Tyrosine-specific protein kinase activity associated with p105 of avian sarcoma virus PRCII

A protein kinase activity was found to be associated with the transformation-specific polyprotein (p105) of avian sarcoma virus PRCII. The kinase was detected in immune complexes with antisera reactive with the gag sequences of p105. Addition of [γ-³²P]ATP to these complexes resulted in phosphorylation of p105 and, with some sera, phosphorylation of immunoglobulin heavy chain. Phosphoamino acid analysis showed that phosphotyrosine was the major product of the in vitro kinase reaction and the major phosphoamino acid of p105 extracted from ³²P-labeled cells. The same tryptic peptides of p105 were found to be labeled by the in vitro kinase reaction and by ³²P labeling of PRCII-transformed cells. These phosphopeptides were not found in Pr76^gag Thus, like Rous sarcoma virus, PRCII has an associated tyrosine-specific protein kinase which may be responsible for its transforming activity.     

Avian sarcoma virus PRCII: Conditional mutants temperature sensitive in the maintenance of fibroblast transformation

Avian sarcoma virus PRCII was mutagenized with 5-azacytidine, and from single foci of transformed cells induced by this virus preparation three conditional mutants were isolated that are temperature sensitive for oncogenic transformation of fibroblasts. Two of these, LA42 and LA47, are not affected in their replication potential at the nonpermissive temperature (41.5°) nor are the virions heat labile. The third mutant, LA46, is coordinately temperature sensitive in replication and transformation. A shift of the incubation temperature from 36° to 41.5° at any time after infection causes transformed cells by any one of the three mutants to revert to normal morphology.     

Esh avian sarcoma virus codes for a gag-linked transformation-specific protein with an associated protein kinase activity

Esh sarcoma virus, initially isolated from a spontaneous tumor of a chicken, transforms fibroblasts in vitro and induces fibrosarcomas in vivo. It is defective for replication, and infectious viral stocks consist of a mixture of a sarcomagenic virus (ESV) and an a avian leukosis virus of subgroup A (EAV) which serves as helper. Cloned stocks of infectious ESV contain two RNA components of M_r, 3 and 1.5 × 10⁶, respectively, as determined by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The component of M_r 1.5 × 10⁶ appears to be the genome of defective ESV, since it is not detected in preparations of the helper virus EAV. The size of the ESV genome suggests major deletions of replicative genes, and ESV-transformed nonproducer cells fail to express functional translation products of the gag, pol, and env genes. ESV-transformed producer and nonproducer clones also do not express pp60^src but contain a gag-related protein of M_r 80,000 (p80). Two-dimensional analyses of the [³⁵S]methionine-labeled tryptic peptides of p80 indicate that this protein contains part of the sequences of gag-p19 covalently linked to additional sequences unrelated to gag, pol, and env gene products. These ESV-specific sequences are also unrelated to pp60^src and to gag-linked polyproteins found in cells transformed by defective avian sarcoma viruses PRCII and Fujinami or defective leukemia viruses AEV, MC29, and MH2. P80 is phosphorylated in vivo at two major sites, one involving phosphoserine and the other phosphotyrosine residues. Immunoprecipitates containing ESV-p80 are associated with a protein kinase activity that is specific for tyrosine residues of several acceptor molecules including p80 itself, rabbit immunoglobulin H chain of the immune complex and exogenously added α-casein. p80 is phosphorylated in vitro at the same tyrosine site as in vivo suggesting that the enzyme activity detected in vitro is of physiological significance. The p80-associated protein kinase activity is strictly dependent on the presence of Mg²⁺ or Mn²⁺ but was found independent of known effectors of cellular protein kinases Ca²⁺, cAMP, or cGMP.     

