-coded proteins. HC designates the immunoglobulin heavy chain, visualized by staining.
greater than that seen in the same number of uninfected chicken cells assayed in parallel (Figure 1 A, lane 1). Furthermore, infection with PRC II, FSV or Y73 did not induce a detectable increase in the amount of 35S-methionine-labeled p60”“” immunoprecipitated from cells with TBR serum (data not shown). Some immunoglobulins present in TBR sera may possess, it appears, a suitable phosphorylation site for these protein kinases, perhaps without specifically binding them, while those in most other types of sera tested do not. The immunoglobulins phosphorylated in TBR immunoprecipitates from Y73-infected cells had a slightly faster electrophoretic mobility than those phosphorylated in precipitates from PRC Il-infected or FSV-infected cells made with the same serum (Figure 1 A, lanes 2, 3 and 41, suggesting different kinases may be involved in these systems. The possibility cannot be ruled out, however, that the “autophosphorylation” and the immunoglobulin phospho-
rylation reactions are due to different protein kinases, either viral-coded kinases or induced cellular kinases, present in the immune complexes. Protein kinase activity associated with immunoprecipitates containing FSV P140 appeared to be temperature-sensitive. While protein kinase activity was readily detectable in immunoprecipitates containing FSV P140 made from cells grown at 36’C, very little activity was observed when these cells were grown at 41 “C (Figures 1 A and 1 B, lanes 4 and 5). Furthermore, this protein kinase activity was readily reversible in temperature-shift experiments (Figures 1 A and 1 B, lanes 6 and 7). One hour after cultures were shifted from 36°C to 41 “C, kinase activity had decreased to a level slightly above that in uninfected cells. Substantial kinase activity (20-30% of maximal) was regained 1 hr after a shift from 41 “C to 36°C. This temperaturedependent protein kinase activity was not the result of degradation or loss of immunoprecipitability of
Cdl 148
Figure 2. Phosphoamino Isolated Proteins
Acid Composition
of
32P-labeled proteins were isolated and partially hydrolyzed with HCI, and the phosphoamino acids ware separated by two-dimensional electrophoresis at pH 1.9 (from right to left) and at pH 3.5 (from bottom to top). Unlabeled phosphoserine (P-ser). phosphothreonine (P-thr) and phosphotyrosine (Ptyr) were run as internal markers and visualized by ninhydrin staining. (A) TBR immunoglobulin heavy chain phosphorylated in an immunoprecipitate containing PRC II P105. The following proteins were isolated from cells labeled with “P for 18 hr: (6) PRC II Pi 05; (C) Y73 P90; and (D) PRC II-associated virus p19.
P140 at 41 “C, since approximately equivalent amounts of 35S-methionine-labeled P140 were precipitated from cells at 36°C and at 41 “C (Figure 1 D, lanes 4 and 5). Identification of Phosphoamino Acids of ASV Proteins The other viral proteins associated with tyrosine-specific protein kinase activity have phosphotyrosine residues in vivo (Hunter and Sefton, 1980; Barbacid et al., 1980a; Sefton et al., manuscript submitted for publication), so the identity of the phosphorylated amino acids of the ASV proteins was investigated. Tyrosine was the major phosphorylated amino acid found in PRC II Pi05 labeled in vivo, comprising approximately two thirds of the radioactivity, while the remaining one third was in phosphoserine, as displayed in Figure 28. Some of the phosphoserine residues are likely to be those in the pi9 sequences of P105, since RSV p19 is phosphorylated at serine residues (Erikson et al., 1977). Direct analysis of phosphoamino acids of p19 encoded by the PRC IIassociated virus revealed phosphoserine to be its major phosphoamino acid (Figure 2D). Feldman et al. (1980) have reported a ratio of two phosphotyrosines to one phosphoserine for FSV P140. Both PRC II P105 and FSV P140 therefore contained phosphotyrosine as the major phosphoamino acid. Analysis of
Y73 P90 shown in Figure 2C revealed both phosphotyrosine and phosphoserine, but at a ratio of approximately 1:3. Structural Homology between FeSV, PRC II and FSV Proteins Two lines of investigation have been used to determine possible structural relationships between the transforming proteins of the seven different viruses associated with tyrosine-specific protein kinase activity. The first approach was immunological and made use of different antisera that recognize RSV p60”“, STFeSV P85 and GA-FeSV P95. To investigate relationships with p60s”, I used TBR serum, which was capable of cross-reacting with ~60”” from many different RSV strains (Sefton et al., 1980a). While TBR serum precipitated all three gag-related ASV proteins, this effect appeared to be caused by antibodies against viral structural proteins that were also present (Brugge et al., 1978). Preincubation of the serum with an excess of disrupted RSV virions led to a large decrease in the amount of immunoprecipitable ASV proteins, but did not diminish the precipitation of ~60”” (data not shown), which suggests that the proteins of PRC II, FSV and Y73 have little homology with p60src. In addition, the TBR serum failed to precipitate AbMuLV Pi 20 and ST-FeSV P85 (data not shown). Both PRC II P105 and FSV P140 were immunopre-
Retroviruses 149
and Tyrosine
Phosphorylation
cipitated by antisera raised in goats by injection of autologous cells infected with either ST-FeSV or GAFeSV (Figures 1 C and 1 D). This immunoprecipitation was substantially decreased by preincubation of the antisera with solubilized mink lung cells transformed nonproductively by ST-FeSV, but not by preincubation with uninfected ceils (data not shown). When protein kinase assays were performed with PRC II P105 or FSV P140 immunoprecipitated with antisera against the FeSV-infected cells, autophosphorylation was observed (data not shown). There was, however, no detectable immunoprecipitation of Y73 P90 or RSV ~60”” with either of the antisera against FeSV-infected cells (data not shown). Ab-MuLV P120 was immunoprecipitated with both of these antisera, but this immunoprecipitation was due to homology between the structural protein sequences of Ab-MuLV and FeSV proteins. When the sera were preincubated with disrupted Rauscher MuLV virions, the immunoprecipitation of P120 was effectively blocked (data not shown). Tryptic Peptide Comparisons of Transforming Proteins To examine the apparent homology between proteins of PRC II, FSV and ST-FeSV by another method and to investigate the possibility of homology between AbMuLV P120 and the ASV proteins, a series of tryptic peptide maps were prepared with 35S-methionine-labeled proteins. The peptides were separated by electrophoresis at pH 4.7 on cellulose thin-layer plates, followed by chromatography. The two-dimensional analysis was performed on six of the viral transforming proteins associated with tyrosine protein kinases, on the gag proteins p19 and p27 and on Pr76, the gag precursor polypeptide. Homologous peptides were identified by mapping mixtures of digests. Figure 3 shows the existence of a high degree of homology between the methionine-containing tryptic peptides of PRC II P105 and FSV P140. This homology encompassed both the gag and the transformation-specific domains of each protein. P105 and P140 each contained four peptides shared with pl9 (numbered 7, 8, 9 and lo), one peptide derived from p27 (11) and one other peptide (12) present in Pr76 and coded by the gag gene sequences. In addition, these proteins contained 7 to 9 methionine peptides encoded by transformation-specific regions of the viral genomes. The two proteins shared six non-gag peptides (numbered 1, 2, 3, 4, 5 and 6), while P105 had one unique peptide (13), and P140 had three peptides not shared with P105 (14, 15 and 16). When peptide maps of PRC II P105, FSV PI 40 and ST-FeSV P85 were compared, two of the three methionine-containing tryptic peptides of P85 (numbered 1 and 2) had mobilities identical to those of peptides 1 and 2 in both PRC II P105 and FSV P140. These peptides are marked with arrows in the mixtures dis-
