Interspecies Antigenic Determinants Research Articles

Major structural polypeptides of retrovirus produced by a Suncus murinus mammary tumor cell line and type D retrovirus share interspecies antigenic determinants

Constitutive polypeptides of retrovirus produced by a Suncus murinus mammary tumor cell line (Sm-MTV) were characterized by immunofluorescence and immunoblotting. The major structural polypeptide of Sm-MTV, molecular weight 44 43 K , immunologically cross reacted with the structural polypeptides of Mason-Pfizer monkey virus (MPMV) (molecular weights 27K and 12 10 K ). Sm-MTV constitutive polypeptides did not show antigenic relatedness with those of the following mammalian retroviruses: murine mammary tumor virus, Rausher murine leukemia virus, Molony murine leukemia virus, Osborn-Mendel rat leukemia virus, feline leukemia virus, RD114 virus, simian sarcoma virus 1, and human T-cell leukemia virus type I. Among the Sm-MTV structural polypeptides, those with molecular weights of 72 69 K and 47K were glycosylated.

Detection of group and interspecies reactivities of mammalian C-type virus p30 proteins by an enzyme-linked immunosorbent assay (ELISA): Enhancement of interspecies reactivity by denaturation

The applicability of the enzyme immunoassay technique to the detection of group and interspecies determinants of C-type retroviral proteins was tested. For this purpose four groups of C-type retroviruses (MuLV, FeLV, SiSV/GaLV, and BaEV/RD114) were assayed for group-specific and interspecies reactivities of their p30 proteins by an enzyme-linked immunosorbent assay (ELISA). We found that the ELISA can detect group-specific as well as interspecies determinants with sensitivity and reproducibility in purified p30 proteins, disrupted viruses, and cell extracts if an anti-p30 interspecies antiserum is used. If monospecific antisera against MuLV p30, SiSV p30, or BaEV p30 were used, only group-specific reactivities were detected reproducibly, whereas the detectability of interspecies determinants depended on the antisera used and varied even with the same antisera. In assays in which the reactivity of native and denatured p30 proteins was compared the detectability of sodium dodecyl sulphate-denatured MuLV p30 was better than that of native MuLV p30 suggesting that some of the most broadly cross-reactive sequences are localized inside the protein molecule and are freed by the denaturation process. Antisera raised against native and denatured p30 proteins showed identical spectra of reactivity.

Immunochemistry of the moloney MuLV gp71 molecule—I. Distribution of antigenic determinants on the products of digestion by Staphylococcus aureus protease

The antigenic characterization of Moloney murine leukemia virus gp71 was carried out by cleavage with Staphylococcus aureus protease. The whole molecule and two fragments of 43,000 and 32,000 daltons were immunoprecipitated with monospecific anti-type, group and interspecies antiserum. The three native molecules were recognized by anti-type antibodies, but only the gp71 and the 32,000-dalton molecule seemed to retain group-specific antigenic determinants. No interspecies determinants were detected on the two products of digestion. A similar pattern of precipitation was found with SDS-denatured antigens. The results suggest that sequential structures contribute to the type-specific antigenic determinants, because they were not destroyed by SDS unfolding or by the change in the protein conformation that follows enzymatic cleavage. Interspecies determinants seem to be mainly conformational, since they disappeared from the whole molecule and the two peptides after the same treatment. Group-specific determinants appear to be intermediate: they disappeared after the conformational change on the native 43K but not on the 32K peptide and the monospecific anti-group antibody showed an intermediate ability to precipitate the SDS-unfolded antigens.

55,000-dalton, retrovirus-associated, cell membrane glycoprotein: purification and quantitative measurements of expression in viruses, cells, and tissues.