An avian sarcoma virus mutant which produces an aberrant transformation affecting cell morphology

ST 529 is a temperature-sensitive mutant of Rous sarcoma virus (RSV) strain SR-A, which causes an unusual pattern of phenotypic changes in cells that it transforms. At 35°, ST 529-transformed cells exhibit an elongated, fusiform morphology (morph^f), and also possess an aberrant and “unlinked” phenotypic pattern of transformation-related properties. ST529-transformed cells, at 35°, resemble classically transformed cells in respect to density-independent growth, sugar uptake, protease levels, and ability to form soft agar colonies. However, they differ from classically transformed cells in respect to cell morphology, fibronectin levels, adhesiveness, and organization of actin stress fibers. At a nonpermissive temperature (42°), ST529-infected cells appear phenotypically normal. pp60^src isolated from ST529-infected cells at 42° possesses little or no associated protein kinase activity. Kinase activity becomes detectable rapidly, however, within 30 min after a shift to 35°, and reaches maximal levels within 3 hr after the shift. It is probable that an unusual, mutant src gene product is responsible for the novel pattern of transformation-related changes observed in ST529-infected cells. Previous studies utilizing mutants or variants of RSV have suggested that there are probably at least two biologically significant targets for pp60^src. The present experiments provide additional evidence for a multifunctional src gene product.     

Alterations in the genomes of avian sarcoma viruses

We have identified polypeptides specific to region Elb (map position [mp] 4.6–112) of adenovirus 2 (Ad2) that are synthesized in six lines of Ad-transformed rat or human cells (F17, F4, T2C4, 8617, 5RK clone I, 293), and in Ad2 early infected KB cells. [³⁵S]Methionine-labeled polypeptides were immunoprecipitated using antisera against F17 cells, an Ad2-transformed rat cell line that retains only El. To determine whether they are viral coded, these polypeptides were compared by tryptic peptide mapping with polypeptides translated in vitro from Ela-specific mRNA (mp 1.3–4.5) and Elb-specific mRNA. Polypeptides of 19,000 daltons early infected KB cells. The 19K, 20K, and 53K could be translated from Elb-specific mRNA and thus are coded by Elb. The 19K was precipitated from all transformed cell lines, the 20K was immunoprecipitated from F4, 8617, and T2C4 cells, and the 53K was immunoprecipitated from F4, 8617, T2C4, and 293 cells. These results suggest that the 19K, and perhaps the 20K and 53K, may be important in adenovirus-induced cell transformation. The 20K and 53K share methionine-containing tryptic peptides with each other, but not with the 19K. These results, together with the Ad2 Elb DNA sequence (T. Gingeras and R. Roberts, personal communication), suggest that 19K is translated in a different reading frame from 53K and 20K.     

Helper viruses associated with avian acute leukemia viruses inhibit the cellular immune response

The cellular immune competence of lymphocytes obtained from birds infected with acute or chronic leukemia viruses was studied. The in vitro blastogenic response of thymus-derived lymphocytes (T cells) to the mitogens phytohemagglutinin (PHA) and connanavalin A (Con A) correlates with the ability of birds to elicit a cellular immune response (C. K. Naspitz and M. Richter, 1968, Prog. Allergy12, 1–85; P. Toivanen and A. Toivanen, 1973, J. Immunol.111, 1602–1603). The PHA response of splenic lymphocytes from birds infected with the avian acute leukemia viruses, myelocytomatosis virus strain 29 (MC29), avian erythroblastosis virus (AEV), avian myeloblastosis virus (AMV), or reticuloendotheliosis virus (REV-T), was completely suppressed within 1 week after virus infection of chickens by avian acute leukemia virusus. Spleen cells from chickens infected with the chronic leukemia viruses, Rous-associated viruses (RAVs) types 1 and 3, exhibited PHA responses similar to those of uninfected birds. In contrast, lymphocytes from RAV-2 infected birds had suppressed PHA responses. Acute leukemia viruses are replication-defective and are associated with replication-competent nontransforming helper viruses. When chickens were infected with helper viruses isolated from the acute leukemia virus stocks by endpoint dilution, the T-cell response to various T-cell mitogens was completely suppressed. The helper viruses, therefore, contribute to the immunosuppression which occurs during the development of avian acute leukemia. The rapid lethality of the acute leukemia viruses could be, in part, the result of the immunosuppressive activity of the associated helper viruses. Though all the acute leukemia viruses severely immunosuppress their hosts, the mechanism by which the immunosuppression was induced by these viruses differed. REV-T was the only acute leukemia virus which induced a population of splenic suppressor cells. All the viruses of the leukosis-sarcoma complex tested which had subgroup B specificity were immunosuppressive while those of subgroup A were not. These results suggest that immunosuppression by these viruses may be related to subgroup specificity.     

