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.     

Gene order of mouse mammary tumor virus precusor polyproteins and their interaction leading to the formation of a virus.

Mouse mammary tumor virus (MMTV) proteins are synthesized as two major precursor polyproteins; gPr75^env containing gp52 and gp36, and Pr75^gag containing p27, pp20, p14, and p10. The gene order for gPr75^env has been previously shown to be H₂N-gp52-gp36-COOH (Schochetman, et al., 1977). gag polyproteins undergo intracellular cleavage in cat cells infected with MMTV and GR mammary tumor cells. Based on immunoprecipitation studies with antisera against intermediate MMTV cleavage products we now report the gene order for Pr75^gag is H₂N-p10-pp20-p27-p14-COOH. These results were further substantiated by analyzing the binding to ssDNA of the intermediate cleavage products which contain p14. To analyze the interaction of MMTV proteins with the cell membrane leading to budding of a virus particle, we used (i) lactoperoxidase-catalyzed iodination of MMTV cell surface proteins, (ii) galactose oxidase-catalyzed radiolabeling of carbohydrates on cell surface MMTV glycoproteins, (iii) serum cytotoxicity based on [⁵¹Cr] release with monospecific MMTV antisera, and (iv) membrane immunofluorescence with monospecific MMTV antisera. Analysis of ¹²⁵I-labeled MMTV cell surface antigens by immune precipitation with MMTV anti-gp52, gp36, p27, p14, and p10 sera followed by SDS-PAGE revealed only ¹²⁵I-gp52. In contrast, cell surface glycoprotein labeling revealed [³H]gp52 and [³H]gp36, indicating that, although the protein portion of gp36 was buried, some carbohydrate regions were exposed. EDTA treatment of cells to alter cell membranes prior to iodination resulted in the labeling of both Pr75^gag and gp52 but not gPr75^env. Furthermore, anti-p10 but not anti-p27 serum was cytotoxic against EDTA-treated cells. Similar results were obtained when the same antisera were tested by membrane immunofluorescence, ruling out the possibility that anti-p27 serum was not cytotoxic because it was unable to fix complement. These results show that Pr75^gag molecules, presumably as MMTV cores, interact with cell membrane sites containing gp52 and gp36 via the hydrophobic p10 portion of the molecule.     

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.     

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.     

Cell-free synthesis of Rauscher murine leukemia virus "gag" and "env" gene products from separate cellular mRNA species.

RNA from cells infected with Rauscher murine leukemia virus (R-MuLV) has been translated in an mRNA-dependent cell-free protein synthesizing system. It was found that a cellular RNA species of about 35 S in size codes for polypeptides of approximately 65,000 MW (Pr65^gag) and 200,000 MW (Pr200^gag) which are immunoprecipitable with antisera directed against the R-MuLV gag proteins p30, p15, p12, and p10. The methionine-containing-tryptic peptides of the 65,000 MW polypeptide translated from cellular 35 S RNA were identical to those of authentic Pr65^gag. Translation of RNA in the 25–35 S size class suggests that while Pr65^gag can be translated by RNA throughout this size range, Pr200^gag-pol translation is restricted to mRNA which sediments at 35 S. Antiserum directed against the R-MuLV envelope protein gp69/71 recognized a polypeptide of 68,000 MW, designated Pr68^env, which was coded for by RNA which sedimented at about 22 S in sucrose gradients and which had a minimum size of about 1.25 × 10⁶ daltons as estimated by agarose gel electrophoresis. Tryptic maps of Pr68^env showed it to contain all of the methionine-labeled tryptic peptides and most of the tyrosine-containing tryptic peptides characteristic of gPr90^env the authentic R-MuLV glycosylated envelope precursor.     

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.     

Two-dimensional analysis of murine leukemia virus gag-gene polyproteins.

The processing of gag translational products in a Gross Murine Leukemia virus (MuLV)-induced leukemia (E λ G2) was studied with two-dimensional gel electrophoresis, combining separation based upon charge in the first dimension and separation based upon size in the second dimension. In most experiments, the gag species were compared to the env species; gag species were precipitated from labeled cells or virus with antisera to the virion gag proteins p30 or p10, whereas env species were precipitated from labeled cells or virus with anti-gp70 serum. Three viral proteins were detected on the surface of E λ G2 cells with [¹²⁵I] lactoperoxidase labelings: these included gp70 and two glycosylated gag gene species (gpP95^gag and gpP85^gag). Neuraminidase treatment of [¹²⁵I] lactoperoxidase-labeled cells did not affect the antigenicity of gp70, gpP95^gag, or gpP85^gag. However, the neuraminidase treatment caused gp70, gpP95^gag, and gpP85^gag to migrate as more basic species, indicating that all three glycoproteins contain terminal sialic acid. The cytoplasmic gag-gene products were studied with [³⁵S]methionine labelings of E λ G2 cells; seven relatively stable gag species were identified. In general, none of the gag intermediates were single proteins; rather, each of the species exhibited multiple, specific modifications that resulted in complex yet reproducible patterns in the two-dimensional gel system. The core polyproteins Pr75^gag and Pr65^gag were formed rapidly after pulse-labelings, with Pr65^gag being processed into Pr55^gag involving cleavage of p10. The smaller gag species (Pr45^gag and p30) also appeared to result from processing of Pr65^gag. In contrast, Pr75^gag was directly processed to form gpP95^gag. A protein of approximately 58,000 daltons, designated P58^gag, qualified as a gag species since it was specifically precipitated by anti-p30 serum. However, P58^gag did not appear to be a precursor of p30 since it was long-lived in the cytoplasm. Multiple forms of p30 were precipitated from the cytoplasm and from the virion, with unique forms of p30 present in both the cytoplasm and the virion. Comparisons of the gag species from several AKR leukemias indicated that similar, but not identical gag gene products were present in the various leukemias.     

