Antigenic determinants of the 70,000 molecular weight glycoprotein of woolly monkey type C RNA virus.

The 70,000 molecular weight glycoprotein (gp70) of a type-C RNA virus originally isolated from a woolly monkey has been partially purified and immunologically characterized. Evidence that this viral protein is viral coded was derived from studies showing its antigenic properties to be unaltered by virus passage in cells of different species. A broadly reactive competition immunoassay was developed utilizing antiserum prepared against feline leukemia virus to precipitate 125I-labeled woolly monkey virus gp70. Gibbon and woolly viruses, as well as feline and several mouse type-C viruses, all reacted with equal efficiency in this assay. In contrast, an endogenous virus of the baboon failed to cross-react, suggesting that viruses of this latter group are less immunologically related to the others. In a homologous competition immunoassay for the woolly viral glycoprotein, the woolly virus was readily distingusihed from otherwise colsely related viruses of gibbon apes. These findings demonstrate the pronounced type-specific antigenic dterminants possessed by this viral protein. The antigenic determinants of gp70 responsible for neutralization have also been investigated.     

Translation of type C viral RNAs in Xenopus laevis oocytes: evidence that the 120,000-molecular-weight polyprotein expressed in Abelson leukemia virus-transformed cells is virus coded.

The genomic RNA of Abelson leukemia virus (AbLV) has been purified and translated in Xenopus laevis oocytes. The primary AbLV-specific protein synthesized is a polyprotein corresponding in molecular weight and immunological properties to a previously described p15 and p12 containing 110,000- to 130,000-molecular-weight polyprotein expressed in AbLV-transformed cells. In contrast, translation of woolly monkey sarcoma virus genomic RNA resulted in symthesis of a 55,000-molecular-weight polyprotein consisting of woolly helper virus p30, p15, and p12. These findings demonstrate the value of the X. laevis oocyte in vitro system for studies of translational products of replication-defective transforming viruses and establish the virus-coded nature of the nonstructural component of the 110,000- to 130,000-molecular-weight polyprotein expressed in AbLV-transformed cells.     

Search for mammalian type-C retrovirus polypeptides on infected animal cells and human tumor cell lines using interspecies-reacting antibodies

Cells producing endogenous and exogenous type-C retroviruses of murine, feline and primate origin were evaluated for expression of those virus-specific cell-surface antigens which cross-react with antibodies to interspecies determinants of mammalian type-C viral polypeptides. Surface polypeptides of cells replicating endogenous and exogenous type-C retroviruses were iodinated by the lactoperoxidase method. Labelled antigens were immunoprecipitated and analyzed in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. This method detected gp71 and less frequently p15E/p12E on the surface of virus-producing cells; in addition, p27 was found on F422 cells replicating feline leukemia virus. Antibodies to the membrane protein p15E displayed the broadest cross-reactivity but only antibodies to gp71 mediated complement-dependent interspecies cell lysis. The pattern of cross-reactivity reflected known genetic relationships among these mammalian viruses. Although antiserum to the simian sarcoma virus complex (SSV) was strongly cytotoxic to some human tumor cell lines, this reactivity could not be attributed to antibodies cross-reacting with SSV gp71.     

Expression of the Major Internal Viral Polypeptide in Cells Transformed by Wild-Type and Temperature-Sensitive Murine Sarcoma Virus

Phenotypic expression of the murine intraspecies and interspecies antigenic determinants of the major type C viral structural 30,000-dalton polypeptide, p30, was measured by radioimmunoassay inhibition in cell lines from different species. Uninfected normal rat kidney (NRK) cells did not contain detectable levels of murine intraspecies and interspecies p30 antigen, whereas rat cells transformed by and producing murine sarcoma virus (MSV)-Moloney leukemia virus (M-MSV-MuLV) contained high levels of both murine intraspecies and interspecies p30 antigen. Significant amounts of murine intraspecies and interspecies p30 antigen were detected in wild-type MSV-transformed nonproducer NRK cells. The control of p30 antigen expression was examined in temperature-sensitive MSV-transformed nonproducer cells [NRK(MSV-1b)] which are cold sensitive for maintenance of the transformed phenotype. Both murine intraspecies and interspecies p30 antigens were detected in NRK(MSV-1b) cells when grown at the permissive (39 C) or nonpermissive (33 C) temperature, suggesting that p30 antigen expression is not correlated with maintenance of the transformed phenotype. The results demonstrate that previously undetectable p30 antigens are expressed in MSV-transformed nonproducer NRK cells, and suggest that the expression of p30 antigen may be a useful marker for viral gene expression in mammalian cells.     

Comparative restriction endonuclease maps of proviral DNA of the primate type C simian sarcoma-associated virus and gibbon ape leukemia virus group.