Chemical crosslinking of proteins in avian sarcoma and leukemia viruses

We have investigated the nearest neighbor relationships of proteins in avian sarcoma and leukemia viruses by means of bifunctional crosslinking agents. In intact virions dimethyl suberimidate and dithiobispropionimidate induce the formation of covalently linked multimeric species of all four internal structural proteins (gag proteins). By immunological tests and by two-dimensional polyacrylamide gel electrophoresis the major species were identified as homotypic dimers of p27, p19, p15, and p12, and homotypic tetramers of p27. No recognizable heterotypic multimers of gag proteins are formed. We conclude that the major gag protein-protein interactions in virions occur between like species. The crosslinking agents also introduce links into the env protein dimer, gp85-gp37, and form a higher multimer of this dimer.     

The expression of pp60src and its associated protein kinase activity in cells infected with different transformation-defective temperature-sensitive mutants of Rous sarcoma virus

A set of five transformation-defective temperature-sensitive mutants of Rous sarcoma virus has been used to investigate the relation between pp60^src its associated protein kinase activity, and expression of the transformed phenotype. In radioimmune competition experiments, the levels of pp60^src induced by the mutants did not vary by more than a factor of two, either among the mutants at a given temperature or between nonpermissive and permissive temperatures for a given mutant. The mutants fell into two distinct classes with respect to the temperature conditional expression of pp60^src-associated kinase activity. Three mutants (GI 201, GI 202, and GI 251) induced two- to fivefold higher levels of pp60^src-associated kinase activity at the permissive temperature. The other mutants GI 203 and 253 induced only very low levels of pp60^src-associated kinase at either temperature. The pp60^src-associated kinase activity induced by GI 201, 202, and 251 at the permissive temperature was significantly more heat labile in vitro than that of the wild type. Furthermore, downshift of the mutant-infected cells to the permissive temperature resulted in a rapid increase (within 15 min) in the pp60^src-associated kinase activity only with mutants GI 201, GI 202, and GI 251, i.e., only with those mutants having an elevated activity at the permissive temperature. The results taken as a whole suggest that there is not a simple relationship between pp60^src, pp60^src-associated kinase activity, and transformation and support the idea of multifunctionality of the src gene product.     

Temperature-sensitive B mutants of SV40 disassemble intracellular encapsidation particles at elevated temperature

We have investigated the effect of elevated temperature on intracellular SV40 nucleoprotein complexes. Shifting cell cultures infected with strains 777, Rh911, or established tsB mutants to 41° for 3 hr before extracting viral nucleoprotein from cell nuclei, led to the selective, irreversible loss of viral encapsidation-oriented structures, including mature virions, from nuclear extracts. Returning cells to 37° after a shift to elevated temperature allowed for encapsidation to resume with newly synthesized nucleoprotein being normally processed into virions. Encapsidation complexes lost during the shift to elevated temperature were not recoverable in nuclear extracts. Using a simplified complementation assay, we found mutation in the B region to be responsible for this response. Electron microscopic investigation showed that shift of intact cells infected with tsB mutants to elevated temperature, led to massive disassembly of capsids. No effect on capsid integrity was noted on extracted nucleoprotein shifted to elevated temperature in vitro. Attachment of virus both before and after temperature shift to a filamentous nuclear structure supports a role for it in the disassembly response.     

