(polyprotein) p80 of Eshavian sarcoma virus (ESV) has been com- pared by tryptic peptide mapping with the homologous protein p90 of Yamaguchi 73 avian ...
Proc. Natl. Acad. Sci. USA Vol. 78, No. 4, pp. 2611-2615, April 1981
Microbiology
A third class of avian sarcoma viruses, defined by related transformation-specific proteins of Yamaguchi 73 and Esh sarcoma viruses (polyproteins/protein kinase/peptide mapping)
JACQUES GHYSDAEL, JAMES C. NEIL,
AND
PETER K. VOGT
Department of Microbiology, University of Southern California, School of Medicine, 2011 Zonal Avenue, Los Angeles, California 90033
Contributed by Peter K. Vogt, January 12, 1981
ABSTRACT The gag-linked transformation-specific protein (polyprotein) p80 of Esh avian sarcoma virus (ESV) has been compared by tryptic peptide mapping with the homologous protein p90 of Yamaguchi 73 avian sarcoma virus (Y73). p80 of ESV and p90 of Y73 were found to share all four of their major nonstructural, transformation-specific, methionine-containing peptides and to have at least seven cysteine-containing transformation-specific peptides in common. Two nonstructural cysteine-containing peptides unique for ESV p80 and three specific for Y73 p90 were also identified. None of these peptides were found in the transforming gene product pp60frc of Rous sarcoma virus (RSV) or in the transformation-specific polyproteins p105 of avian sarcoma virus PRCII (PRCII) or p140 of Fujinami sarcoma virus (FSV). ESV p80 and Y73 p90 are phosphorylated, and their tryptic phosphopeptides appear to be identical. In each polyprotein two major phosphopeptides were demonstrated, one containing phosphoserine, the other phosphotyrosine. The latter serves as phosphoacceptor for the protein kinase activities (ATP:protein phosphotransferase, EC 2.7.1.37) associated with p80 and p90. These protein kinase activities were found to be functionally indistinguishable but could be easily distinguished from the activities associated with PRCII p105 and FSV p140 on the basis of their cation requirement and target site specificity. On that basis also, p80/p90-associated protein kinases were found to be more similar to the enzymatic activity of pp6Osrc than to those associated with the PRCII and FSV transformation-specific polyproteins. These results document a close genetic relationship between the two independently isolated sarcoma viruses Y73 and ESV. On the basis ofthe relatedness of transformation-specific proteins, ESV and Y73 constitute class III of avian sarcoma viruses, with class I containing the various strains of RSV and class II encompassing FSV and PRCH.
Esh sarcoma virus (ESV) was isolated in Pennsylvania from a spontaneous fibrosarcoma in a chicken (1). Recent biological and biochemical studies have shown that viral stock preparations of ESV are a mixture of a replication-competent Esh-associated helper virus and a replication-defective, transforming virus, ESV (2). Chicken embryo cells nonproductively transformed by ESV do not synthesize any of the helper virus gene products Pr76gag gPr90env or Pr1809'9 , nor do they produce pp6Osrc, the transforming gene product of Rous sarcoma virus (RSV). Instead, they synthesize a Mr 80,000 gag-related phosphoprotein (p80) composed of gag sequences that represent part ofviral protein p19 and are covalently linked to nonstructural transformation-specific sequences (2). In these properties ESV p80 resembles the transformationspecific proteins of Fujinami sarcoma virus (FSV) (3, 4), avian sarcoma virus PRCII (5, 6), and Yamaguchi 73 avian sarcoma
virus (Y73) (7). Cells nonproductively transformed by these viruses also do not express detectable levels of pp6OSrc but synthesize gag-related polyproteins (FSV p140, PRCII p105, or Y73 p90). Studies on transformation-specific proteins and genomic RNA have shown that the genetic structure of these agents is similar to that of avian acute leukemia viruses, with extensive deletions in the gag, pol, and env genes and substitution of part of this information by cell-derived, transformation-specific sequences unrelated to src. The pp6Osrc of RSV is a protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) specific for tyrosine residues (8-11). This enzymatic activity is labile in RSV mutants that are