Extrachromosomal DNA was purified from canine thymus cells acutely infected with different strains of infectious primate type C viruses of the woolly monkey (simian) sarcoma helper virus and gibbon ape leukemia virus group. All DNA preparations contained linear proviral molecules of 9.1 to 9.2 kilobases, at least some of which represent complete infectious proviral DNA. Cells infected with a replication-defective fibroblast-transforming sarcoma virus and its helper, a replication-competent nontransforming helper virus, also contained a 6.6- to 6.7-kilobase DNA. These proviral DNA molecules were digested with different restriction endonucleases, and the resultant fragments were oriented to the viral RNA by a combination of partial digestions, codigestion with more than one endonuclease, digestion of integrated proviral DNA, and hybridization with 3'- and 5'-specific viral probes. The 3'- and 5'-specific probes each hybridized to fragments from both ends of proviral DNA, indicating that, in common with those of other retroviruses, these proviruses contain a large terminal redundancy at both ends, each of which consists of sequences derived from both the 3' and 5' regions of the viral RNA. The proviral sequences are organized 3',5'-unique-3',5'. Four restriction enzymes (KpnI, SmaI, PstI, and SstI) recognized sites within the large terminal redundancies, and these sites were conserved within all the isolates tested. This suggests that both the 3' and 5' ends of the genomic RNA of these viruses are extremely closely related. In contrast, the restriction sites within the unique portion of the provirus were not strongly conserved within this group of viruses, even though they were related along most of their genomes. Whereas the 5' 60 to 70% of the RNA of these viruses was more closely related by liquid hybridization experiments than was the 3' 30 to 40%, restriction sites within this region were not preferentially conserved, suggesting that small sequence differences or point mutations or both exist throughout the entire unique portion of the genome among these viruses.     

Arrangement of Integrated Avian Sarcoma Virus DNA Sequences Within the Cellular Genomes of Transformed and Revertant Mammalian Cells

We have examined the arrangement of integrated avian sarcoma virus (ASV) DNA sequences in several different avian sarcoma virus transformed mammalian cell lines, in independently isolated clones of avian sarcoma virus transformed rat liver cells, and in morphologically normal revertants of avian sarcoma virus transformed rat embryo cells. By using restriction endonuclease digestion, agarose gel electrophoresis, Southern blotting, and hybridization with labeled avian sarcoma virus complementary DNA probes, we have compared the restriction enzyme cleavage maps of integrated viral DNA and adjacent cellular DNA sequences in four different mouse and rat cell lines transformed with either Bratislava 77 or Schmidt-Ruppin strains of avian sarcoma virus. The results of these experiments indicated that the integrated viral DNA resided at a different site within the host cell genome in each transformed cell line. A similar analysis of several independently derived clones of Schmidt-Ruppin transformed rat liver cells also revealed that each clone contained a unique cellular site for the integration of proviral DNA. Examination of several morphologically normal revertants and spontaneous retransformants of Schmidt-Ruppin transformed rat embryo cells revealed that the internal arrangement and cellular integration site of viral DNA sequences was identical with that of the transformed parent cell line. The loss of the transformed phenotype in these revertant cell lines, therefore, does not appear to be the result of rearrangement or deletions either within the viral genome or in adjacent cellular DNA sequences. The data presented support a model for ASV proviral DNA integration in which recombination can occur at multiple sites within the mammalian cell genome. The integration and maintenance of at least one complete copy of the viral genome appear to be required for continuous expression of the transformed phenotype in mammalian cells.     

Serological analysis of an oncornavirus (PMF virus) detected in malignant permanent human cell lines.

Immunodiffusion analysis of the PMF virus which was detected in malignant permanent human cell lines revealed positive reactions with antisera against the Mason-Pfizer monkey virus (MPMV). No cross-reactivity was demonstrated with murine leukemia virus (MuLV), rat leukemia virus (RaLV), hamster leukemia virus (HaLV), feline leukemia virus (FeLV), simian (woolly monkey) sarcoma virus (SSV-1) and mouse mammary tumor virus (MTV). The cross-reactive antigens of the PMF virus and the MPMV are considered as evidence for the human origin of the PMF virus.     