Class II defective avian sarcoma viruses: Comparative analysis of genome structure

The genomes of class II avian sarcoma viruses PRCII, PRCII-p, PRCIV, and Fujinami sarcoma virus (FSV), were studied by oligonucleotide fingerprinting, heteroduplex mapping, and nucleic acid hybridization. All of these viruses are genetically defective and have a small RNA genome between 4.5 and 6.1 kilobases (kb) in length. They contain helper-related sequences at both the 5′- and 3′-ends, but most of the retroviral sequences in the middle of the genome are deleted. In place of this deleted information, a contiguous stretch of transformation-specific sequences, termed fps, is found. These putative oncogenic sequences are about 1.2 kb in PRCII, and those in PRCII-p and PRCIV are roughly 2.9 kb. From the analysis of oligonucleotides, it appears that the fps sequences of PRCII represent a subset of those of PRCII-p. Most of the additional sequences present in PRCII-p but absent from PRCII are at the 5′-half of fps. The helper-related sequences in PRCII and PRCII-p are almost indistinguishable, except that PRCII-p contains slightly more retroviral information at the 3′-end of the genome. Therefore, it is possible that PRCII has been derived by deletion from PRCII-p. By contrast, PRCII-p and PRCIV were found to contain identical fps sequences, but their helper-related sequences have diverged substantially. These two sarcoma viruses either represent two independent isolates or, if derived from a single isolate, they have undergone extensive mutation and recombination with diverse avian retroviruses. FSV was found to differ to a greater extent from other class II sarcoma viruses in both helper-related and fps sequences. The difference in fps sequences is localized in the 5′-half of that region. Considering the variation in fps among all members of class II avian sarcoma viruses, it appears that the 3′-half of that genetic region is more conserved than the 5′-half.     

Temperature-sensitive mutants of avian sarcoma viruses: genetic recombination between multiple or coordinate mutants and avian leukosis viruses.

Five coordinate temperature-sensitive mutants of avian sarcoma viruses which fail to transform or produce infectious progeny at 41° have been analyzed by genetic recombination. Four, namely LA334, 336, 338, and 343, carry multiple mutations. One of these mutations is always in the src gene affecting initiation and maintenance of transformation. The other mutations have not been mapped, but our data suggest that in LA338 there is no linkage of the second mutation with env, whereas in LA343 there is some linkage to env. LA336 has a second mutation affecting an early transient function in accordance with physiological data which have shown a thermolabile polymerase in this virus. The data on LA334 are in accord with previous studies which have indicated lesions in src and gag. For LA337 our data confirm the existence of single coordinate lesion segregating from env.     

Transforming proteins of fujinami and PRCII avian sarcoma viruses have different subcellular locations

The subcellular locations of transforming proteins encoded by the related avian sarcoma viruses, PRCII and Fujinami sarcoma virus (FSV), were compared by cell fractionation and by indirect immunofluorescence. Whereas both viruses encode gag-fps proteins associated with tyrosine-specific kinase activity, FSV is more highly tumorigenic than PRCII in vivo. Cell fractionation studies showed that the PRCII transforming protein, P105, became associated with the high-speed particulate fraction shortly after synthesis. However, PRCII P105 did not fractionate with the plasma membrane marker, but rather with high-density membranes. It is unique in this subcellular localization among viral tyrosine kinases. This membrane association was found to be relatively insensitive to salt concentration and did not require divalent cations. Immunofluorescent studies, using anti-fps serum, showed that the PRCII protein was present in discrete, large, cytoplasmic patches, as well as in a juxtanuclear location. In contrast, FSV-encoded P130 was found to fractionate with the plasma membrane marker when cells were analyzed in low salt in the presence of magnesium. However, at higher salt concentrations and in the absence of magnesium, the bulk of P130 was found to be soluble. Immunofluorescent staining of FSV P130 revealed a diffuse, cytoplasmic pattern that was distinct from that of the PRCII product. The observed difference in the subcellular localization of these transforming proteins may be the cause of the difference in tumorigenicity between the two viruses.     

