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.     

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.     

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.     

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.     

Partial characterization of a Moloney murine sarcoma virus 85,000-dalton polypeptide whose expression correlates with the transformed phenotype in cells infected with a temperature-sensitive mutant virus

The viral proteins specified by a Moloney murine sarcoma virus (Mo-MuSV) with a temperature-sensitive mutation in its transforming gene were examined. Normal rat kidney cells infected with this replication-defective virus have transformed cell characteristics at 33° but revert to a normal phenotype at 40°. At the temperature permissive for transformation, the cells contained an 85,000-dalton protein (P85) which had antigenic determinants of p15, pp12, and p30, and also tryptic peptides characteristic of p15 and p30 as well as additional unidentified tryptic peptides. P85 was only detectable at the permissive temperature. A 58,000-dalton protein (P58) was also detected. It had both antigenic determinants and tryptic peptides of p15, pp12, and p30. P58 was seen at both temperatures. Phosphorylation experiments indicated that P58 is a phosphoprotein whereas ³²P-labeled P85 was not observed. Temperature shift experiments showed that newly synthesized P85 was first detected between 2 and 3 hr following transfer of cultures to 33°. Morphological and biochemical changes indicative of transformation occurred 8 or more hr after temperature shift. These results are consistent with the interpretation that P85 contains peptide sequences derived from both the gag gene and the MuSV-acquired or src gene sequences.     

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.     

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.     

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.     

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.     

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.     

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 oncovirus proteins expressed on the surface of infected cells

Lactoperoxidase-catalyzed iodination and anti-AMV immunoprecipitation showed that both chicken and duck fibroblasts infected with a sarcoma virus (Rous sarcoma virus PrC) or a leukemia virus (avian myeloblastosis virus; AMV) had on their surface a protein of approximately 120 kilodaltons molecular weight (120K), as well as envelope glycoprotein precursors of 90–92 kd. Uninfected chicken fibroblasts of the gs^?, chf^? phenotype had much lower, but detectable amounts of surface 120K, whereas uninfected duck fibroblasts did not have any, suggesting a relationship between surface 120K and expression of chicken virus information in the cell. 120K is a glycoprotein, since it could be labeled with [³H]mannose and contained a component that bound to a concanavalin A affinity column. The 120K protein was characterized by tryptic fingerprinting after reiodination with chloramine-T. Total and Con A-selected 120K from infected chicken cells and total 120K from infected duck cells had essentially identical fingerprints. Moreover, they were extensively related to the iodinated fingerprint of Pr76^gag, the intracellular precursor of viral core proteins. These results indicate that expression on the cell surface of glycosylated forms of gag polyproteins occurs also in avian oncornavirus infections, similarly to findings in the murine leukemia virus system.     

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.     

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.     

Virus-specific precursor polypeptides in cells infected with Rauscher leukemia virus: Synthesis,identification, and processing

The synthesis of virus-specific polypeptides in JLS-V9 and JLS-V5 cells infected with Rauscher leukemia virus (R-MuLV) was studied in pulse-chase experiments, followed by radioimmunoprecipitation analysis with polyvalent and monospecific antisera against R-MuLV proteins. Two glycosylated polypeptides with molecular weights of about 8 (env-pr82) were identified as precursors of the virion envelope polypeptides gp69/71 and p15(E). On the other hand, virion polypeptides p30 and pl5 are derived from a 75,000-(gag-pr75) and a 65,000-dalton (gag-pr65) precursor polypeptide. These precursor-product relations were confirmed by analysis of chymotryptic digests of virion polypeptides and their precursors. In the presence of the arginine analog canavanine two polypeptides with molecular weights of 82,000 and 72,000 (gag-pr82 and gag-pr72, respectively) were synthesized instead of gag-pr75 and gag-pr65. Processing of precursor polypeptides is reduced in the presence of canavanine. From these results, we conclude that gag-pr82 is possibly the primary gag-gene product and is cleaved into gag-pr75. These studies provided the following additional information: First, we established that immediately after cleavage of their precursors, gp69/71 is found on the outer surface of the cell and p30, p15, and p12 leave the cell as components of budding virions. Therefore, these polypeptides were detected intracellularly in very small amounts only. Polypeptide p15(E) was present within the cell as well as on its outer surface. Second, despite a great similarity in virus-specific (precursor) polypeptides detected in JLS-V9 and JLS-V5 cells, small differences in molecular weights of some of these polypeptides were observed after SDS-PAGE.     

Isolation of a new feline sarcoma virus (HZ1-FeSV): Biochemical and immunological characterization of its translation product

A new strain of feline sarcoma virus, designated HZ1-FeSV, was isolated from a 4-year-old domestic cat with multicentric fibrosarcoma. A primary tumor cell line was established and virus produced from that line was found to induce foci in feline embryonic lung fibroblasts (FLF3) and mink lung fibroblasts (CCL64) in tissue culture and fibro-sarcomas in inoculated 10-week-old kittens. The derivation of transformed nonproducer clones of FLF3 and CCL64 cells containing helper virus-rescuable, focus-forming activity indicated that HZ1-FeSV was defective for replication. The only discernible translation product of the HZ1-FeSV genome in cultured cells was a 100,000-Da polyprotein (P100) which contained amino-terminal sequences of the FeLV gag gene precursor protein covalently linked to a sarcoma virus-specific domain. Immunoprecipitates containing P100 exhibited a protein kinase actvity capable of phosphorylating tyrosine residues of P100. Immunologically, P100 was highly cross-reactive with gag-fes polyproteins encoded by two previously characterized strains of FeSV, the GA- and the ST-FeSV. By comparison of methionine-containing tryptic peptides, the HZ1-FeSV protein was shown to be more closely related to the GA-FeSV protein than to the ST-FeSV protein, but to be distinguishable from both other proteins.     

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.