Effect of interferon on the exogenous Friend murine leukemia virus infection

Interferon treatment (600 U/ml) of NIH/3T3 cells induced greater than 90% reduction in the de novo production of Friend MuLV when measured 24 hr postinfection. Early events in viral replication such as the synthesis of proviral DNA and its subsequent integration into the cell genome were not inhibited by interferon treatment indicating that the suppression of virus production by interferon appears to occur after synthesis of proviral DNA. Analysis of viral RNA species present in controls and interferon-treated cells 24 hr after infection show that the same RNA species were present in the presence and absence of interferon. Synthesis of viral polypeptides was reduced but not blocked in interferon-treated cells when measured within 24 hr after infection while processing of gag precursor, Pr65^gag, and glycoprotein precursor, gPr85^env, to viral proteins was not altered. Phosphorylation of viral protein p12 but not that of the precursor, Pr65^gag, was inhibited in newly infected interferon-treated cells. In contrast to the first replicative cycle, interferon did not inhibit synthesis of viral proteins, and phosphorylation of p12 in those cells chronically infected with F-MuLV.     

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.     

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 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.     

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.     

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.     

Translation of mouse mammary tumor virus RNA: precursor polypeptides are phosphorylated during processing.

The synthesis of viral polypeptides of the mouse mammary tumor virus (MMTV) was studied by pulse-labeling of MMTV-producing cells and by translating MMTV virion RNA in vitro, in Xenopus laevis oöcytes. Virus-related polypeptides were detected by means of immunoprecipitation withm monospecific antisera against the major viral proteins gp49 and p24 and analysis of the immunoprecipitates on polyacrylamide gels. In pulse-labeled MMTV-producing cells (Mm5mt/c1), a precursor polypeptide of 73,000 daltons was immunoprecipitated by anti-p24 serum (Pr73^gag). Pr73^gag co-migrated with the 73,000-dalton glycosylated precursor for the envelope proteins (Pr73^env) immunoprecipitated by anti-gp49 serum.Pr73^gag was, during chase, converted into a 76,000-dalton polypeptide, also reacting with the anti-p24 serum (Pr76^gag). After prolonged incubation, the mature internal protein p24 was synthesized. Pulse-labeling with ³²P and subsequent chasing revealed that phosphate was incorporated into Pr76^gag and not into Pr73^gag. Isolated virion 70 S RNA of MMTV, microinjected into Xenopus oöcytes, gave rise to synthesis of Pr73^gag, Pr76^gag, and p24, all immunoprecipitated by anti-p24 serum, and the viral core proteins p14 and p10, precipitated by polyvalent anti-MMTV serum. 70 S RNA did not instruct synthesis of the viral envelope glycoproteins.     

p65 of Gazdar murine sarcoma viruses contains antigenic determinants from all four of the murine leukemia virus (MuLV) gag polypeptides (p15, p12, p30, and p10) and can be cleaved in vitro by the MuLV proteolytic activity

Thin-section electron micrographs of Gazdar murine sarcoma virus (Gz-MSV) particles showed that 100% of the particles possessed an immature morphology. Correspondingly, p65 (the major 65,000-dalton protein observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis for Gz-MSV particles) possessed antigenic determinants from all four of the murine leukemia virus (MuLV) Pr65^gag polypeptides—that is, p30, p15, p12, and p10. This result is in contrast to earlier observations (A. Pinter and E. deHarven (1979), Virology, 99, 103–110) which reported that p65 lacked antigenic determinants of MuLV p10. It is consistent with the recent finding of Maxwell and Arlinghaus ((1981), J. Virol., 39, 963–967) that Gz-MSV p65, when cleaved in vitro, gives rise to a polypeptide with the size and antigenic determinant of MuLV p10. Thus, we suggest that Gz-MSV p65 should be designated as Gz-MSV Pr65^gag. We also found that Gz-MSV Pr65^gag could be cleaved in vitro by using a partially purified proteolytic factor that had been derived from Moloney murine leukemia virus (M-MuLV) by Sephadex G-75 column chromatography (Y. Yoshinaka and R. B. Luftig (1980), J. Gen. Virol., 48, 329–340). Protein bands were produced that migrated on gels and had the same antigenic determinants as the MuLV intermediates Pr40^gag (p30, p10) and Pr27^gag (p15, p12). Pr55^gag (p15, p12, p30), a minor component, was also produced. Additional incubation of Gz-MSV Pr65^gag led to a breakdown of the intermediate polyproteins into the four MuLV gag polypeptides p30, p10, p15, and p12. The final processing of Pr55^gag and Pr40^gag occurred more rapidly than that of Pr27^gag. It thus seems that in vitro sequentially different processing events are involved in production of the four internal gag antigens from Gz-MSV Pr65^gag.