played in Figures 3D and 3E. None of the ST-FeSV P85 methionine-containing peptides represents FeLV gag gene sequences (Barbacid et al., 1980b). Peptides 1 and 2 from PRC II Pi05 and from ST-FeSV P85 were further compared by electrophoresis at pH 1.9. Comigration was again observed, which strongly suggests sequence homology between these proteins. Comparison of these maps with those of RSV p60”“, Ab-MuLV P120 and Y73 P90 (Figure 4) and analysis of mixtures of digests (data not shown) revealed no significant homologies in transformation-specific peptides. Y73 P90 contained three pl9 peptides (Figure 4A, numbered peptides) also found in the gag-related proteins of PRC II and FSV. None of the peptides derived from transformation-specific sequences, however, was common to Y73 and PRC II or FSV. Similarly, Y73 was found to be unrelated to RSV ~60”” and to share only one out of 17 peptides with AbMuLV (Figure 4, arrows pointing up). Y73 therefore appears to be in a class by itself. Comparison of PRC II P105 and Ab-MuLV P120 revealed one peptide with very similar mobilities (Figure 3, peptide 12); however, this is a gag-derived peptide in P105. RSV ~60’” and Ab-MuLV P120 also had one common peptide (Figures 4B and 4C, arrows pointing left). From analysis at pH 1.9, the peptides shown to comigrate in Figure 4 do not appear to represent identical sequences; furthermore, they do not account for a very large proportion of the methionine peptides of the proteins involved. Comparison of ST-FeSV P85 with Ab-MuLV P120, RSV ~60”” and Y73 P90 revealed no homologous methionine-containing peptides. In summary, PRC II, FSV and ST-FeSV and GAFeSV appeared to represent a single class of sarcoma viruses, which had independently acquired related transformation-specific sequences. There was, however, no evidence to support the existence of any substantial structural homology between the transforming proteins of RSV, Y73, Ab-MuLV and the FSV/ PRC II/FeSV class, all of which are associated with protein kinases specific for tyrosine residues. Discussion Tyrosine Protein Kinases Implicated in Transformation by All Four ASVs Three independently isolated avian sarcoma viruses, PRC II, FSV and Y73, were shown to be associated with tyrosine-specific protein kinase activity, assayed both in vivo by measurement of phosphotyrosine levels in cellular protein and in vitro in immune complexes containing viral proteins. Each of these viruses encodes a single gag-related polypeptide thought to be the viral transforming protein and shown to be phosphorylated in vivo at both serine and tyrosine residues (this report; Feldman et al., 1980). While
Cell 150
Figure
3. Comparison
of Methionine-Containing
Tryptic
Peptides
of gag-Related
Proteins
of FSV, PRC II and ST-FeSV
Tryptic digests were resolved by electrophoresis on cellulose plates at pH 4.7 (from left to right) followed by ascending chromatography. (A) FSV P140. (B) PRC II P105. (C) Schematic drawing of a mixture of digests of FSV P140. PRC II P105 and ST-FeSV P85. Peptides common to all three proteins are marked with arrows here and in (D) and (El. Symbols are: (0) transformation-specific peptides common to FSV P140 and PRC II P105; (0) gag peptides common to FSV P140 and PRC II P105; (0) peptide unique to PRC II P105; (@) peptides unique to FSV P140; and (0) peptide unique to ST-FeSV P85. (D) Mixture of FSV Pi40 and ST-FeSV P85. (E) Mixture of PRC II PI05 and ST-FeSV P85. (F) ST-FeSV P85.
Figure
4. Comparison
of Methionine-Containing
(A) Y73 P90. The gag peptides by arrows.
are numbered
Tryptic as in Figure
Peptides
of Transforming
3. (6) Ab-MuLV
Proteins
of Y73. Ab-MuLV
Pi 20. (0 RSV ~60”~.
Peptides
and RSV
having
the same
mobilities
are denoted
Retroviruses 151
and Tyrosine
Phosphorylation
these transforming proteins have not yet been shown to be tyrosine protein kinases, this identification has been quite well established with purified preparations of RSV ~60”” (Erikson et al., 1979; Collett et al., 1980). Thus it appears that all four ASVs characterized to date may share a common mechanism of neoplastic transformation. Both the cellular protein levels of phosphotyrosine in FSV-infected cells and the levels of protein kinase activity in immunoprecipitates containing FSV Pi 40 were temperature-sensitive. At 41 “C, the levels of cellular phosphotyrosine and of immunoprecipitable kinase activity both were scarcely above the levels in uninfected cells. At 36”C, however, both had increased significantly. This evidence suggests either that the FSV P140 in this stock of virus is a temperature-sensitive protein kinase or that it activates a cellular protein kinase in a temperature-sensitive fashion. Pawson et al. (1980) have also observed that their FSV stock was temperature-sensitive for transformation and for protein kinase activity. Feldman et al. (1980), however, reported no evidence for temperature sensitivity of their stock of FSV. Transforming Proteins of PRC II, FSV and FeSV Are Homologous Shibuya et al. (1980) demonstrated nucleic acid sequence homology between the PRC II RNA genome and a cDNA probe complementary to the transformation-specific sequences of FSV. A smaller amount of homology was observed between this FSV probe and GA-FeSV and ST-FeSV RNA, while no homology was observed with RNA from RSV, Y73, Ab-MuLV or the avian acute leukemia viruses. Using a similar cDNA probe specific for Y73, Yoshida et al. (1980) observed no homology with RSV, FSV, PRC II or the avian acute leukemia viruses. This report confirms and extends these data by analysis of the protein products of those viruses currently thought to be associated with tyrosine-specific protein kinases and shows that the homologous transformation-specific RNA sequences are translated to form homologous proteins. This study shows that the only known virus-coded products of PRC II and FSV are closely related to one another, in that they share 12 out of a total of 13 to 15 methionine-containing tryptic peptides. Both these proteins were also shown by immunological criteria and by tryptic peptide mapping to be related to STFeSV P85 and GA-FeSV P95. The two peptides shown in this report to be common to proteins of FSV, PRC II and ST-FeSV were previously shown to be present also in GA-FeSV P95 (Barbacid et al., 1980b). The close homology between FSV P140 and PRC II Pi 05 observed by analysis of methionine-containing tryptic peptides is particularly striking when compared with peptide maps of ~60’” encoded by different strains of RSV derived from the same original isolate and passaged for many years. When six strains of RSV were
