Characterization of a 105,000 molecular weight gag-related phosphoprotein from cells transformed by the defective avian sarcoma virus PRCII

In cells infected with the replication-defective avian sarcoma virus PRCII a single virus-specific product is detectable, a polyprotein of 105,000 molecular weight (p105). P105 can be precipitated with antisera togag proteins of avian leukosis and sarcoma viruses. By two-dimensional tryptic peptide analysis of [³⁵S]methionine-labeled proteins we have shown that p105 contains peptides of helper viriongag proteins p19 and p27, but not of p15. In addition a number of peptides are present in p105 that are not found in any of the helper virus gene products including gPr95^env and Pr180^gag-pol. These p105-specific peptides are not detectable in the p60^src protein of Rous sarcoma virus (RSV) nor in thegag-related polyproteins encoded by avian myelocytoma and carcinoma viruses MC29 and MH2 or avian erythroblastosis virus AEV. P105 is not detectably glycosylated, but is heavily phosphorylated. In this respect it resembles p60^src of RSV rather than the polyproteins of avian leukemia viruses. Since p105 is the only viral gene product detectable in nonproducing cells transformed by PRCII, this protein may be important in the initiation and maintenance of oncogenic transformation. The nonstructural sequences in p105 would then represent a new class of transforming gene in avian oncoviruses.     

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.     

Recombinant avian oncoviruses. II. Alterations in the gag proteins and evidence for intragenic recombination.

The internal structural (gag) proteins of recombinant avian oncoviruses selected for the env gene of RAV-O (an endogenous chicken virus) and the src gene for PR-RSV-C were examined. Eight of ten clones of such recombinants were found to synthesize altered gag proteins. The gag proteins of one recombinant clone, PR-E-95c, were examined in more detail by gel electrophoresis and tryptic peptide mapping. These methods have allowed us to distinguish between the gag proteins of the two parental viruses and to determine from which virus the proteins of the recombinant virus were derived. PR-E-95c virions were found to contain p27₀, an electrophoretically distinguishable variant of p27 which is found in isolates of RAV-0. This recombinant virus also contains p12/15, which is electrophoretically indistinguishable from the p12/15 of both of the parental viruses. However, tryptic peptide analysis of p15 indicates that PR-E-95c has inherited PR-RSV-C-specific p15 sequences. These observations suggest that at least one cross-over has occurred between p15 and p27 in PR-E-95c. A striking difference between the proteins of PR-E-95c virus and those of the parental viruses is that the recombinant lacks polypeptides migrating in the position of p19 and contains two novel polypeptides termed p19α (MW 20,000) and p19β (MW 15,000). Both of these polypeptides are phosphorylated and share antigenic determinants and some tryptic peptides with parental p19. As determined by peptide analysis and radioimmunoassay, these p19-related proteins contain information from both parental viruses, suggesting that PR-E-95c has another cross-over within p19. The altered p19 proteins bind to viral RNA specifically and are associated with genomic RNA in the virion. Neither the stability nor the specific infectivity of the recombinant viruses appears to be significantly affected by the altered proteins.     

Biochemical properties of avian type-C virus gag gene-coded proteins: comparison with structural proteins coded for at analogous positions within the mammalian type-C viral genome.

The biochemical properties of avian type-C viral gag gene-coded proteins of 27,000 (p27), 19,000 (p19), 15,000 (p15), and 12,000 (p12) molecular weight were examined. These properties were compared to those obtained by a similar analysis of mammalian type-C viral structural proteins. The results indicate striking similarities between proteins mapping at corresponding positions within the respective genomes of the two groups of viruses and thus raise the possibility that these viruses may be evolutionarily related.     

Avian oncovirus MH2 is defective in Gag, Pol, and Env.

The defectiveness of avian oncovirus MH2 is characterized further by genetic experiments and by tryptic peptide mapping. The results show that MH2 lacks full function of all three viral genes necessary for replication, namely, gag, env, and pol. The polyprotein MH2 p100 made in MH2 nonproducer cells contains the tryptic peptides of gag proteins p19 and p27.     

Immunological analysis of v-myc gene products using antibodies against a myc-specific synthetic peptide

Rabbit antisera were prepared against a synthetic peptide corresponding to the carboxyterminal amino acid sequences of the transforming protein p110^gag-myc of avian oncovirus MC29. Analysis of immunoprecipitates formed with these sera (anti-mycC) demonstrated that the gag-myc hybrid proteins encoded by the avian leukemia viruses MC29, CMII, and OK10 were recognized by the anti-peptide sera, but not the gag or pot precursor proteins of helper viruses or the MH2-encoded p100^gag-mil protein. In cells transformed by OK10 or MH2, putative v-myc proteins of 60K (OK10) and 59/61K (MH2) were also precipitated by anti-mycC. In addition, the anti-peptide sera reacted specifically with the gag-myc proteins encoded by three partially transformation-defective mutants of MC29, td10A, td10C, and td10H, and by MC29 variant HBI.     

