The transforming protein of avian sarcoma virus UR2, p68v-s, has an associated tyrosine-specific protein kinase activity similar to that of p6O-SrC and several other oncogene products. However, this activity has not been linked unequivocally to transformation, and the physiological action of these proteins remains in doubt. We now have found that immunoprecipitated p68v-s also is associated with phosphatidylinositol (PtdIns) kinase (22,23), tyrosine kinase activity shows little substrate specificity in vitro, and the phosphorylation stoichiometries observed are often too low to be of obvious regulatory significance (21). The possibility that the physiological substrate of the oncogene kinases might not be tyrosine was raised by recent observations that p6fV-src can phosphorylate glycerol (24,25). Because of (i) the similarities between the activity of oncogene kinases and that of growth factor receptors (16-18) and (ii) the implication of P-inositide turnover in cell proliferation, it occurred to us to test the phosphorylating activity of one of these kinases, p68vr's, towards phosphatidylinositol (PtdIns). The protein p68v-ros is the oncogene product of avian sarcoma virus UR2 (26, 27); although unrelated to the p60vsrc of Rous sarcoma virus (28), it will phosphorylate itself and exogenous protein substrates on tyrosine residues (27). We found that immunoprecipitated p68v-ros is also capable of phosphorylating PtdIns to Ptdlns 4-phosphate (PtdIns4P). Viruses and Antisera. The origins of UR2 and some of the properties of its transforming protein have been described (26,27). A temperature-sensitive (ts) mutant, UR2-R0200, has been isolated by one of us (P.B.). This mutant produces full expression of the transformed phenotype at 37°C but only partial expression at 42°C. Anti-gag antiserum was obtained from E. J. Smith.Cell Cultures and Conditions. Chicken embryo fibroblast cultures were prepared and maintained as described (26). Infected cells were passaged 2 or 3 times until extensive morphological conversion was apparent.Immunoprecipitation of p68vros. Cell lysates were prepared with either RIPA buffer or a buffer containing 1% NP-40 (or Triton X-100), 1% aprotinin, 10 mM Tris, 100 mM NaCl, and 1 mM EDTA (pH 7.4). Plates (10 cm) containing about 107 cells were washed three times in Tris/glucose, and

Two onc genes of avian sarcoma viruses unrelated to the src gene have recently been identified: fps of Fujinami sarcoma virus/PRCII/UR1 and yes of Y73/Esh sarcoma virus. In the first part of this study we demonstrated that UR2, the most recently isolated avian sarcoma virus, contains in its genome a unique sequence, ros, nonhomologous to src, fps, and yes sequences or to transforming genes of avian acute leukemia viruses. Using cDNAs specific to the inserts of avian sarcoma virus genomes, we examined the existence and the transcription of cellular nucleotide sequences related to the three new onc genes of avian sarcoma virus (fps, yes and ros) in various cells. The progenitor cellular sequences for these onc genes (c-onc) were present in uninfected chicken DNA in one or few copies per haploid genome. These c-onc sequences were detectable in cellular DNA of a wide variety of vertebrates, and the homology between viral and cellular onc was inversely related to the phylogenetic distance of animal species.

