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.     

Mutants of Rous sarcoma virus with extensive deletions of the viral genome.

Deletion mutants of Rous sarcoma virus (RSV) have been isolated from a stock of Prague RSV which had been irradiated with ultraviolet light. Quail fibroblasts were infected with irradiated virus and transformed clones isolated by agar suspension culture. Three clones were obtained which did not release any virus particles. Analysis of DNA from these non-producer clones with restriction endonucleases and the Southern DNA transfer technique indicated that the clones carry defective proviruses with deletions of approximately 4 × 10⁶ daltons of proviral DNA. The defective proviruses, which retain the viral transformation (src) gene, contain only 1.7–2.0 × 10⁶ daltons of DNA. Multiple species of viral RNA containing the sequences of the src gene were detected in these clones; some of these RNAs may contain both viral and cellular sequences. The protein product of the src gene, p60^src (Brugge and Erikson, 1977), was also synthesized in the nonproducer clones. However these clones did not contain the products of the group-specific antigen (gag), DNA polymerase (pol), or envelope glycoprotein (env) genes, nor did they contain the 35 and 28 S RNA species which are believed to represent the messengers for these viral gene-products. The properties of these mutants indicate that expression of the src gene is sufficient to induce transformation. These clones may represent useful tools for the study of the expression of this region of the genome.     

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.     

A nonconditional replication-defective mutant of the Schmidt-Ruppin strain of Rous sarcoma virus.

A nonconditional mutant of the Schmidt-Ruppin strain of Rous sarcoma virus (subgroup A) that fails to synthesize infectious progeny virus is described. Nonproducing cells transformed by this mutant LA7365 have functional viral src and env genes. The gag gene of LA7365 is defective. There is no complementation with a gag ts mutant, and pulse-chase experiments show that the proteolytic processing of the gag precursor pr76 is strongly inhibited in mutant-infected cells. Whether there is an additional lesion in the pol gene of LA 7365 could not be determined from the available data.     

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.     

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.     

Genetic analysis of the defectiveness in strain MC29 avian leukosis virus.

Avian myelocytomatosis virus MC29 is defective in replication. The extent of this defectiveness was analyzed in complementation experiments. Continuous nonproducer quail cell lines transformed by MC29 were superinfected with different helper viruses. Infectious MC29 pseudotypes were formed only with helper viruses which belonged to the avian leukosis-sarcoma virus complex, e.g., Rous-associated virus type 1 (RAV-1) or ring-necked pheasant virus (RPV). Helper viruses of other retroviral species, such as reticuloendotheliosis virus (REV), golden pheasant virus (GPV), or an amphotrophic murine leukemia virus (MuLV-1313), could not rescue MC29 from these nonproducer cells but complemented the envelope-defective Bryan high titer strain of Rous sarcoma virus. Host range and interference patterns of MC29 rescued from nonproducer cells indicated that the envelope specificity of the pseudotypes was determined exclusively by the activating helper virus. Cocultivation or Sendai virus-induced fusion of MC29 nonproducer cells with quail or chicken cells transformed by the defective Bryan high titer strain of Rous sarcoma virus did not result in complementation of the defects in either of the two viruses. Fluorescent antibody staining failed to detect virus-specific antigens at the surface of MC29 nonproducer cells. Temperature-sensitive mutants of Rous sarcoma virus with lesions in the genes coding for the RNA-directed DNA polymerase (pol gene) or the nonglycosylated structural proteins (gag gene) did not acquire wild-type characteristics when grown in MC29 nonproducer cells. It is concluded that MC29 is defective in all three genes essential for the replication of retroviruses, namely, env, pol, and gag.     

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.     

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.     

Recombinant avian oncoviruses. I. Alterations in the precursor to the internal structural proteins.

The synthesis of the gag precursor protein (Pr76) was studied in a number of recombinant avian oncoviruses, which were selected for recombination between the env and pol genes or the env and src genes. Such studies show that the electrophoretic mobility of the gag precursor protein of recombinant viruses (ΔPr76) was greater than that of the parental gene product (Pr76) in 16 of 24 cases. Viruses derived from recombination between endogenous (RAV-0) and exogenous viruses (RSV), as well as between two exogenous viruses, showed the ΔPr76 phenotype. In an mRNA-dependent rabbit reticulocyte translation system, 35 S RNA isolated from PR-RSV-C directed the synthesis of Pr76, while RNA isolated from a recombinant between PR-RSV-C and RAV-0 directed the synthesis of ΔPr76. These observations show that the synthesis of ΔPr76 is due to an alteration in the genome related to recombination. An analysis of the RNase T₁-resistant oligonucleotides demonstrated a crossover near the 5′ end of the genome (which may be within the gag gene) in two recombinant virus clones which synthesize ΔPr76 in infected cells; but no crossover was detected near the 5′ end of the genome in a third recombinant virus clone which synthesizes Pr76 in infected cells. Our data suggest that the synthesis of ΔPr76 is a consequence of recombination near the 5′ end of the genome.     