An avian oncovirus mutant deficient in genomic RNA: characterization of the packaged RNA as cellular messenger RNA.

SE 21Qlb is a recombinant avian oncovirus which is continuously produced by a line of transformed quail cells. These cells produce no detectable infectious viral progeny. Virions of SE 21Q1b contain 1–2% of the normal amounts of RSV-specific RNA found in PR-RSV-C virions and substantial quantities of heterogeneously sedimenting nonviral RNA (M. Linial, E. Medeiros, and W. S. Hayward, 1978b, Cell, 15:1371–1381). RNA extracted from purified virions of SE 21Q1b is as efficient a template for amino acid incorporation in a mRNA-dependent reticulocyte lysate as oligo(dT)-cellulose purified cellular mRNA. Virion RNA from SE 21Q1b serves as a template in vitro for a small amount of the precursor to the internal structural proteins (gag proteins). But unlike RNA from other avian oncoviruses, SE 21Q1b RNA makes, in addition, a number of proteins whose sizes on sodium dodecyl sulfate (SDS)-polyacrylamide gels resemble those of a number of uninfected quail fibroblast proteins. The proteins range in size from 15,000–220,000 daltons. Immunoprecipitation of proteins synthesized in vitro from SE 21Qlb virion RNA with antisera against the gag, pol, and env gene products shows that this RNA serves as a template for only one viral gene product, the recombinant gag gene precursor of 72,000 daltons (ΔPr76) (R. Shaikh, M. Linial, J. Coffin, and R. Eisenman, 1978, Virology87, 326–338). Addition of lysed virus to proteins made in vitro from PR-C RNA or RNA from PR-E-95c, a recombinant whose gag gene structure is similar to that of PR-E-21Qlb, results in the cleavage of gag protein precursors (K. von der Helm, 1977, Proc. Nat. Acad. Sci. USA74, 911–915) into several of the internal structural proteins. On the other hand, products of translation of PR-E-21Q1b RNA are not detectably cleaved into the internal structural proteins, suggesting that a very low amount of PR-E-21Q1b translation products are gag related. A protein of 37,000 daltons constitutes about 15% of the total protein synthesized from SE 21Qlb RNA. Data from serological and peptide mapping studies indicate that this protein is unrelated to the virion gag, pol, or env proteins. However, the major tryptic peptide of this protein appears to be identical to the major peptide of a prominent quail cell protein having the same apparent molecular weight as the in vitro translation product. Thus, several lines of evidence suggest that SE 21Q1b virions contain substantial amounts of functional cellular messenger RNAs.     

Biological properties and translational products of three independent isolates of feline sarcoma virus

Three transforming retroviruses have been isolated from naturally occurring tumors of outbred cats. In the present report, the biological properties of the SM, GA, and ST strains of feline sarcoma virus (FeSV) have been characterized in tissue culture. Like previous mammalian transforming viruses, each FeSV strain was demonstrated to be replication defective, requiring a type-C RNA virus as a helper. Reproducible differences were demonstrated in the morphology of transformed foci induced by each FeSV strain. These findings were independent of helper virus or assay cell utilized, suggesting that the transforming activities of the three FeSV strains are distinguishable. Nonproducer transformants of SM-FeSV expressed feline leukemia virus (FeLV) gag gene products, p15, p12, and p30. In contrast, the GA-and ST-FeSV genomes coded only for p15 and p12 proteins, further distinguishing SM-FeSV from the other FeSV strains. Each remained stable with respect to viral protein expression upon transmission to new cells, demonstrating the stable association of FeLV gag gene information with each FeSV genome. Analysis of the FeLV structural proteins translated in nonproducer transformants of the three FeSV strains revealed the presence of precursor molecules, whose sizes could not be accounted for solely by the number of FeLV gag proteins present in them.     

The NH2-terminal sequence of the avian oncovirus gag precursor polyprotein (Pr76gag).