temperature sensitive for the maintenance of transformation, a fact that supports the possibility that kinase activity may be essential to transformation (9-12). Immunoprecipitates containing the gag-linked polyproteins of replication-defective sarcoma viruses FSV, PRCII, ESV, and Y73 are also associated with protein kinase activities specific for tyrosine residues (2, 7, 13, 14). This observation suggests that all avian sarcoma viruses may induce transformation by similar mechanisms. Comparisons of the transformation-specific genetic sequences (3, 4, 15, 16) and their products (6, 17) in RSV, FSV, PRCII, and Y73 have led to the recognition of three classes of avian sarcoma viruses: a first including the various strains of RSV and avian sarcoma virus B77, a second composed of PRCII and FSV, and a third consisting of Y73. To this class III, ESV may now be added. Our initial characterization of ESV showed that the nonstructural transformation-specific sequences of ESV p80 were unrelated to those of FSV, PRCII, the avian acute leukemia viruses MC29, MH2, and avian erythroblastosis virus (AEV) and to pp6osrc (2). The experiments reported here indicate that ESV p80 is structurally related to the transformationspecific protein p90 of Y73 and that the protein kinase activities associated with p80 and p90 are functionally indistinguishable. The p80- and p90-associated protein kinase activities bear some similarity in function to the kinase activity of the pp6Osrc but are quite distinct from those associated with the gag-linked polyproteins of class II avian sarcoma viruses. MATERIALS AND METHODS Cells and Viruses. Sources of ESV, FSV, and PRCII have been described (2, 4, 5, 14). Y73 was a kind gift of K. Toyoshima, M. Yoshida, and S. Kawai. Chicken embrvo fibroblasts were Abbreviations: RSV, Rous sarcoma virus; FSV, Fujinami sarcoma virus; PRCII, PRCII avian sarcoma virus; ESV, Esh sarcoma virus; Y73, Yamaguchi 73 sarcoma virus; TosPheCH2Cl, L-tosvlamido-2-phenvlethyl chloromethyl ketone. Retrovirus genes and their encoded proteins are: gag, internal virion proteins; pol, reverse transcriptase (RNA-dependent DNA polymerase); env, virion surface glycoproteins; src, transforming protein pp6Osrc
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Microbiology: Ghysdael et al.
prepared according to standard techniques and infected at a multiplicity of infection of 0.1-1.0 focus-forming unit/cell with ESV, Y73, PRCII, or FSV. They were used as secondary cultures in all biochemical experiments. Tryptic Peptide Mapping. Labeling of cells with L[35S]methionine, L-['S]cysteine, or [32P]orthophosphate and immunoprecipitation procedures have been described (2). After sodium dodecyl sulfate/polyacrylamide gel electrophoresis and autoradiography, the proteins were located on the gel, eluted, and digested with trypsin treated with L-tosylamido-2-phenylethyl chloromethyl ketone (TosPheCH2Cl). Two-dimensional separation of [3S]methionine or [35S]cysteine tryptic peptides, of phosphopeptides, and of phospho amino acids followed published procedures (2). In Vitro Phosphotransfer Reactions. Procedures for cell lysis, immunoprecipitation, and kinase reaction were included in a previous publication (2). [32P]Phosphate-labeled proteins were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, localized by autoradiography of the dried gel, and excised. Radioactivity in each band was quantitated by liquid scintillation counting after solubilization of the 32P-labeled proteins in NCS (Amersham) for 16 hr at 500C. RESULTS Two-Dimensional Analysis of ESV p80 and Y73 p90 Tryptic Peptides. Chicken embryo cells infected with either ESV or Y73 and their respective helper viruses were labeled for 16 hr with L-[35S]methionine or L-['S]cysteine and then lysed. The gagrelated viral polypeptides were allowed to react with a rabbit antiserum directed against virion gag protein p19 and then precipitated by adsorption of the immune complexes to a suspension of Staphylococcus aureus. ESV p80 and Y73 p90 were separated from Pr769'9 and Pr1809ag-Po by slab gel electrophoresis in the presence of sodium dodecyl sulfate (Fig. 1, lanes C and D). Each was then eluted from the gel, and tryptic peptide maps were prepared. It is known that p80 