Transformation-defective virus mutants in a class of morphologic revertants of sarcoma virus transformed nonproducer cells

In studies of the viral and cellular functions involved in expression of transformation by murine sarcoma virus, selective methods have led to the isolation of morphologic revertants following mitomycin C mutagenization of nonproductively transformed mouse cells. The revertants exhibit normal growth properties, yet still contain the sarcoma virus. Further, they are as susceptible as normal cells to exogenous sarcoma virus infection. In the present studies, these revertants are shown to contain levels of sarcoma viral RNA quantitatively and qualitatively indistinguishable from that present in the parental transformed clone. Following rescue with helper leukemia virus, they release low levels of wild-type transforming virus and a large excess of transformation-defective sarcoma virus as measured by molecular hybridization. The defective viruses can be transmitted to new cells in the absence of morphologic alteration. These results provide strong evidence that the revertants contain mutant viruses defective in transforming functions. The release of wild-type sarcoma virus by cells in a revertant culture appears to occur concomitantly with the spontaneous appearance of retransformed cells. This suggests that the reversion of mutant virus to wild-type within the cell occurs as a result of reversion of a point mutation in the integrated sarcoma viral genome. The present sarcoma virus mutants appear to be the first obtained by spontaneous or chemically-induced genetic alteration of stably integrated virus in eucaryotic cells.     

A serologic comparison of Moloney lymphoma cell surface and Moloney oncornavirus antigens.

Moloney lymphomas and Moloney sarcomas share strong tumor antigens. In this report we analyze the cell-surface antigens on a Balb/c Moloney lymphoma, LSTRA, using hyperimmune sarcoma regressor sera (alphaMo) as a primary reagent. We also use heterologous anti-viral p30 and gp70 sera for a direct analysis of virion protein antigens on the LSTRA surface. Using radiolabeled alphaMo-binding assays, we demonstrate that LSTRA tumor antigens detected by these sera are all Moloney viral antigens; approximately 1/3 of these antigenic determinants are expressed on the intact virus, and the other determinants are revealed by detergent lysis of the virus. The major viral antigens expressed on the LSTRA cell surface are viral env gene products, whereas gag gene products are only sparsely represented. We conclude that alphaMo sera detect almost exclusively viral antigens on LSTRA cells, and these antigens are almost exclusively virion env gene products.     

The specificity of H-2-restricted cytotoxic T lymphocytes directed to AKR/Gross leukemia virus-induced tumors. III. Coordinate alterations in viral gp70 antigen expression and restoration of CTL-susceptibility to insusceptible variant tumors

Two variant subclones, called cl.18-5 and cl.18-12, were derived from the AKR.H-2bSL1 tumor cell line that were, in contrast to the parental cells, selectively insusceptible to H-2-restricted anti-AKR/Gross virus cytotoxic T lymphocytes (CTL). Cell surface expression of viral envelope (env) and group-specific antigens (gag) on these CTL-resistant variants were analyzed and compared with the expression of these antigens on AKR.H-2bSL1 and two other CTL-susceptible clones, cl.1 and cl.5, also derived from AKR.H-2bSL1. Although normal levels of gag-encoded and H-2 antigens were displayed on the CTL-resistant variants, the expression of five distinct determinants of viral gp70 antigen as defined by monoclonal antibodies was significantly decreased on these CTL-resistant variants relative to their expression on the CTL-susceptible cell lines. However, similar dramatic changes in cell surface gp70 antigen expression were undetectable as defined by anti-gp70-specific antiserum. Immunoprecipitation and gel electrophoretic analysis revealed that gp70 molecules from cl.18-5 cells had a lower m.w. than those of AKR.H-2bSL1, but there were no differences in the m.w. of gp70 antigens from AKR.H-2bSL1, cl.5, and cl.18-12 cells. Expression of the five gp70 antigenic determinants mentioned above was completely restored by exposure of cl.18-5 and cl.18-12 cells to the halogenated pyrimidine, iododeoxyuridine (IudR). Treatment of cl.18-5 and cl.18-12 cells with IudR simultaneously restored CTL susceptibility of these cells to anti-AKR/Gross virus CTL without affecting gag and H-2 antigen expression. Viral gp70 antigen immunoprecipitated from IudR-treated cl.18-5 cells had a mobility slightly lower, but different from that of untreated cl.18-5 cells. Pulse-labeling with [35S]-methionine showed that IudR treatment of cl.18-5 cells caused the expression of an additional high m.w. gp70 precursor protein originally absent in untreated cl.18-5 cells but present on parental AKR.H-2bSL1 cells. Collectively, these results pointed to the involvement of viral gp70 antigenic determinants in the recognition of AKR/Gross virus-induced tumor targets by anti-AKR/Gross virus CTL.     

RD-114, baboon, and woolly monkey viral RNA's compared in size and structure.