analyzed, only four out of a total of 19 peptides were common to all strains (Beemon et al., 1979). PRC II P105 and FSV P140, though probably derived from different recombinational events, appear to have undergone much less divergence from their cellular progenitor than has RSV p60”“. Diversity of Viral and Cellular Tyrosine Protein Kinases Uninfected feline cells contain DNA sequences homologous to the transformation-specific sequences of ST-FeSV and GA-FeSV (Frankel et al., 1979). Furthermore, Barbacid et al. (1980a) demonstrated that the transformation-specific sequences of the ST-FeSV and GA-FeSV proteins were homologous to a normal cell phosphoprotein NCP92. FSV and PRC II were probably both derived by recombination of an ALV with a normal avian cellular gene homologous to the feline cellular progenitor of these FeSVs. No direct evidence for such a cognate gene, however, has yet been obtained (Frankel et al., 1979). Since there is no evidence for homology between the transforming sequences of RSV, Y73, Ab-MuLV and the FSV/PRC II/FeSV class, these viruses may well have been generated by recombinational events involving a minimum of four unrelated cellular genes. RSV p60 ST’ is structurally very similar to its cellular homolog p60”“” (Sefton et al., 1980a), which is also associated with tyrosine protein kinase activity (Hunter and Sefton, 1980). Ab-MuLV P120 has a normal cell homolog NCPI 50 (Witte et al., 1979b), while no homolog of Y73 P90 has yet been identified. If all these viral-coded transforming proteins and their cellular homologs are tyrosine-specific protein kinases, there must be a minimum of four distinct cellular proteins with the ability to phosphorylate tyrosine and to transform cells when overproduced or modified, or both. If the substrate specificities of the four different virus-associated protein kinases are found to be somewhat distinct but overlapping, it may be possible to limit the number of potential substrates essential for cellular transformation to those that are shared by several viruses. Experimental Procedures Cells and Viruses Chicken cells productively infected with PRC II were provided by P. K. Vogt (Breitman et al., 1981; Neil et al., 1981). FSV-infected chicken cells were provided by H. Temin; K. Toyoshima provided chicken cells infected with Y73 (Kawai et al., 1980). Infection with SR-RSV-A and growth of all chicken cells were as described (Beemon and Hunter, 1978). Mink lung cells nonproductively transformed by ST-FeSV were provided by M. Barbacid (Porzig et al., 1979). The ANN-l nonproducer line of NIH-Swiss 3T3 cells infected with AbMuLV was obtained from W. Raschke (Scher and Siegler, 1975). Radiolabeling and lmmunoprecipitation Procedures for labeling cells with %-methionine orthophosphate (ICN) and immunoprecipitation
(Amersham) or3*Phave been described
Cl?ll 152
(Sefton et al., 1978). Eppendorf tubes (1.5 ml; Sarstedt) were used, however, for the entire immunoprecipitation procedure, and centrifugation was carried out in a microcentrifuge (Brinkmann) at 10,000 g for 20 sec. TBR serum was obtained by injection of SR-RSV-D into newborn rabbits (Brugge and Erikson, 1977). Rabbit antiserum against AMV pi9 was provided by D. P. Bolognesi., Goat antisera raised by injection of autologous cells infected by ST-FeSV or GAFeSV and anti-FeLV p30 were provided by M. Barbacid (Barbacid et al., 1980b). Goat antiserum against Rauscher MuLV ~15, used to precipitate Ab-MuLV P120, and Rauscher MuLV used in blocking experiments were provided by the National Cancer Institute. Phosphoamino Acid Analysis Analysis of phosphoamino acids in whole cells and in individual proteins was performed as described (Hunter and Sefton, 19801, except that partial acid hydrolysis was for 1 hr in 6 N HCI at 11O’C. 32P-labeled cells were lysed in RIPA buffer (0.15 M NaCI, 10 mM sodium phosphate, pH 7.0, 1% NaDOC, 1% NP40, 0.1% SDS 1% Trasylol, 2 mM EDTA), proteins were extracted into phenol, precipitated with trichloroacetic acid, washed with chloroform-methanol and hydrolyzed with acid. Phosphoamino acids were separated by twodimensional electrophoresis on cellulose thin-layer plates at pH 1.9 and pH 3.5. 32P-labeled proteins were immunoprecipitated with specific antisera, resolved on SDS-polyacrylamide gels, electroeluted from gels, precipitated with trichloroacetic acid and hydrolyzed with HCI as described above. Protein Kinase Assay Assay of protein kinase activity in immune complexes was basically as described by Collett and Erikson (1978). lmmunoprecipitates prepared in RIPA buffer and adsorbed onto fixed Staphylococcus aureus (Calbiochem) were incubated for 10 min at 24°C in 20 ~1 of buffer containing 0.01 M sodium phosphate, pH 6.8, 0.005 M MgC12 and 1 pCi of Y-~‘P-ATP (NEN, spec. act. >2500 Ci/mmole). Samples were assayed by SDS-polyacrylamide gel electrophoresis. autoradiography and scintillation counting. Gel Electrophoresis and Tryptic Peptide Analysis Procedures for SDS-polyacrylamide gel electrophoresis were as described (Sefton et al., 1978). 35S-methionine-labeled proteins were immunoprecipitated from cells, subjected to electrophoresis on preparative gels 2 mm thick, electroeluted from the gels in 0.1% SDS, 0.05 M NH.,HC03 and 5% P-mercaptoethanol (W. Welch and B. Sefton, manuscript in preparation), precipitated with trichloroacetic acid and subsequently treated as described by Beemon and Hunter (1978). After digestion with TPCK-trypsin (Millipore), samples were subjected to electrophoresis on cellulose thin-layer plates (E. M. Reagents) at 1 kV for 27 min in a pH 4.7 buffer containing butanol. pyridine. acetic acid and water at a ratio of 2:1:1:36. Ascending chromatography was in a buffer containing these same reagents at a ratio of 97:75:15:60. In some cases, peptides were also subjected to electrophoresis in a buffer at pH 1.9, containing formic acid, acetic acid and water at a ratio of 25:78:897, at 1 kV for 20 min. After drying, the plates were dipped into molten P-methylnaphthalene containing 0.4% diphenyloxazole and exposed to preflashed film (Kodak XR-5) at -70°C (Banner and Stedman, 1978). Acknowledgments I thank E. A. McNelly for expert assistance; P. K. Vogt, H. Temin. K. Toyoshima, M. Barbacid and W. Raschke for infected cells: M. Barbacid and D. Bolognesi for antisera: and M. Barbacid, G. S. Martin, J. Neil, T. Hunter, T. Patschinsky, J. Cooper and 8. Adkins for helpful discussions and communication of unpublished results. This work was supported by a research grant from the National Cancer Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact.
Received
October
23, 1980;
revised
January
9. 1981
References Barbacid, M.. Beemon, K. and Devare, S. G. (1980a). Origin and functional properties of the major gene product of the Snyder-Theilen strain of feline sarcoma virus. Proc. Nat. Acad. Sci. 77, 5158-5162. Barbacid, M., Lauver, A. V. and Devare. S. G. (1980b). Biochemical and immunological characterization of polyproteins coded for by the McDonough. Gardner-Arnstein. and Snyder-Theilen strains of feline sarcoma virus. J. Virol. 33, 196-207. Beemon, K. and Hunter, virus src gene products
T. (1978). Characterization of Rous sarcoma synthesized in vitro. J. Virol. 28, 551-566.
Beemon, K., Hunter, T. and Sefton, B. M. (1979). Polymorphism avian sarcoma virus src proteins. J. Virol. 30, 190-200.
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Banner. W. M. and Stedman. J. D. (1978). ‘H and “C on thin layers. Anal. Biochem.
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Efficient fluorography 89, 247-256.
Breitman. M. L.. Neil, J. C.. Moscovici, C. and Vogt. P. K. (1981). The pathogenicity and defectiveness of PRCII. Virology 108, l-l 2. Brugge, J. S. and Erikson, R. L. (1977). Identification mation-specific antigen induced by an avian sarcoma 269, 346-348.
of a transforvirus. Nature
Brugge, J. S., Erikson, E. and Erikson. R. L. (1978). Antibody to virion structural proteins in mammals bearing avian sarcoma virusinduced tumors. Virology 84, 429-433. Carr, J. G. and Campbell, J. G. (1958). Three sarcomata. Brit. J. Cancer 12, 631-635.