and p90 are composed of p19-specific gag sequences that are covalently linked to sequences unrelated to any helper virus gene product (2, 17). As shown in Fig. 2 A and B, the two-dimensional maps of the [3S]methionine-labeled tryptic peptides of p80 and p90 are very similar. In fact, p80 and p90 share all of their non-gag [3S]methionine-containing peptides (indicated by an arrow in Fig. 2 A) and three out of four of their p19-derived peptides. The fourth p19-derived peptide of p80 and p90 (arrowheads in A and B) migrated identically in electrophoresis but behaved differently upon chromatography, suggesting a small primary sequence change. The [3S]cysteine-containing tryptic peptides of p80, p90, and Pr769ag of the Y73 helper virus are compared in Fig. 2 C, D, and E. The results of this comparison, corroborated by a mixing experiment (Fig. 2F), are summarized in the diagram of Fig. 2G. The p19-derived cysteine peptides are two closely migrating spots that are shared by both p80 and p90. The relatedness of the non-gag portion of p80 and p90 is demonstrated by the fact that the polyproteins shared seven cysteine peptides. However, the nonstructural sequences of p80 and p90 are not identical, because two cysteine peptides unique to p80 and three specific for p90 were also detected. We conclude from these experiments that p80 and p90 have similar primary structure: both contain part of the p19 gag sequences linked to transformation-specific sequences that are related but not identical. Two-Dimensional Analysis of p80 and p90 Phosphopeptides and Phospho Amino Acids. We previously reported that ESV p80 was heavily phosphorylated in vivo at two major sites different from those of virion gag phosphoprotein p19 (2). These
Proc. Natl. Acad. Sci. USA 78 (1981)
A B C D
E
FG H
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Ink FIG. 1. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of immunoprecipitates. Chicken embryo cells productively transformed by ESV (lanes A and C) and Y73 (lanes B and D) were labeled for 16 hr with L-[35S]methionine, a lysate was prepared, and viral proteins were precipitated by an antiserum to gag protein p19 (lanes C and D) or normal rabbit serum (lanes A and B). Immunoprecipitates of chicken embryo cells transformed by Y73 (lanes E and G) and ESV (lanes F and H) with anti-p19 serum (lanes E and F) or control rabbit serum (lanes G and H) were incubated in the presence of [y32P]ATP as described in the legend of Table 1.
sites will be referred to as a and b (Fig. 3A). The phosphopeptides of Y73 p90 were also analyzed after immunoprecipitation from [32P]orthophosphate-labeled Y73-transformed cells, sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and TosPheCH2Cl-trypsin digestion. As shown in Fig. 3B and confirmed by the mixing experiment in Fig. 3C, the two major phosphopeptides of p90 are indistinguishable from those of p80. Fig. 3 D and E shows that p90 phosphopeptide b contains exclusively phosphotyrosine, whereas p90 phosphopeptide a contains exclusively phosphoserine. Identical results have been obtained for p80 phosphopeptides a and b (not shown). Phosphopeptide b appears also identical to the phosphopeptide derived from the polyproteins after in vitro phosphorylation at tyrosine residues by the respective associated protein kinase activities (2, 17). We conclude that the homology between ESV p80 and Y73 p90 extends to their sites of phosphorylation and is reflected in the probable identity ofthe two predominant phosphopeptides. The site containing tyrosine also appears to act as target in vitro for the polyprotein-associated kinase activities. Divalent Cation Requirements of the in Vitro Phosphorylation Reactions. Immunoprecipitates containing the gag-linked polyproteins of several defective sarcoma viruses are associated with protein kinase activities specific for tyrosine residues (refs. 2, 7, 13, 14; Fig. 1 E-H). ESV p80 and Y73 p90-associated kinases share other general features with those seen with the poly-
Microbiology: Ghysdael et al.
Proc. Natl. Acad. Sci. USA 78 (1981)
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Microbiology: Ghysdael et al.
2614
A
Proc. Natl. Acad. Sci. USA 78 (1981)
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