We analyzed the genetic structure and gene products of the newly isolated avian sarcoma virus UR1, which recently has been shown to be replication defective and to contain no sequences homologous to the src gene of Rous sarcoma virus. The sizes of the genomic RNAs of UR1 and its associated helper virus, UR1AV, were determined to be 29S and 35S (5.9 and 8.5 kilobases), respectively, by gel electrophoresis and sucrose gradient sedimentation. RNase T1 oligonucleotide mapping of purified viral RNAs indicated that UR1 RNA contains eight unique oligonucleotides in the middle of the genome and shares four 5'-terminal and three 3'-terminal oligonucleotides with UR1AV RNA. The unique sequences of UR1 and Fujinami sarcoma virus were found to be closely related to each other by molecular hybridization of UR1 RNA with DNA complementary to the unique sequence of Fujinami sarcoma virus RNA, but minor differences were found by oligonucleotides fingerprinting. In the regions flanking the unique sequences, UR1 and Fujinami sarcoma viral RNAs contain distinct oligonucleotides, which are shared with oligonucleotides of the respective helper viral RNAs. Cell transformed with UR1 produce a single 29S RNA species which contains a UR1 unique sequence; this species is most likely the mRNA coding for the transforming protein. In UR1-transformed cells, a phosphoprotein fo 150,000 daltons (p150) was detected by immunoprecipitation with antiserum against gag proteins. p150 was associated with a protein kinase activity that was capable of phosphorylating p150 itself, immunoglobulin G of antiserum, and a soluble substrate, alpha-casein. This enzyme transferred phosphate exclusively to tyrosine residues of substrates in vitro, but p 150 labeled in vivo with 32P contained both phosphoserine and phosphotyrosine. The in vitro kinase reaction was not affected by the presence of cyclic AMP or cyclic GMP and strongly preferred Mn2+ over Mg2+. Thus, the properties of UR1 protein are almost identical to those of Fujinami sarcoma virus protein.

UR2 is a newly characterized avian sarcoma virus whose genome contains a unique sequence that is not related to the sequences of other avian sarcoma virus transforming genes thus far identified. This unique sequence, termed ros, is fused to part of the viral gag gene. The product of the fused gag-ros gene of UR2 is a protein of 68,000 daltons (P68) immunoprecipitable by antiserum against viral gag proteins. In vitro translation of viral RNA and in vivo pulse-chase experiments showed that P68 is not synthesized as a larger precursor and that it is the only protein product encoded in the UR2 genome, suggesting that it is involved in cell transformation by UR2. In vivo, P68 was phosphorylated at both serine and tyrosine residues. Immunoprecipitates of P68 with anti-gag antisera had a cyclic nucleotide-independent protein kinase activity that phosphorylated P68, rabbit immunoglobulin G in the immune complex, and a-casein. The phosphorylation by P68 was specific to tyrosine of the substrate proteins. P68 was phosphorylated in vitro at only one tryrosine site, and the tryptic phosphopeptide of in vitro-labeled P68 was different from those of Fujinami sarcoma virus P140 and avian sarcoma virus Y73-P90. A comparison of the protein kinases encoded by UR2, Rous sarcoma virus, Fujinami sarcoma virus, and avian sarcoma virus Y73 revealed that UR2-P68 protein kinase is distinct from the protein kinases encoded by those viruses by several criteria. Our results suggest that several different protein kinases encoded by viral transforming genes have the same functional specificity and cause essentially the same cellular alterations.

We have recently shown that a newly isolated avian sarcoma virus, UR2, is defective in replication and contains no sequences homologous to the src gene of Rous sarcoma virus. In this study, we analyzed the genetic structure and transforming sequence of UR2 by oligonucleotide fingerprinting. The sizes of the genomic RNAs of UR2 and its associated helper virus, UR2AV, were determined to be 24S and 35S, respectively, by sucrose gradient sedimentation. The molecular weight of the 24S UR2 genomic RNA was estimated to be 1.1 x 106, corresponding to 3,300 nucleotides, by gel electrophoresis under the native and denatured conditions. RNase T1 oligonucleotide mapping indicated that UR2 RNA contains seven unique oligonucleotides in the middle of the genome and shares eight 5'-and six 3'-terminal oligonucleotides with UR2AV RNA. From these data, we estimated that UR2 RNA contains a unique sequence of about 1.2 kilobases in the middle of the genome, and contains 1.4 and 0.7 kilobases of sequences shared with UR2AV RNA at the 5' and 3' ends, respectively. Partial sequence analysis of the UR2-specific oligonucleotides by RNase A digestion revealed that there are no homologous counterparts to these oligonucleotides in the RNAs of other avian sarcoma and acute leukemia viruses studied to date. UR2-transformed non-virus-producing cells contain a single 24S viral RNA which is most likely the message coding for the transforming protein of UR2. On the basis of the uniqueness of the transforming sequence, we concluded that UR2 is a new member of the defective avian sarcoma viruses.