Structural similarities of proteins encoded by three classes of avian sarcoma viruses

The structure and location of the phosphorylation sites of a number of avian sarcoma virus polyproteins have been examined by protease cleavage analysis. The PRCIIp and PRCII polyproteins, P170^gag-fps and P105^gag-fps yield indistinguishable cleavage fragments from an N-terminal region of 65,000 molecular weight, including the gag/non-gagjunction. This provides strong support for the view that PRCII arose directly from PRCIIp by a genomic deletion. For P909^agag-yes, P800^gag-yes, and P105^gag-fps the major tyrosine phosphorylation sites are on C-terminal fragments of 27,000, 26,500, and 36,000 molecular weight, respectively. Further similarities have been shown by partial sequence analysis of the tyrosine phosphorylation sites; the positions of trypsin and staphylococcal V8 protease cleavage sites largely correspond in the src, fps, and yes gene products. The homology between the src and yes products is particularly striking. They yield C-terminal V8-resistant fragments of similar size, containing the major tyrosine phosphorylation sites which are indistinguishable after further cleavage with several proteases. These results suggest structural and functional relatedness between the src, fps, and yes gene products despite the lack of hybridization between their DNA sequences.     

Isolation of an avian sarcoma virus-specific protein kinase from virus particles

Avian sarcoma viruses (ASV) of the Schmidt-Ruppin strain (SR) contain a protein kinase activity which specifically phosphorylates the IgG of sera from tumor-bearing rabbits (TBR). The amount is comparable with that from transformed cells. The activity is thermolabile in two mutants with a temperature-sensitive lesion in the sarcoma (src) gene. Ion-exchange column chromatography on DEAE-cellulose and phosphocellulose allowed a 250-fold purification of enzymatically active protein kinase with a molecular weight of 60,000 (60K). It phosphorylated casein and the heavy chain of IgG of TBR sera but not of control sera. Phosphorylation of casein could be completely inhibited by TBR serum and resulted in phosphorylation of IgG. Purification of the protein kinase from a mutant virus, OS122, and its wild-type SR-D revealed a threefold higher thermolability for the mutant enzyme. Partial proteolytic digest of the [³⁵S]methionine-labeled 60K protein obtained from the phosphocellulose column by immune precipitation was indistinguishable from that of pp60^STC precipitated from SR-D-transformed cells.     

The transformation-specific proteins of avian (Fujinami and PRC-II) and feline (Synder--Theilen and Gardner--Arnstein) sarcoma viruses are immunologically related

The detection of protein kinase activity associated with transformation-specific gene products of Rous sarcoma virus (RSV), Abelson murine leukemia virus (Ab-MuLV), the Snyder-Theilen (ST), and Gardner-Arnstein (GA) strains of feline sarcoma virus (FeSV) and the recently characterized Fujinami (FSV) and PRCII avian sarcoma viruses suggests that these viruses may induce oncogenic transformation by similar mechanisms. In spite of their similar enzymatic properties, the transformation-specific proteins of RSV or Ab-MuLV were not found to share common antigenic determinants with those of the ST or the GA strains of FeSV. These results suggest that vertebrates contain multiple unrelated genes that code for proteins with associated tyrosine-specific protein kinase activity and oncogenic properties when incorporated into the genome of a retrovirus. In contrast, the transformation-specific proteins of ST and GA-FeSV were shown to be immunologically related to those of the FSV and PRCII avian sarcoma viruses. The antigenic determinants shared by these viral proteins have been mapped within their respective sarcoma virus-specific regions, suggesting that the cellular insertions present in these avian and feline sarcoma viruses are related. These observations indicate that potentially oncogenic sequences have been conserved during the evolution of feline and avian genomes and have been independently acquired by two sets of sarcoma viruses.     