Pr76^gag, the polyprotein precursor to avian oncovirus internal structural proteins (gag proteins) was isolated by immunoprecipitation from a mRNA-dependent in vitro translation system programmed with genomic (35 S) RNA purified from the Prague strain of Rous sarcoma virus (PR-RSV-C). Attempts to sequence Pr76 were initially unsuccessful due to the presence of a blocked NH₂-terminus. However, when virion 35 S RNA was translated under conditions that prevent NH₂-terminal acetylation, the sequence: Met-Glu-Ala-Val-Ile-Lys-Val-Ile-X-X-Ala-X-Lys was obtained by automated Edman degradation. Since the NH₂-terminal methionine is derived from Met-tRNA_f^Met we conclude that this sequence repesents a primary translation product. Sequence analysis of the supernatant remaining after immunoprecipitation suggests that no viral-related proteins are synthesized which are not precipitated by anti-gag serum. Previous studies employing pactamycin mapping of viral proteins synthesized in infected chick cells indicated that the virion protein p19 was located close to the NH₂-terminus of Pr76^gag (Vogt, et al., J. Mol. Biol.96, 471–493, 1975). We therefore prepared tryptic peptides of p19 purified from virions, separated them by cation exchange chromatography, and determined the amino acid composition of the 19a and 19d peptides. The amino acid composition of the 19a, but not the 19d, peptide correlated with the first six amino acids determined by sequencing. In addition, 19a was the onyl p19-derived peptide not susceptible to digestion by leucine amino peptidase, indicating the presence of a blocked NH₂-terminus. We conclude that the NH₂-terminal sequence of Pr76^gag represents the NH₂-terminus of p19. Comparison of the amino acid sequence with the previously published nucleotide sequence of the 5′ end of 35S RNA from PR-RSV-C (Haseltine et al., Proc. Nat. Acad. Sci. USA74, 989–993, 1977; Shine et al., Proc. Nat. Acad. Sci. USA74, 1473–1477, 1977) shows that synthesis of Pr76^gag is not initiated within the first 119 nucleotides of the viral genome.     

Analysis of cells transformed by defective leukemia virus OK10: production of noninfectious particles and synthesis of Pr76gag and an additional 200,000-dalton protein

Nonproducer cells transformed by the defective leukemia virus (DL V), OK10, have been analyzed. Unlike nonproducer cells transformed by the other avian defective leukemia viruses examined so far, the OK10-transformed cells were found to release noninfectious particles. Analysis of these particles indicated that they contained the viral gag gene proteins but lacked env and pol gene products. In agreement with these results analysis of [³⁵S]methio-nine-labeled cell extracts of these nonproducer clones by immune precipitation showed that of the three viral structural protein precursors Pr769^gag, gPr95^env, and Pr180^gag-pol only Pr76^gag could be detected. In addition, a 200,000 molecular weight protein (OK10-200K) was identified in the cell extracts which by using specific antisera, was shown to be related to the gag and pol gene products but not to the product of the env gene. Tryptic peptide analysis of the OK10-200K protein confirmed the immunological data in that the OK10-200K protein was shown to contain all but one of the Pr1809^gag-pol methionine tryptic peptides plus unique peptides which were specific for OK10 and not related to the env gene product. One of these OK10-specific peptides was also shown to comigrate with one of the putative mac gene product tryptic peptides of the MC29-110K protein. These data indicate a novel gene order for a DLV.     

The defectiveness of Mill Hill 2, a carcinoma-inducing avian oncovirus.

The avian carcinoma virus Mill Hill 2 (MH2) transforms fibroblasts and macrophages in tissue culture. It is defective in replication and dependent on a helper virus, MH2 AV, for the production of infectious progeny. MH2 AV contains both subgroup C and A envelope determinants; its helper functions are required by MH2 in the env, pol, and probably also the gag genes. The defects of MH2 can be complemented only by helper viruses of the chicken leukosis group, further suggesting that MH2 is defective in gag or pol, or both. MH2 transformed nonproducer cells synthesize an aberrant viral polyprotein of 100,000 daltons. This MH2p100 carries antigenic determinants of the gag protein p27, but not of the env protein gp85. It is not cleaved into smaller, functional proteins and is not glycosylated.     

Murine leukemia virus gag polyproteins: the peptide chain unique to Pr80 is located at the amino terminus.

The gag gene-encoded precursor polyproteins Pr80^gag and Pr65^gag of Rauscher leukemia virus were analyzed by chemical fragmentation followed by immune precipitation with antisera specific to viral structural proteins and one-dimensional peptide mapping. Peptides of similar antigenic determinants and size were obtained from the carboxyl-terminal region of the two polyproteins. Precipitation of cleavage products with antiserum known to react with the amino-terminal region of Pr65^gag resulted in the appearance of two distinct fragments: an ～33,000-dalton peptide generated from Pr80^gag and an ～18? to 20,000-dalton fragment generated from Pr65^gag. The data provide evidence that the unique peptide chain (13–15,000 daltons) which distinguishes Pr80^gag is located at the amino terminus and that the carboxyl ends of these two polyproteins may be identical.     