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fowl
Collett, M. S. and Erikson, R. L. (1978). Protein kinase activity associated with the avian sarcoma virus gene product. Proc. Nat. Acad. Sci. 75, 2021-2024. Collett. M. S.. Purchio, A. F. and Erikson, R. L. (1980). Avian sarcoma virus-transforming protein, pp60src shows protein kinase activity specific for tyrosine. Nature 285, 167-l 69. Erikson, E.. Brugge, J. S. and Erikson, R. L. (1977). Phosphorylated and non-phosphorylated forms of avian sarcoma virus polypeptide ~19. Virology 80, 177-I 85. Erikson. R. L., Collett, M. S., Erikson. E. L. and Purchio, A. F. (1979). Evidence that the avian sarcoma virus transforming gene product is a cyclic AMP-independent protein kinase. Proc. Nat. Acad. Sci. 76, 6260-6264. Feldman, R. A., Hanafusa. T. and Hanafusa. H. (1980). Characterization of protein kinase activity associated with the transforming gene product of Fujinami sarcoma virus. Cell 22, 757-765. Frankel, A. E., Gilbert, J. H., Porzig. K. J., Scolnick, E. M. and Aaronson, S. A. (1979). Nature and distribution of feline sarcoma virus nucleotide sequences. J. Viral. 30, 821-827. Fujinami, A. and Inamoto, K. (1914). Uber Geschwtilste bei japanischen Haushuhnern, insbesondere ijbereinen transplantablen tumor. Z. Krebsforsch 14, 94-119. Hanafusa. T., Wang, L.-H., Anderson, S. M., Karess, R. E., Hayward, W. S. and Hanafusa, H. (1980). Characterization of the transforming gene of Fujinami sarcoma virus. Proc. Nat. Acad. Sci. 77, 3009301 3. Hunter, T. and Sefton, B. M. (1980). Rous sarcoma virus phosphorylates 77,1311-1315.
Transforming gene product of tyrosine. Proc. Nat. Acad. Sci.
Itohara. S., Hirata, K.. Inoue, M., Hatsuoka, M. and Sato, A. (1978). Isolation of a sarcoma virus from a spontaneous chicken tumor. Gann 69, 825-830. Kawai. S., Yoshida, M.. Segawa. K., Sugiyama, H., Ishizaki, R. and Toyoshima, K. (1980). Characterization of Y73. a newly isolated avian sarcoma virus. Proc. Nat. Acad. Sci. 77, 6199-6203. Lee. W. H., Bister. K., Pawson, A., Robbins. T., Moscovici. C. and Duesberg. P. H. (1980). Fujinami sarcoma virus: an avian RNA tumor virus with a unique transforming gene. Proc. Nat. Acad. Sci. 77, 2018-2022.
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Levinson, A. D., Oppermann, H., Levintow, L.. Varmus, H. E. and Bishop, J. M. (1978). Evidence that the transforming gene of avian sarcoma virus encodes a protein kinase associated with a phosphoprotein. Cell 75, 561-572. Neil, J. C., Breitman, M. L. and Vogt, P. K. (1981). Characterization of a 105,000 molecular weight gag-related phosphoprotein from cells transformed by the defective avian sarcoma virus PRCII. Virology, 108, 98-l 10. Pawson, T., Guyden. J., Kung. T-H., Radke, K., Gilmore, T. and Martin, G. S. (1980). A strain of Fujinami sarcoma virus which is temperature-sensitive in protein phosphorylation and cellular transformation. Cell 22, 767-775. Porzig, K. J., Barbacid, M. and Aaronson, S. A. (1979). properties and translational products of three independent feline sarcoma virus. Virology 92, 91-l 07. Scher, C. D. and Siegler. R. (1975). by Abelson murine leukemia virus.
Biological isolates of
Direct transformation of 3T3 cells Nature 253, 729-731.
Sefton, B. M.. Beemon. K. and Hunter, T. (1978). Comparison of the expression of the src gene of Rous sarcoma virus in vitro and in vivo. J. Virol. 28, 957-971. Sefton. B. M.. Hunter, T. and Beemon. polypeptide products of the transforming and the homologous gene of vertebrates. 2059-2063.
K. (1980a). Relationship of gene of Rous sarcoma virus Proc. Nat. Acad. Sci. 77,
Sefton, B. M., Hunter, T., Beemon, K. and Eckhart, W. (1980b). Evidence that the phosphorylation of tyrosine is essential for cellular transformation by Rous sarcoma virus. Cell 20, 807-816. Shibuya, M.. Hanafusa. T., Hanafusa. H. and Stephenson, J. R. (1980). Homology exists among the transforming sequences of avian and feline sarcoma viruses. Proc. Nat. Acad. Sci. 77, 6536-6540. Van de Ven, W. J. M.. Reynolds, F. H. and Stephenson, J. R. (1980). The nonstructural components of polyproteins encoded by replication-defective mammalian transforming retroviruses are phosphorylated and have associated protein kinase activity. Virology 701, 185197. Witte, 0. N., Dasgupta. A. and Baltimore, leukemia virus protein is phosphorylated rosine. Nature 283, 826-831.
D. (1979a). Abelson murine in vitro to form phosphoty-
Witte, 0. N.. Rosenberg, N. and Baltimore, D. (1979b). A normal cell protein cross-reactive to the major Abelson murine leukaemia virus gene product. Nature 287, 396-398. Yoshida. M., Kawai, S. and Toyoshima, K. (1980). cells contain structurally unrelated progenitors genes. Nature 287, 653-654.
Uninfected avian of viral sarcoma
53, April 1981,
Copyright
0 1981
by MIT
Transforming Proteins of Some Feline and Avian Sarcoma Viruses Are Related Structurally and Functionally Karen Beemon Tumor Virology Laboratory The Salk institute P. 0. Box 85800 San Diego, California 92138
Summary Transformation of chicken cells by Fujinami sarcoma virus (FSV), PRC II or Y73 (three independently isolated avian sarcoma viruses that are replication-defective and lack the Rous sarcoma virus src gene) resulted in significant elevation (4-13 fold) of phosphotyrosine levels in cellular protein. The gag-related proteins encoded by these avian sarcoma viruses (ASVs) were all associated with tyrosine-specific protein kinase activity when assayed in immune complexes and were phosphorylated at both tyrosine and serine residues in vivo. Both the phosphotyrosine level in protein of FSVinfected cells and the protein kinase activity assayed in immune complexes containing the FSV protein P140 were temperature-sensitive. The presumed transforming proteins of these ASVs were compared with those of Rous sarcoma virus (RSV), Abelson murine leukemia virus and the Snyder-Theilen and Gardner-Arnstein strains of feline sarcoma virus (FeSV), which have previously been associated with tyrosine-specific protein kinase activity. FSV and PRC II proteins were shown to be structurally related to one another and to the FeSV proteins by tryptic peptide mapping and by immunological studies. No homology was observed, however, between the transforming proteins of RSV, Y73, Abelson murine leukemia virus and the FSV/PRC II/ FeSV class, suggesting there may be at least four classes of retroviruses whose transformation mechanisms involve aberrant phosphorylation of cellular protein at tyrosine residues. Introduction Fujinami sarcoma virus (FSV), PRC II and Y73 are three replication-defective avian sarcoma viruses (ASVs) that induce sarcomas in birds and transform fibroblasts in culture; however, they lack the Rous sarcoma virus (RSV) src gene. Each of these viruses was isolated independently from a spontaneous chicken fibrosarcoma (Fujinami and Inamoto, 1914; Carr and Campbell, 1958; itohara et al., 1978). The organization of the genomes of these ASVs is also different from that of RSV, but resembles that of avian acute leukemia viruses and certain mammalian RNA tumor viruses. The approximately 5 kilobase (kb) FSV genome shares sequences at its 5’ terminus (which include part of the viral gag gene coding for the internal structural proteins) and at its 3’ terminus (from