ts Transformation mutants of avian sarcoma virus PRCII: lack of strict correlation between transforming ability and properties of the P105-associated kinase

Three mutants of avian sarcoma virus PRC-II, LA42, LA46, and LA47, have a temperature-sensitive (ts) lesion affecting cellular transformation in vitro. At the nonpermissive temperature (41.5°) they do not induce focus formation in fibroblast cultures. LA46 also fails to induce colonies in soft agar at 41.5°, while LA42 and LA47 have retained this ability. The mutations appear to be located in the transformation-specific insert of the defective sarcoma virus genomes, since association with different wild-type (wt) helper viruses does not lead to changes in the transforming phenotypes. The transformation-specific protein P105 of PRCII is detectable at the nonpermissive temperature in moderately reduced quantity in wt- and LA42-infected cells, while the amounts of P105 precipitable from LA47-infected cultures under these conditions are significantly decreased. LA46 made barely detectable quantities of P105 at 41.5°. This temperature sensitivity of LA46 in the synthesis of P105 may reflect the greatly reduced levels of transformation-specific RNA in LA46-infected cells at 41.5°. Intracellular phosphorylation of P105 was not found to be ts in the mutants or in wt PRCII at both serine and tyrosine acceptor sites. P105 extracted from wt-, ts mutant- or wt-revertant-infected cells at permissive and nonpermissive temperatures did not vary significantly in the specific activity of its associated protein kinase as assayed in vitro by phosphorylation of P105 itself. However, preincubation of P105 in vitro at 41.5° revealed greater instability of protein kinase reactions measured in P105 immunoprecipitates from mutant- as compared to wt-infected cells. Also the elevation of cellular phosphotyrosine, characteristics of PRCII-transformed cells, was greatly reduced in ts mutant-infected cells at the nonpermissive temperature but was restored to wt levels in genetic revertants derived from the ts mutants. These observations suggest that there is no direct correlation between in vivo or in vitro phosphorylation of P105 and the induction of all parameters of oncogenic transformation. The increase of total cellular phosphotyrosine appears to be correlated with focus formation, but not with the ability to induce agar colonies.     

The structure and protein kinase activity of proteins encoded by nonconditional mutants and back mutants in the sec gene of avian sarcoma virus

We have characterizedsrc proteins encoded by approximately 30 nonconditional transformation-defective mutants of avian sarcoma virus (ASV) and by several back mutants which reestablish a transformed phenotype. We used gel electrophoresis of immunoprecipitated proteins labeled with³²PO₄ or [³⁵S]methionine to assess size, stability, and phosphorylation; partial digestion with staphylococcal V8 protease to determine structure; and an immune complex assay to measure protein kinase activity. The mutants were all isolated as phenotypic revertants of the B31 line of B77-ASV transformed rat cells, each revertant cell bearing a single provirus without appreciable deletions, as described in the accompanying report (Varmuset al., 1980). In several instancesm the mutant proteins were examined both in the revertant rat cells and in chicken cells infected with transformation-defective viruses rescued from the nonpermissive rat cells. In addition, secondary mutations to restore a transformed phenotype (back mutations) occurred in some cases, in the original rat cells and/or chicken cells infected with rescued viruses. Three categories of mutants were identified by this survey. The largest group (Class I) encodedsrc proteins of normal size (60,000M_r); these proteins were hypophosphorylated and exhibited little or no protein kinase activity.Class II mutants displayed immunoprecipitablesrc proteins of less than normal size. In three cases, the shortsrc related proteins were mapped to the amino terminus of wild-type pp60^src and may be the result of nonsense mutations; in two cases, the short proteins were mapped to the car?yl terminus. Most of Class II mutants lacked protein kinase activity, but the 45,000M_r protein in line 000 exhibited moderate levels of activity, thereby mapping the enzymatically active site to the car?yl terminal three-fourths of pp60^src. The smallest group of mutants (Class III) did not produce detectablesrc proteins. Some of the mutant proteins behaved differently in permissive and nonpermissive hosts; in particular, the product of mutant L produced fusiform transformation and was highly phosphorylated and associated with wild-type levels of protein kinase activity in chicken cells, but was nontransforming, hypo-phosphorylated, and associated with low levels of protein kinase activity in rat cells. In all cases, back mutation to a transformed phenotype was accompanied by a restoration of wild-type (or near wild type) levels of protein kinase activity, further documenting the functional significance of the enzymatic activity. Some of the back mutants, however, encoded proteins of atypical size, either smaller or larger than pp60^src. The active proteins larger than pp60^src ranged up to 68,000M_r in size and were altered at or near the amino terminus. In one case (a retransformed derivative of the Class II revertant 000), the generation of a functionalsrc protein of 68,000M_r coincided with the appearance of an insert of ca. 200 base pairs into the ASV provirus, within or adjacent to the coding region for the amino terminus ofsrc. The diversity of reagents, both mutants and back mutants, derived from the single provirus in B31 cells indicates that this system will be useful for correlation of functional and structural attributes ofsrc.     