Characteristics of avian sarcoma virus strain PRCIV and comparison with strain PRCII-p

Avian sarcoma virus strain Poultry Research Centre IV (PRCIV) (J. G. Carr and J. G. Campbell (1958)Brit. J. Cancer12, 631–635) is highly oncogenic in chickens and transforms chicken embryo fibroblasts in culture. It is defective in replication, unable to code for complete functional products of the three essential viral genes gag, pol, or env. Nonproducing cells transformed by PRCIV lack the transforming protein of Rous sarcoma virus, pp6^src but synthesize a new transformation-specific protein of 170,000 MW. This P170 contains partial gag sequences, probably at its N-terminus and transformation-specific, cell-derived sequences, probably in the C-terminal portion of the molecule. In two-dimensional tryptic peptide maps P170 of PRCIV is indistinguishable from the transformation-specific protein P170 of the previously described PRCII-p (M. L. Breitman, J. C. Neil, C. Moscovici, and P. K. Vogt (1981)Virology108, 1–12). Both P170 proteins also contain all but one tryptic peptide of the transformation-specific gag-linked protein P105 found in cells transformed by avian sarcoma virus PRCII. The P170 proteins of PRCIV and PRCII-p have an associated tyrosine-specific kinase activity similar to P105 of PRCII. All three agents, PRCII, PRCII-p, and PRCIV, belong to class II of avian sarcoma viruses. In cells transformed by PRCIV or PRCII-p secondary gag-linked transformation-specific proteins of less than 170,000 MW are sometimes seen, but limited in vivo and in vitro passage of the viruses shows the P170 to be a stable characteristic of PRCIV and PRCII-p transformation. The genomic RNA of PRCIV and PRCII-p sediments at 30 S corresponding to a size of about 6.1 kilobases.     

Recovered src genes are polymorphic and contain host markers

Analysis of recovered sarcoma viruses (rASV) and their parental sarcoma virus SR-D by oligonucleotide fingerprinting revealed multiple differences in the src region of the viral genomes. This heterogeneity was further investigated by tryptic peptide mapping of the in vitro translated products of rASV and SR-D RNA. No differences were found in the pr76^gag proteins encoded by the various rASVs or SR-D, but the p60^src proteins showed considerable variation. The p60^src proteins of rASV could be distinguished from that of SR-D on the basis of their mobility in SDS-polyacrylamide gels. Furthermore, two peptides which were absent from SR-D but consistently found in rASV p60^src proteins were also demonstrated in a tryptic peptide map of the cellular src-related protein, p60^sarc. These results provide strong support for the hypothesis that rASV arose by recombination of residual viral src sequences with cellular src-related sequences.     

Identification of a 39,000-dalton protein in cells transformed by the FBJ murine osteosarcoma virus

Two components of the FBJ murine osteosarcoma virus complex have been isolated separately in tissue culture; the FBJ murine leukemia virus (FBJ-MLV) by dilution and the FBJ murine sarcoma virus (FBJ-MSV) by the establishment of nonproducer transformed rat cells. Analysis of these cells using MLV antisera indicated that there were no new proteins related to viral structural proteins specifically associated with the presence of the FBJ-MSV genome. The FBJ-MSV nonproducer cells were used to induce tumors in syngeneic and allogeneic F₁ rats. Sera from tumor-bearing rats were examined for activity against FBJ-MSV-specific antigens. A number of sera were found to precipitate a 39,000-dalton protein, p39, from several producer and nonproducer FBJ-MSV transformed rodent cells, but not from cells transformed by other strains of MSV or cells infected with MLV. Precipitation of p39 was not blocked by the presence of excess viral proteins, indicating that p39 is not related to the viral structural proteins. This conclusion was confirmed by methionine tryptic peptide analysis which showed that the fingerprint of p39 was distinct from those of the viral gag or env gene proteins. The data demonstrate the presence of a unique antigenic protein, unrelated to the MLV proteins, in FBJ-MSV transformed cells.     

Immunoselection and characterization of moloney murine leukemia virus-infected cell lines deficient in surface gag antigen expression

NIH/3T3 cells infected with Moloney murine leukemia virus (M-MuLV) which were deficient in gag surface antigen were selected by incubation with anti-serum to the major gag virion protein, p30, in the presence of complement. Survivors of the selection were cloned and characterized with respect to intracellular production of gag and env gene products, gag surface antigen expression as revealed by indirect immunofluorescence, and virus production. Nineteen clones tested were all positive for env gene products in cytoplasmic extracts. Seventeen of the nineteen were positive for gag gene products, and two were negative. The gag-positive clones all produced Pr65^gag (the precursor to the internal structural proteins) and they also produced gPr80^gag (the precursor to the cell-surface gag antigen). The selected clones were all deficient in the presence of surface gag antigen as measured by immunofluorescent microscopy or flow microfluorimetry, and they all processed Pr65^gag to mature p30 more slowly than the parental cells. In addition, all of the surface gag-deficient clones produced virus at a reduced rate. The nature of the defect in one gag-deficient clone was studied by infection of progeny virus onto uninfected NIH/3T3 cells. The resultant cells showed normal gag surface fluorescence and virus production. This suggests that the defect was cellular rather than viral.