the noncoding “common” region), with its associated avian leukosis virus (ALV) helper. The internal 3 kb region contains transformation-specific sequences, probably derived from the cell (Lee et al., 1980; Hanafusa et al., 1980). PRC II and Y73 appear to have similar genomic organizations. Each virus encodes a large phosphoprotein containing sequences from the N terminus of the gag gene linked to transformation-specific sequences. The presumed transforming proteins encoded by these viruses are FSV P140 (Lee et al., 1980; Hanafusa et al., 1980), PRC II P105 (Neil et al., 1981) and Y73 P90 (Kawai et al., 1980). In contrast, RSV encodes a phosphorylated transforming protein, p60”“, which does not contain gag sequences (Brugge and Erikson, 1977; Sefton et al., 1978). The protein p60 “’ is associated with protein kinase activity as assayed both in immune complexes (Collett and Erikson, 1978; Levinson et al., 1978) and in partially purified preparations (Erikson et al., 1979). A tyrosine residue is phosphorylated by this protein kinase activity in vitro (Hunter and Sefton, 1980; Collett et al., 1980). Furthermore, transformation of cells with RSV increases the level of phosphotyrosine found in cellular protein 6-10 times, suggesting this protein kinase activity is involved in transformation by RSV (Sefton et al., 1980b). Cells infected with a large number of other transforming viruses, including Moloney and Kirsten murine sarcoma viruses, avian myelocytomatosis virus MC29, SV40 and polyoma virus, as well as cells transformed by some chemical carcinogens, have been screened for increased levels of phosphotyrosine in cellular proteins. None of these transformed cells showed more phosphotyrosine in protein than the corresponding nontransformed cells (Sefton et al., 1980b). Both Abelson murine leukemia virus (Ab-MuLV) and Snyder-Theilen feline sarcoma virus (ST-FeSV), however, induce an elevation of 5-10 times in the level of phosphotyrosine in cellular protein (B. M. Sefton, T. Hunter and W. Raschke, manuscript submitted for publication; Barbacid et al., 1980a). Furthermore, the gag-related proteins encoded by these viruses, AbMuLV P120 and ST-FeSV P85, are associated with tyrosine-specific protein kinase activity when assayed in immune complexes (Witte et al., 1979a; Barbacid et al., 1980a). The Gardner-Arnstein strain of FeSV (GA-FeSV) encodes a protein, P95, structurally related to P85 (Barbacid et al., 1980b), which also has associated kinase activity (Van de Ven et al., 1980). This study examines the structure and function of the gag-related proteins encoded by PRC II, FSV and Y73. Although none of these viruses contains RSV src gene sequences, the pathogenic similarity of these viruses to RSV suggests they might transform cells by a mechanism similar to that of RSV. Furthermore, all gag-related ASV proteins are phosphorylated, as are RSV p60src, Ab-MuLV P120 and ST-FeSV P85. In
Cell 146
addition, tyrosine-specific protein kinase activity has been detected in immunoprecipitates containing FSV P140 (Feldman et al., 1980; Pawson et al., 1980) and Y73 P90 (Kawai et al., 1980). I have extended the comparison between RSV and the other ASVs by asking whether these viruses also induce elevated phosphorylation of tyrosine residues in cellular protein. In addition, I have investigated possible structural relationships between the presumptive transforming proteins of these ASVs and those of other retroviruses. Results Cells Infected with PRC II, FSV and Y73 Have Elevated Levels of Phosphotyrosine To analyze the levels of tyrosine phosphorylation in cellular protein after infection with PRC II, FSV or Y73, well transformed cultures were labeled with 32P-orthophosphate for 18 hr; cellular protein was extracted and partially hydrolyzed with HCI, and the acid-stable phosphoamino acids were resolved by two-dimensional electrophoresis. Table 1 indicates the relative amount of phosphotyrosine recovered in each case. The relative levels of the major phosphoamino acids, phosphothreonine and phosphoserine, were essentially unchanged by transformation by these viruses. Cells infected with PRC II showed a dramatic increase in phosphotyrosine to a level 13 times that seen in protein of uninfected chicken embryo fibroblasts (Table 1). Y73-infected cells showed a ninefold increase in relative phosphotyrosine levels (Table l), similar to that previously reported for cells transformed by RSV. Infection only with ALVs, similar to the helper viruses present here, has been demonTable 1. Effect Cellular Protein Infecting
Virus
of ASV Infection
Growth ture
on the Level of Phosphotyrosine
Tempera-
in
Relative Abundance of Phosphotyrosinea
None
38°C
0.021
PRC II
38°C
0.282
None
37°C
0.030
Y73
37oc
0.266
None
41 “C
0.068
FSV
41 “C
0.079
FSV
36°C
0.303
Chicken cells were labeled with 32P-orthophosphate for 18 hr at the temperatures shown, and proteins were extracted and partially hydrolyzed with acid. Approximately 2 X 1 O6 cpm of each hydrolysate plus 0.5 pg of each phosphoamino acid marker were analyzed by two-dimensional electrophoresis as in Fig. 2. a The radioactivity in phosphotyrosine is expressed as a percentage of the total radioactivity recovered in phosphoserine, phosphothreonine and phosphotyrosine. Phosphothreonine represented approximately 6%. and phosphoserine approximately 94%, of the recovered phosphoamino acids in all cases.
strated to have no effect on phosphotyrosine levels in cellular protein (Hunter and Sefton, 1980). When FSV-infected ceils were grown at 36°C the phosphotyrosine level in cellular protein increased fourfold, while there was essentially no increase over the level in uninfected cells when these cells were grown at 41 “C (Table 1). FSV-induced transformation also appeared to be temperature-sensitive by the criteria of cellular morphology and of focus formation in agar (data not shown). The rate of 3H-labeled 2-deoxyglucose uptake by cells infected with either FSV or PRC II also was measured to provide a more quantitative assay of transformation. FSV-infected cells grown at 36°C had an uptake rate three times that of FSV-infected cells grown at 41 “C, while cells infected with PRC II had an elevated rate of uptake at both temperatures (T. Ryden and K. Beemon, unpublished results). Furthermore, a striking difference was observed in the amount of protein kinase activity assayed in immunoprecipitates containing FSV P140 derived from cells grown at different temperatures (see below). In Vitro Protein Kinase Activity Tyrosine-specific protein kinase activity has recently been demonstrated in immunoprecipitates containing Y73 P90 and FSV Pi 40 (Kawai et al., 1980; Feldman et al., 1980; Pawson et al., 1980). In this report, immunoprecipitates containing PRC II P105 were also assayed for protein kinase activity. In addition, the protein kinase activities associated with different ASV proteins were compared with regard to phosphate acceptors. lmmunoprecipitates containing PRC II P105, FSV P140 or Y73 P90 all exhibited protein kinase activity, as demonstrated in Figure 1. The substrate that was phosphorylated, however, depended on the antiserum used for immunoprecipitation. When antiserum against the N-terminal viral gag protein pl9 was used, phosphorylation of a protein comigrating with the respective ASV-coded protein was observed (Fig. 1 B). When sera from rabbits bearing RSV-induced tumors (TBR sera) that contain antibodies to the viral structural proteins as well as to ~60’” (Brugge et al., 1978; Sefton et al., 1978) were used, phosphorylation of the immunoglobulin heavy chain was observed, as well as of the ASV proteins (Fig. 1A). No kinase activity was apparent in immunoprecipitates made from infected cells with sera from nonimmunized animals (data not shown). Analysis of in vitro phosphorylation products in immunoprecipitates of P105 revealed that all of the phosphate incorporated into the immunoglobulin heavy chain (Figure 2A) and into Pi05 (data not shown) was at tyrosine residues. Phosphorylation of the immunoglobulin heavy chain in this assay probably is not due to the activity of cellular p60”““, which is also recognized by the broadly cross-reactive TBR serum used, because the amount of phosphorylation observed was vastly
Retroviruses 147
and Tyrosine
Phosphorylation
2345
1234567123456712341
HC-
Figure
1. Assay
of Protein
Kinase
Activity
and lmmunoprecipitation
of ASV Proteins
Protein kinase activity was assayed in immune complexes made with (A) TBR serum or (6) anti-p1 9 serum from cells that were (lane 1) uninfected or infected with (lane 2) Y73. (lane 3) PRC II, (lane 4) FSV at 36”C, (lane 5) FSV at 41 “C, (lane 6) FSV shifted from 36’C to 41 ‘C for 1 hr before lysis or (lane 7) FSV shifted from 41 “C to 36’C for 1 hr. Bands comigrating with P105 and P140 were visible in lanes 3 and 4 of A when the autoradiogram was exposed for a longer time. for 2 hr. and lysates were immunoprecipitated with (lane 1) (C) PRC II-infected or (D) FSV-infected cells were labeled with “S-methionine nonimmune serum, (lane 2) anti-GA-FeSV, (lane 3) anti-ST-FeSV, (lanes 4 and 5) anb-pl9. FSV-infected cells in lanes 1, 2, 3 and 4 of D were grown at 36’C and in lane 5 of D at 41 “C. Samples were subjected to electrophoresis on a 12.5% acrylamide gel, which was fluorographed. Numbers on the sides represent molecular weight (x 1 O-? of viral-coded proteins. HC designates the immunoglobulin heavy chain, visualized by staining.