A comparison of avian and murine retrovirus polyprotein cleavage activities

The murine leukemia virus Pr65^gag proteolytic activity (MuLV-PF) which processes Pr65^gag to murine gag-specific polypeptides and the avian tumor virus p15-associated protease (AMV-p15) which likewise processes the avian gag polyprotein, differ substantially in their detergent, pH, and salt requirements for optimal activity. These differences are consonant with reports that MuLV-PF is associated with a serine protease (Y. Yoshinaka and R. B. Luftig, 1977, Cell12, 709–719) while AMV-p15 has a thiol protease-like activity (K.J. Dittmar and K. Moelling, 1978, J. Virol.28, 106–118). In spite of these differences, in vitro cleavage of MuLP Pr65^gag (NH₂-p15-P12-p30-p10-COOH) by AMV-p15 can be achieved. The initial cleavage products observed are polypeptides of M_r 45,000 daltons (45K) and M_r 15K. The 15K polypeptide cross-reacts with MuLV p15 antisera while the 45K polypeptide possesses antigenic determinants of p30 and p12 but not p10. The 45K polypeptide thus differs from Pr40^gag, the intermediate cleavage product obtained after treatment of Pr65^gag with the murine leukemia virus proteolytic activity; Pr40^gag contains only p30 and p10 determinants (Y. Yoshinaka and R.B. Luftig, 1977, Biochem. Biophys. Res. Commun.79, 319–325). AMV-p15 further cleaves the 45K polypeptide to one with M_r 39K which has the group-specific antigenic determinant of p30 but not p12. At higher concentrations of AMV-p15, a more complete breakdown to polypeptides of M_r 12–15K, without any buildup of p30, is observed. These results suggest that there are at least four thiol protease-like sites on Pr65^gag: one very near the COOH terminus at p15 which makes it a possible in vivo cleavage site, and three other sites at the interior of polypeptides p10, p12, and p30.     

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.     

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.     

Gene order of murine mammary tumor virus gag proteins and env proteins

The gene orders of murine mammary tumor virus (MuMTV) gag proteins and env proteins have been determined by pactamycin mapping techniques. A MuMTV producing cell line, Mm5mt, was pulse-labeled with [³¹S]methionine in the presence or absence of 5 × 10^?7 M pactamycin, an inhibitor of initiation of protein synthesis. Both pactamycin and the labeled amino acid were removed after the pulsing period and cells were further incubated in normal growth medium. Virus was harvested from the medium after 24 hr, and the individual protein constituents of the virus were analyzed by SDS-gel electrophoresis. For each protein, the ratio of the radioactivity incorporation in the presence of pactamycin and in its absence was determined. The lower this ratio, the closer would be the protein to the amino terminal of the precursor polyprotein. The following gene orders were derived from these experiments: gag, NH, p10(p28,pp23)p14 COOH, and env, NH₂ gp52gp36 COOH.     

Endogenous murine leukemia virus-encoded proteins in radiation leukemias of BALB/c mice

To explore the role of endogenous retroviruses in radiation-induced leukemogenesis in the mouse, we have examined virus-encoded proteins in nine BALB/c leukemias by pulse-chase labeling procedures and serological typing with monospecific and monoclonal antibodies. The major gag precursor protein, Pr65^gag, was observed in all cases, but only three leukemias expressed detectable amounts of the glycosylated gag species, gP95^gag, or its precursor, Pr75^gag. No evidence was found for synthesis of gag-host fusion proteins. None of the leukemias released infectious xenotropic or dualtropic virus, but all nine expressed at least one env protein with xenotropic properties. In two instances a monoclonal antibody, $\text{35}\text{56}$, which is specific for the MuLV G_IX antigen, displayed a distinctive reactivity with this class of env protein, although this antibody is unreactive with replicating xenotropic viruses. An ecotropic/xenotropic recombinant env protein with the same $\text{35}\text{56}$ phenotype was observed in a leukemia induced by a strongly leukemogenic virus isolated from a BALB/c radiation leukemia.     

Genome structure of the defective avian sarcoma virus PRCIV

The genome of PRCIV, a defective avian sarcoma virus of class II, was studied by nucleic acid hybridization and oligonucleotide mapping. It has a length of about 6.1 kilobases (kb). A stretch of retrovirus replicative sequences encompassing part of the gag, all of the pol, and most of the env region, in all about 5 kb, is deleted. In its place a 2.9 kb substitution that is transformation-specific for PRCIV was found. Flanking the PRCIV-specific substitution are sequences shared with the helper virus, a stretch of 2.0 kb on the 5′-side, including part of the gag region, and a stretch of up to 1 kb on the 3′-side, including the C-region and possibly some env sequences. A PRCIV-specific cDNA probe representing the transformation-specific sequences was prepared. This probe hybridizes only with RNAs of other class II sarcoma viruses but fails to hybridize with either class I or class III sarcoma viruses.