Transformation parameters of chick embryo fibroblasts transformed by Fujinami, PRCII, PRCII-p, and Y73 avian sarcoma viruses

The transformation parameters of chicken embryo fibroblasts transformed by replication defective avian sarcoma viruses were compared with those of cells transformed by Rous sarcoma virus (RSV). Two classes of avian sarcoma virus with transforming genes distinct from the src gene of RSV were examined: Fujinami sarcoma virus (FSV), PRCII, and PRCII-p were used as representatives of one class, and Y73 as a representative of the other class. The transformation parameters examined included morphological markers: cell surface morphology, the disappearance of microfilament bundles, and the loss of cell surface fibronectin; alterations in growth properties: growth to high densities and growth in suspension; and metabolic changes: increased uptake of hexose, release of plasminogen activator, and increased synthesis of hyaluronic acid. In general, all of the viruses induced a phenotype which was qualitatively similar to that of RSV-transformed cells, although the extent of each change was less than that observed with RSV. One strain of FSV was found to be coordinately temperature sensitive for the induction of all phenotypic markers examined, whereas another strain of FSV and the other viruses examined were temperature resistant. PRCII-transformed cells had a lower efficiency of colony formation in agar suspension and showed only partial disruption of microfilament bundles; this partial transformation phenotype may be correlated with the low oncogenicity of this virus. These findings in conjunction with previous studies on protein phosphorylation in ASV-transformed cells are consistent with the idea that the replication-defective avian sarcoma viruses transform cells by a mechanism similar to that involved in transformation by RSV.     

Feline sarcoma virus-specific transformation-related proteins and protein kinase activity in tumor cells

Polyproteins (gag-fes) encoded by the Synder-Theilen (ST) and the Gardner-Arnstein (GA) strains of feline sarcoma virus (FeSV) were previously shown to be associated with mink or rat cells that were nonproductively transformed in vitro. In the present study we demonstrated that the same gag-fes proteins were found in cat cells transformed in vitro. Of greater importance, these transformation-related proteins were also in cells taken from fresh biopsies of FeSV-induced tumors. Cells from fibrosarcomas induced with ST-FeSV had gag-fes proteins that were characteristic of this strain. Fibrosarcomas and melanomas were induced with GA-FeSV and both types of tumors contained the protein that is characteristic of cells transformed in vitro with this virus. Expression of these proteins in cultured tumor cells appeared to be independent of the passage level. Based on two-dimensional tryptic peptide analysis, the gag-fes proteins of cat tumor cells appeared to be indistinguishable from those found in cells transformed in vitro. The polyproteins of the cat tumor cells have a closely associated protein kinase activity, as demonstrated in the in vitro assay, and phosphorylated tyrosine residues. Gag-fes proteins of either the ST or GA class were not present in cell cultures initiated from five spontaneous cat tumors.