greater than that seen in the same number of uninfected chicken cells assayed in parallel (Figure 1 A, lane 1). Furthermore, infection with PRC II, FSV or Y73 did not induce a detectable increase in the amount of 35S-methionine-labeled p60”“” immunoprecipitated from cells with TBR serum (data not shown). Some immunoglobulins present in TBR sera may possess, it appears, a suitable phosphorylation site for these protein kinases, perhaps without specifically binding them, while those in most other types of sera tested do not. The immunoglobulins phosphorylated in TBR immunoprecipitates from Y73-infected cells had a slightly faster electrophoretic mobility than those phosphorylated in precipitates from PRC Il-infected or FSV-infected cells made with the same serum (Figure 1 A, lanes 2, 3 and 41, suggesting different kinases may be involved in these systems. The possibility cannot be ruled out, however, that the “autophosphorylation” and the immunoglobulin phospho-
rylation reactions are due to different protein kinases, either viral-coded kinases or induced cellular kinases, present in the immune complexes. Protein kinase activity associated with immunoprecipitates containing FSV P140 appeared to be temperature-sensitive. While protein kinase activity was readily detectable in immunoprecipitates containing FSV P140 made from cells grown at 36’C, very little activity was observed when these cells were grown at 41 “C (Figures 1 A and 1 B, lanes 4 and 5). Furthermore, this protein kinase activity was readily reversible in temperature-shift experiments (Figures 1 A and 1 B, lanes 6 and 7). One hour after cultures were shifted from 36°C to 41 “C, kinase activity had decreased to a level slightly above that in uninfected cells. Substantial kinase activity (20-30% of maximal) was regained 1 hr after a shift from 41 “C to 36°C. This temperaturedependent protein kinase activity was not the result of degradation or loss of immunoprecipitability of
Cdl 148
Figure 2. Phosphoamino Isolated Proteins
Acid Composition
of
32P-labeled proteins were isolated and partially hydrolyzed with HCI, and the phosphoamino acids ware separated by two-dimensional electrophoresis at pH 1.9 (from right to left) and at pH 3.5 (from bottom to top). Unlabeled phosphoserine (P-ser). phosphothreonine (P-thr) and phosphotyrosine (Ptyr) were run as internal markers and visualized by ninhydrin staining. (A) TBR immunoglobulin heavy chain phosphorylated in an immunoprecipitate containing PRC II P105. The following proteins were isolated from cells labeled with “P for 18 hr: (6) PRC II Pi 05; (C) Y73 P90; and (D) PRC II-associated virus p19.
P140 at 41 “C, since approximately equivalent amounts of 35S-methionine-labeled P140 were precipitated from cells at 36°C and at 41 “C (Figure 1 D, lanes 4 and 5). Identification of Phosphoamino Acids of ASV Proteins The other viral proteins associated with tyrosine-specific protein kinase activity have phosphotyrosine residues in vivo (Hunter and Sefton, 1980; Barbacid et al., 1980a; Sefton et al., manuscript submitted for publication), so the identity of the phosphorylated amino acids of the ASV proteins was investigated. Tyrosine was the major phosphorylated amino acid found in PRC II Pi05 labeled in vivo, comprising approximately two thirds of the radioactivity, while the remaining one third was in phosphoserine, as displayed in Figure 28. Some of the phosphoserine residues are likely to be those in the pi9 sequences of P105, since RSV p19 is phosphorylated at serine residues (Erikson et al., 1977). Direct analysis of phosphoamino acids of p19 encoded by the PRC IIassociated virus revealed phosphoserine to be its major phosphoamino acid (Figure 2D). Feldman et al. (1980) have reported a ratio of two phosphotyrosines to one phosphoserine for FSV P140. Both PRC II P105 and FSV P140 therefore contained phosphotyrosine as the major phosphoamino acid. Analysis of
Y73 P90 shown in Figure 2C revealed both phosphotyrosine and phosphoserine, but at a ratio of approximately 1:3. Structural Homology between FeSV, PRC II and FSV Proteins Two lines of investigation have been used to determine possible structural relationships between the transforming proteins of the seven different viruses associated with tyrosine-specific protein kinase activity. The first approach was immunological and made use of different antisera that recognize RSV p60”“, STFeSV P85 and GA-FeSV P95. To investigate relationships with p60s”, I used TBR serum, which was capable of cross-reacting with ~60”” from many different RSV strains (Sefton et al., 1980a). While TBR serum precipitated all three gag-related ASV proteins, this effect appeared to be caused by antibodies against viral structural proteins that were also present (Brugge et al., 1978). Preincubation of the serum with an excess of disrupted RSV virions led to a large decrease in the amount of immunoprecipitable ASV proteins, but did not diminish the precipitation of ~60”” (data not shown), which suggests that the proteins of PRC II, FSV and Y73 have little homology with p60src. In addition, the TBR serum failed to precipitate AbMuLV Pi 20 and ST-FeSV P85 (data not shown). Both PRC II P105 and FSV P140 were immunopre-
Retroviruses 149
and Tyrosine
Phosphorylation