The molecular weights, subunit compositions, and secondary structure patterns of the RNAs from an endogenous baboon virus and from a woolly monkey sarcoma virus were examined and compared to the properties of the RNA of RD-114, an endogenous feline virus. The high molecular weight RNA extracted from each of these three viruses has a sedimentation coefficient of 52S, and a molecular length, measured by electron microscopy, of 16-20 kb (kb=kilobase, 1000 nucleotides). Each such RNA is a dimer, containing two monomer subunits of 8-10 kb in length (molecular weight 3 X 10(6) daltons). The two monomer subunits are joined at their non-poly(A) ends in a structure called the dimer linkage structure. The appearance of this structure is somewhat different for the different viruses. The dimer linkage dissociates at temperature estimated to be 87 degrees C in aqueous 0.1M Na+ for RD-114 and baboon viral RNAs, but at the lower temperature of 66 degrees C for woolly monkey RNA. All three viral RNAs have two large loops of similar size and position symmetrically placed on either side of the dimer linkage structure. Since the baboon virus is partially related to RD-114, and the woolly monkey virus is unrelated to either of the other two, the dimer linkage and symmetrical loops are surprisingly similar and may well be common features of type C virus RNAs.     

Antibodies to murine leukemia virus gp70 and p15(E) in sera of BALB/c mice immunized with syngeneic chemically induced sarcomas

The sera of BALB/c mice immunized with syngeneic methylcholanthrene-induced sarcomas commonly contain antibodies that react with the immunizing tumor line and also with other chemically induced sarcomas. Three lines of evidence suggest that the cross-reactive antibodies are directed against antigenic determinants of gp70 and p15(E), envelope proteins of endogenous murine leukemia virus (MuLV). 1) The antibodies bind only to sarcomas expressing MuLV antigens, 2) they are removed by absorption with purified MuLV antigens, and 3) they precipitate proteins identified as gp70 and p15(E) from 125I-labeled sarcoma cell lysates.     

Cellular changes in the thymuses of preleukemic AKR mice: correlation with changes in the expression of murine leukemia viruses.

Thymus cells of preleukemic and leukemic AKR mice express on their cell surface elevated levels of antigens associated with the murine leukemia virus (MuLV) proteins gp70 and p30. The gp70 antigenicity is contained in a 70,000 dalton polypeptide that corresponds to the viral envelope protein, while the p30 antigenicity is contained in two polypeptides of 85,000 and 95,000 daltons that correspond to glycosylated forms of the polyprotein product of the gag gene.The expression of these viral coded proteins on the cell surface of thymocytes varies both quantitatively with the age of the mouse and qualitatively with the cellular populations that express these antigens. Four discrete stages in the leukemic pathway can be identified. First, low numbers of cells from the thymuses of young (2 month old) AKR mice express p30 (<0.25%) and gp70 (2–7%) antigens. Expression of gp70 antigen is restricted to large cells in the subcapsular region of the thymus. Second, thymuses of 6 month old AKR mice show a selective depletion of cortical thymocytes with a concomitant increase in the medullary region of the thymus. Thymus cells of these mice contain elevated numbers of cells that express an increased concentration of p30 and gp70 antigens. Viral antigens are found on the surface of all large cells of the subcapsular region of the thymus, and in variable numbers (2–85%) of small cells of the cortical and medullary regions. Third, the thymuses of some 8 month old AKR mice demonstrate selective hypertrophy of a single thymic lobe. The enlarged lobe contains a population of cells that are intermediate in size between the small cortical cells and leukemic blast cells. This new cell population expresses elevated levels of p30 and gp70 viral antigens. These cells, which are not leukemic (since transfer of high numbers of these cells to syngeneic hosts does not induce transplantable disease), may represent preleukemic thymocytes. Fourth, thymuses of mice with overt leukemia contain primarily leukemic blast cells. These cells express extremely high levels of viral antigens on their cell surfaces, and upon transfer of these cells to syngeneic hosts, they rapidly induce transplantable leukemias.The increased expression of viral antigens on the surface of thymus cells is correlated with an increased production of infectious ecotropic and xenotropic MuLV in the thymus. During aging, the percentage of cells producing ecotropic MuLV increases 10-fold, while the percentage of cells producing xenotropic MuLV increases 100 fold.     

Primate origin of the cell-derived sequences of simian sarcoma virus

We sought to identify the species of origin of the cell-derived (sis) sequences of simian sarcoma virus. A molecular clone comprised of sis DNA detected related nucleotide sequences at low copy numbers in normal cellular DNAs of species as diverse as humans and quail. The extent of hybridization and degree of base-pair matching with sis DNA were greatest with New World primate DNAs. The thermal denaturation curve midpoints of hybrids formed between sis and woolly monkey DNAs were indistinguishable from homologous sis DNA hybrids, establishing the woolly monkey (Lagothrix spp.) as the source of sis sequences. In comparative studies, sis was shown to be more conserved among mammalian species than unique-sequence woolly monkey cellular DNA. There was no detectable homology between sis and the cell-derived sequences of other fibroblast-transforming retroviruses. These findings indicate that sis is likely to be a unique onc gene among transforming retroviruses.