We have purified to homogeneity and characterized a 55,000-dalton rat cell membrane glycoprotein, gp55. This protein was originally identified in preparations of a defective pseudotype of the Kirsten sarcoma virus and shown to be present in several rodent retrovirus particles. The gp55 was purified from this defective virus by concanavalin A and heparin affinity chromatography, as well as by preparative sodium dodecyl sulfate-gel electrophoresis. Both preparations displayed similar purity and antigenic characteristics. The 125I-labeled gp55 was precipitated by antisera against rodent retroviruses, but not by monospecific antisera against purified type C virus structural proteins, thus indicating that gp55 was retrovirus associated, but unrelated to known retrovirus structural proteins. Competition radioimmunoassay with an anti-rat virus serum which recognized rodent group-specific antigens on gp55 indicated: the presence of gp55 antigens in 15 rodent cell lines, but not 10 nonrodent cell lines; no effect of viral infection or cell transformation on the amount of gp55 expressed; up to 100-fold increases in the concentration of the gp55 antigens in nine rodent retroviruses, but not in five nonrodent viruses, as compared to cells; the presence of gp55 in rodent sera, especially of the NZB mouse, where anti-gp55 antibody was also detected; a lymphoid and epithelial tissue distribution of gp55 in rats and mice. Additional competition radioimmunoassays with a broad-reacting antivirus serum also detected the presence of gp55 in nonrodent, mink, and human cells and thus distinguished rat type, rodent group, and interspecies antigenic determinants on gp55. In conclusion, gp55 is a cell membrane glycoprotein associated in high concentration with retroviruses.

55,000-Dalton, Retrovirus-Associated, Cell Membrane Glycoprotein: Purification and Quantitative Measurements of Expression in Viruses, Cells, and Tissues

We have purified to homogeneity and characterized a 55,000-dalton rat cell membrane glycoprotein, gp55. This protein was originally identified in preparations of a defective pseudotype of the Kirsten sarcoma virus and shown to be present in several rodent retrovirus particles. The gp55 was purified from this defective virus by concanavalin A and heparin affinity chromatography, as well as by preparative sodium dodecyl sulfate-gel electrophoresis. Both preparations displayed similar purity and antigenic characteristics. The 125I-labeled gp55 was precipitated by antisera against rodent retroviruses, but not by monospecific antisera against purified type C virus structural proteins, thus indicating that gp55 was retrovirus associated, but unrelated to known retrovirus structural proteins. Competition radioimmunoassay with an anti-rat virus serum which recognized rodent group-specific antigens on gp55 indicated: the presence of gp55 antigens in 15 rodent cell lines, but not 10 nonrodent cell lines; no effect of viral infection or cell transformation on the amount of gp55 expressed; up to 100-fold increases in the concentration of the gp55 antigens in nine rodent retroviruses, but not in five nonrodent viruses, as compared to cells; the presence of gp55 in rodent sera, especially of the NZB mouse, where anti-gp55 antibody was also detected; a lymphoid and epithelial tissue distribution of gp55 in rats and mice. Additional competition radioimmunoassays with a broad-reacting antivirus serum also detected the presence of gp55 in nonrodent, mink, and human cells and thus distinguished rat type, rodent group, and interspecies antigenic determinants on gp55. In conclusion, gp55 is a cell membrane glycoprotein associated in high concentration with retroviruses.

Feline sarcoma virus P115-associated protein kinase phosphorylates tyrosine. Identification of a cellular substrate conserved during evolution.

The Gardner strain of feline sarcoma virus (FeSV) has previously been shown to encode as its major translational product, a phosphoprotein of molecular weight (Mr) = 115,000 (P115) with an associated protein kinase activity and probable transforming function. In the present study, we demonstrate that this enzyme specifically phosphorylates tyrosine and that Gardner FeSV-transformed mink and rat cells exhibit constitutively higher levels of phosphotyrosine than nontransformed control cells. In addition, we identify a Mr = 150,000 cellular substrate for the FeSV P115-associated protein kinase activity. This latter protein, designated P150, is expressed in cells of a number of mammalian species including cat, mink, dog, rat, mouse and human, possesses broadly reactive interspecies antigenic determinants, and exhibits binding affinity for FeSV P115. Although P150 exhibits an associated protein kinase activity, this activity differs from the FeSV P115-associated enzyme in that it phosphorylates serine and threonine rather than tyrosine. On the basis of immunologic criteria and tryptic peptide analysis, the possibility that P150 is structurally related to Gardner FeSV P115 is excluded. As a consequence of its affinity for FeSV P115, P150 is efficiently incorporated into FeSV pseudotype virions, providing a source for its purification and preparation of antisera for study of its expression in mammalian cells.