cipitated by antisera raised in goats by injection of autologous cells infected with either ST-FeSV or GAFeSV (Figures 1 C and 1 D). This immunoprecipitation was substantially decreased by preincubation of the antisera with solubilized mink lung cells transformed nonproductively by ST-FeSV, but not by preincubation with uninfected ceils (data not shown). When protein kinase assays were performed with PRC II P105 or FSV P140 immunoprecipitated with antisera against the FeSV-infected cells, autophosphorylation was observed (data not shown). There was, however, no detectable immunoprecipitation of Y73 P90 or RSV ~60”” with either of the antisera against FeSV-infected cells (data not shown). Ab-MuLV P120 was immunoprecipitated with both of these antisera, but this immunoprecipitation was due to homology between the structural protein sequences of Ab-MuLV and FeSV proteins. When the sera were preincubated with disrupted Rauscher MuLV virions, the immunoprecipitation of P120 was effectively blocked (data not shown). Tryptic Peptide Comparisons of Transforming Proteins To examine the apparent homology between proteins of PRC II, FSV and ST-FeSV by another method and to investigate the possibility of homology between AbMuLV P120 and the ASV proteins, a series of tryptic peptide maps were prepared with 35S-methionine-labeled proteins. The peptides were separated by electrophoresis at pH 4.7 on cellulose thin-layer plates, followed by chromatography. The two-dimensional analysis was performed on six of the viral transforming proteins associated with tyrosine protein kinases, on the gag proteins p19 and p27 and on Pr76, the gag precursor polypeptide. Homologous peptides were identified by mapping mixtures of digests. Figure 3 shows the existence of a high degree of homology between the methionine-containing tryptic peptides of PRC II P105 and FSV P140. This homology encompassed both the gag and the transformation-specific domains of each protein. P105 and P140 each contained four peptides shared with pl9 (numbered 7, 8, 9 and lo), one peptide derived from p27 (11) and one other peptide (12) present in Pr76 and coded by the gag gene sequences. In addition, these proteins contained 7 to 9 methionine peptides encoded by transformation-specific regions of the viral genomes. The two proteins shared six non-gag peptides (numbered 1, 2, 3, 4, 5 and 6), while P105 had one unique peptide (13), and P140 had three peptides not shared with P105 (14, 15 and 16). When peptide maps of PRC II P105, FSV PI 40 and ST-FeSV P85 were compared, two of the three methionine-containing tryptic peptides of P85 (numbered 1 and 2) had mobilities identical to those of peptides 1 and 2 in both PRC II P105 and FSV P140. These peptides are marked with arrows in the mixtures dis-
played in Figures 3D and 3E. None of the ST-FeSV P85 methionine-containing peptides represents FeLV gag gene sequences (Barbacid et al., 1980b). Peptides 1 and 2 from PRC II Pi05 and from ST-FeSV P85 were further compared by electrophoresis at pH 1.9. Comigration was again observed, which strongly suggests sequence homology between these proteins. Comparison of these maps with those of RSV p60”“, Ab-MuLV P120 and Y73 P90 (Figure 4) and analysis of mixtures of digests (data not shown) revealed no significant homologies in transformation-specific peptides. Y73 P90 contained three pl9 peptides (Figure 4A, numbered peptides) also found in the gag-related proteins of PRC II and FSV. None of the peptides derived from transformation-specific sequences, however, was common to Y73 and PRC II or FSV. Similarly, Y73 was found to be unrelated to RSV ~60”” and to share only one out of 17 peptides with AbMuLV (Figure 4, arrows pointing up). Y73 therefore appears to be in a class by itself. Comparison of PRC II P105 and Ab-MuLV P120 revealed one peptide with very similar mobilities (Figure 3, peptide 12); however, this is a gag-derived peptide in P105. RSV ~60’” and Ab-MuLV P120 also had one common peptide (Figures 4B and 4C, arrows pointing left). From analysis at pH 1.9, the peptides shown to comigrate in Figure 4 do not appear to represent identical sequences; furthermore, they do not account for a very large proportion of the methionine peptides of the proteins involved. Comparison of ST-FeSV P85 with Ab-MuLV P120, RSV ~60”” and Y73 P90 revealed no homologous methionine-containing peptides. In summary, PRC II, FSV and ST-FeSV and GAFeSV appeared to represent a single class of sarcoma viruses, which had independently acquired related transformation-specific sequences. There was, however, no evidence to support the existence of any substantial structural homology between the transforming proteins of RSV, Y73, Ab-MuLV and the FSV/ PRC II/FeSV class, all of which are associated with protein kinases specific for tyrosine residues. Discussion Tyrosine Protein Kinases Implicated in Transformation by All Four ASVs Three independently isolated avian sarcoma viruses, PRC II, FSV and Y73, were shown to be associated with tyrosine-specific protein kinase activity, assayed both in vivo by measurement of phosphotyrosine levels in cellular protein and in vitro in immune complexes containing viral proteins. Each of these viruses encodes a single gag-related polypeptide thought to be the viral transforming protein and shown to be phosphorylated in vivo at both serine and tyrosine residues (this report; Feldman et al., 1980). While
Cell 150
Figure
3. Comparison
of Methionine-Containing
Tryptic
Peptides
of gag-Related
Proteins
of FSV, PRC II and ST-FeSV
Tryptic digests were resolved by electrophoresis on cellulose plates at pH 4.7 (from left to right) followed by ascending chromatography. (A) FSV P140. (B) PRC II P105. (C) Schematic drawing of a mixture of digests of FSV P140. PRC II P105 and ST-FeSV P85. Peptides common to all three proteins are marked with arrows here and in (D) and (El. Symbols are: (0) transformation-specific peptides common to FSV P140 and PRC II P105; (0) gag peptides common to FSV P140 and PRC II P105; (0) peptide unique to PRC II P105; (@) peptides unique to FSV P140; and (0) peptide unique to ST-FeSV P85. (D) Mixture of FSV Pi40 and ST-FeSV P85. (E) Mixture of PRC II PI05 and ST-FeSV P85. (F) ST-FeSV P85.
Figure
4. Comparison
of Methionine-Containing
(A) Y73 P90. The gag peptides by arrows.
are numbered
Tryptic as in Figure
Peptides
of Transforming
3. (6) Ab-MuLV
Proteins
of Y73. Ab-MuLV
Pi 20. (0 RSV ~60”~.
