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.     

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.     

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.     

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.     

Comparative peptide analysis of avian oncoviral structural protein markers

The internal structural proteins of certain avian oncoviruses show strain-specific variation in their electrophoretic mobilities. The primary structures of these gag protein markers were compared by proteolytic digestion and two-dimensional peptide analysis. Specific differences in the peptide fingerprints of homologous p27, p19, and p15 proteins were detected. These differences were consistent with the observed electrophoretic properties of strain-specific structural protein markers and segregated with the markers in genetic recombinants. It is concluded that the altered electrophoretic properties of avian oncoviral gag :markers are due to small differences in amino acid sequences of corresponding proteins.     

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.     

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.     

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.     

Vicinal relationships between the major structural proteins of murine mammary tumor virus.

Spatial relationships between the major structural proteins of murine mammary tumor virus (MuMTV), a type-13 RNA tumor virus, were investigated. Untreated virions, when analyzed by diagonal gel electrophoresis, contained two major species of disulfide-linked protein oligomers, i.e., a high-molecular-weight aggregate of p18 and a dimer composed of gp34 and p28. These complexes are termed “native crosslinked oligomers.” In addition, induced crosslinking by dithiobis succinimidyl propionate (DTSP) of vicinal proteins of whole virions or rosettes (viral membranes with attached spikes or projections) was also investigated. Diagonal gel electrophoretic analyses of DTSP-crosslinked virions revealed dimers of gp55 · gp55 and gp55 · gp34 as well as the native crosslinked oligomers. No DTSP-induced crosslinking of internal viral proteins was observed. Similar studies with rosettes indicated the formation of DTSP-induced crosslinked dimers consisting of gp55·gp55 and gp34 · gp34 in addition to gp34 · p28. From these studies, we conclude that the MuMTV projection is a homooligomer of gp55 which is directly apposed to the transmembrane protein, gp34, on the external surface. In addition, a portion of the gp34 transmembrane protein interacts internally with the major core shell protein (p28) as a native crosslinked oligomer. The latter observation most probably accounts for the eccentric location of the MuMTV nucleoid. A model which derives from these studies is discussed vis-à-vis virus assembly and previous studies of MuMTV structure.     

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.     

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.     

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.     

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 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.     

Reduced infectivity of human immunodeficiency virus type 1 produced in the presence of a truncated Gag protein containing p7 gag and p6 gag

Summary.  The C-terminal portion of human immunodeficiency virus type 1 p55gag protein, p15gag, contains two functional proteins; p6gag which is required for incorporation of Vpr into the virion, and p7gag which binds to viral RNA and is necessary for packaging of genomic RNA into virions. p7gag protein overexpressed in trans may compete with wild type p55gag for binding to genomic viral RNA, thereby inhibiting incorporation of RNA into the virions. To investigate if overexpression of the C-terminal portion of p55gag could interfere with generation of infectious virus, a plasmid producing a protein consisting of p2gag, p7gag and p6gag, termed p15gag^*, was generated and cotransfected with an infectious proviral human immunodeficiency virus type 1 clone. Cells overexpressing p15gag^* in trans produced approximately 40 fold less infectious virus than cells lacking exogenous p15gag^*. These results demonstrated that expression of the C-terminal portion of p55gag efficiently reduced virus infectivity. Accepted March 11, 1998     

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.