Peptides
and RSV
having
the same
mobilities
are denoted
Retroviruses 151
and Tyrosine
Phosphorylation
these transforming proteins have not yet been shown to be tyrosine protein kinases, this identification has been quite well established with purified preparations of RSV ~60”” (Erikson et al., 1979; Collett et al., 1980). Thus it appears that all four ASVs characterized to date may share a common mechanism of neoplastic transformation. Both the cellular protein levels of phosphotyrosine in FSV-infected cells and the levels of protein kinase activity in immunoprecipitates containing FSV Pi 40 were temperature-sensitive. At 41 “C, the levels of cellular phosphotyrosine and of immunoprecipitable kinase activity both were scarcely above the levels in uninfected cells. At 36”C, however, both had increased significantly. This evidence suggests either that the FSV P140 in this stock of virus is a temperature-sensitive protein kinase or that it activates a cellular protein kinase in a temperature-sensitive fashion. Pawson et al. (1980) have also observed that their FSV stock was temperature-sensitive for transformation and for protein kinase activity. Feldman et al. (1980), however, reported no evidence for temperature sensitivity of their stock of FSV. Transforming Proteins of PRC II, FSV and FeSV Are Homologous Shibuya et al. (1980) demonstrated nucleic acid sequence homology between the PRC II RNA genome and a cDNA probe complementary to the transformation-specific sequences of FSV. A smaller amount of homology was observed between this FSV probe and GA-FeSV and ST-FeSV RNA, while no homology was observed with RNA from RSV, Y73, Ab-MuLV or the avian acute leukemia viruses. Using a similar cDNA probe specific for Y73, Yoshida et al. (1980) observed no homology with RSV, FSV, PRC II or the avian acute leukemia viruses. This report confirms and extends these data by analysis of the protein products of those viruses currently thought to be associated with tyrosine-specific protein kinases and shows that the homologous transformation-specific RNA sequences are translated to form homologous proteins. This study shows that the only known virus-coded products of PRC II and FSV are closely related to one another, in that they share 12 out of a total of 13 to 15 methionine-containing tryptic peptides. Both these proteins were also shown by immunological criteria and by tryptic peptide mapping to be related to STFeSV P85 and GA-FeSV P95. The two peptides shown in this report to be common to proteins of FSV, PRC II and ST-FeSV were previously shown to be present also in GA-FeSV P95 (Barbacid et al., 1980b). The close homology between FSV P140 and PRC II Pi 05 observed by analysis of methionine-containing tryptic peptides is particularly striking when compared with peptide maps of ~60’” encoded by different strains of RSV derived from the same original isolate and passaged for many years. When six strains of RSV were
analyzed, only four out of a total of 19 peptides were common to all strains (Beemon et al., 1979). PRC II P105 and FSV P140, though probably derived from different recombinational events, appear to have undergone much less divergence from their cellular progenitor than has RSV p60”“. Diversity of Viral and Cellular Tyrosine Protein Kinases Uninfected feline cells contain DNA sequences homologous to the transformation-specific sequences of ST-FeSV and GA-FeSV (Frankel et al., 1979). Furthermore, Barbacid et al. (1980a) demonstrated that the transformation-specific sequences of the ST-FeSV and GA-FeSV proteins were homologous to a normal cell phosphoprotein NCP92. FSV and PRC II were probably both derived by recombination of an ALV with a normal avian cellular gene homologous to the feline cellular progenitor of these FeSVs. No direct evidence for such a cognate gene, however, has yet been obtained (Frankel et al., 1979). Since there is no evidence for homology between the transforming sequences of RSV, Y73, Ab-MuLV and the FSV/PRC II/FeSV class, these viruses may well have been generated by recombinational events involving a minimum of four unrelated cellular genes. RSV p60 ST’ is structurally very similar to its cellular homolog p60”“” (Sefton et al., 1980a), which is also associated with tyrosine protein kinase activity (Hunter and Sefton, 1980). Ab-MuLV P120 has a normal cell homolog NCPI 50 (Witte et al., 1979b), while no homolog of Y73 P90 has yet been identified. If all these viral-coded transforming proteins and their cellular homologs are tyrosine-specific protein kinases, there must be a minimum of four distinct cellular proteins with the ability to phosphorylate tyrosine and to transform cells when overproduced or modified, or both. If the substrate specificities of the four different virus-associated protein kinases are found to be somewhat distinct but overlapping, it may be possible to limit the number of potential substrates essential for cellular transformation to those that are shared by several viruses. Experimental Procedures Cells and Viruses Chicken cells productively infected with PRC II were provided by P. K. Vogt (Breitman et al., 1981; Neil et al., 1981). FSV-infected chicken cells were provided by H. Temin; K. Toyoshima provided chicken cells infected with Y73 (Kawai et al., 1980). Infection with SR-RSV-A and growth of all chicken cells were as described (Beemon and Hunter, 1978). Mink lung cells nonproductively transformed by ST-FeSV were provided by M. Barbacid (Porzig et al., 1979). The ANN-l nonproducer line of NIH-Swiss 3T3 cells infected with AbMuLV was obtained from W. Raschke (Scher and Siegler, 1975). Radiolabeling and lmmunoprecipitation Procedures for labeling cells with %-methionine orthophosphate (ICN) and immunoprecipitation
(Amersham) or3*Phave been described
Cl?ll 152
(Sefton et al., 1978). Eppendorf tubes (1.5 ml; Sarstedt) were used, however, for the entire immunoprecipitation procedure, and centrifugation was carried out in a microcentrifuge (Brinkmann) at 10,000 g for 20 sec. TBR serum was obtained by injection of SR-RSV-D into newborn rabbits (Brugge and Erikson, 1977). Rabbit antiserum against AMV pi9 was provided by D. P. Bolognesi., Goat antisera raised by injection of autologous cells infected by ST-FeSV or GAFeSV and anti-FeLV p30 were provided by M. Barbacid (Barbacid et al., 1980b). Goat antiserum against Rauscher MuLV ~15, used to precipitate Ab-MuLV P120, and Rauscher MuLV used in blocking experiments were provided by the National Cancer Institute. Phosphoamino Acid Analysis Analysis of phosphoamino acids in whole cells and in individual proteins was performed as described (Hunter and Sefton, 19801, except that partial acid hydrolysis was for 1 hr in 6 N HCI at 11O’C. 32P-labeled cells were lysed in RIPA buffer (0.15 M NaCI, 10 mM sodium phosphate, pH 7.0, 1% NaDOC, 1% NP40, 0.1% SDS 1% Trasylol, 2 mM EDTA), proteins were extracted into phenol, precipitated with trichloroacetic acid, washed with chloroform-methanol and hydrolyzed with acid. Phosphoamino acids were separated by twodimensional electrophoresis on cellulose thin-layer plates at pH 1.9 and pH 3.5. 32P-labeled proteins were immunoprecipitated with specific antisera, resolved on SDS-polyacrylamide gels, electroeluted from gels, precipitated with trichloroacetic acid and hydrolyzed with HCI as described above. Protein Kinase Assay Assay of protein kinase activity in immune complexes was basically as described by Collett and Erikson (1978). lmmunoprecipitates prepared in RIPA buffer and adsorbed onto fixed Staphylococcus aureus (Calbiochem) were incubated for 10 min at 24°C in 20 ~1 of buffer containing 0.01 M sodium phosphate, pH 6.8, 0.005 M MgC12 and 1 pCi of Y-~‘P-ATP (NEN, spec. act. >2500 Ci/mmole). Samples were assayed by SDS-polyacrylamide gel electrophoresis. autoradiography and scintillation counting. Gel Electrophoresis and Tryptic Peptide Analysis Procedures for SDS-polyacrylamide gel electrophoresis were as described (Sefton et al., 1978). 35S-methionine-labeled proteins were immunoprecipitated from cells, subjected to electrophoresis on preparative gels 2 mm thick, electroeluted from the gels in 0.1% SDS, 0.05 M NH.,HC03 and 5% P-mercaptoethanol (W. Welch and B. Sefton, manuscript in preparation), precipitated with trichloroacetic acid and subsequently treated as described by Beemon and Hunter (1978). After digestion with TPCK-trypsin (Millipore), samples were subjected to electrophoresis on cellulose thin-layer plates (E. M. Reagents) at 1 kV for 27 min in a pH 4.7 buffer containing butanol. pyridine. acetic acid and water at a ratio of 2:1:1:36. Ascending chromatography was in a buffer containing these same reagents at a ratio of 97:75:15:60. In some cases, peptides were also subjected to electrophoresis in a buffer at pH 1.9, containing formic acid, acetic acid and water at a ratio of 25:78:897, at 1 kV for 20 min. After drying, the plates were dipped into molten P-methylnaphthalene containing 0.4% diphenyloxazole and exposed to preflashed film (Kodak XR-5) at -70°C (Banner and Stedman, 1978). Acknowledgments I thank E. A. McNelly for expert assistance; P. K. Vogt, H. Temin. K. Toyoshima, M. Barbacid and W. Raschke for infected cells: M. Barbacid and D. Bolognesi for antisera: and M. Barbacid, G. S. Martin, J. Neil, T. Hunter, T. Patschinsky, J. Cooper and 8. Adkins for helpful discussions and communication of unpublished results. This work was supported by a research grant from the National Cancer Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact.
Received
October
23, 1980;
revised
January
9. 1981
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