Antibodies against an interspecific erythrokaryocyte antigen in patients with partial red cell aplasta of the bone marrow

Immunofluorescense and cytotoxicity test in vitro were used to demonstrate specific antibodies in sera of 11 out of 19 patients with partial red cell aplasia (PRCA). The antibodies reacted with erythroblast cells from embryos and adult men, with bone marrow cells from a female patient suffering from acute erythroleukemia, with erythrokaryocytes of mouse embryos and cells of Rauscher's viral erythroleukemia. The results of cross adsorption and blockade of the immunofluorescent reaction of the sera of PRCA patients with antibodies against mouse erythroblast antigen bearing an interspecies determinant suggest that in the pathogenesis of PRCA there takes part an autoimmune reaction against specific interspecies antigen to erythrokaryocytes. This antigen is apparently similar to antigen against mouse erythroblast cells.

Effect of chemical modification and fragmentation on antigenic determinants of internal protein p30 and surface glycoprotein gp70 of type C retroviruses.

The effects of protein modification on the antigenic determinants of p30 and gp70 of type C retroviruses were investigated by using solid-phase competition radioimmunoassays. Proteins were modified by reduction with 2-mercaptoethanol and subsequent carboxymethylation of SH groups with iodoacetamide or by amidination of alpha and epsilon amino groups with methylacetimidate. The type-specific determinants of gp70 were found to be conformational in nature, as they were destroyed by these chemical modifications. Group- and interspecies-specific determinants of gp70 antigens, however, appear to be sequential and do not involve residues susceptible to these chemical reagents. Conformation-dependent type-specific determinants of p30 were affected only by methylacetimidate. Group- and interspecies-specific determinants of p30 are similar to those of gp70 in that they also appear to be sequential antigenic sites. Therefore, the broadly reactive group- and interspecies-specific determinants of gp70 and p30 can be followed into small peptides. Accordingly, a cyanogen bromide cleavage fragment derived from the carboxyl-terminal one-third of Rauscher leukemia virus p30 was found to carry group-specific determinants but no detectable interspecies-specific determinants. In contrast, a peptide obtained by limited trypsin cleavage of p30, which was derived from the NH(2)-terminal region of the protein, contained at least one of the interspecies determinants shared with feline leukemia virus p27.

Major structural proteins of type B, type C, and type D oncoviruses share interspecies antigenic determinants.

Interspecies antigenic determinants have been used as markers to study the evolution of oncoviruses. By radioimmunologic techniques, it has been possible to detect crossreactivities among the major structural proteins of prototype viruses representing each of the three major Oncovirinae genera. Two different subsets of interspecies antigenic determinants were demonstrated. One encompasses known type B and type D viruses; the other is shared by type D and mammalian type C viruses. Such determinants were also demonstrated in certain as yet unclassified oncoviruses. These findings provide experimental evidence supporting the concept of an evolutionary relationship among the three major oncovirus genera.

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.

Radioimmunoassays for the 36,000-dalton glycoprotein of murine mammary tumor viruses demonstrate type, group, and interspecies determinants.

Mouse mammary tumor viruses (MMTVs) contain distinct membrane glycoproteins of 52,000 daltons (gp52) and 36,000 daltons (gp36). We report here the development of new radioimmunoassays for gp36, using gp36 purified by hydrophobic chromatography and gel filtration. These assays demonstrate that gp36 has both type-specific and group-specific antigenic determinants. The virus-coded nature of these determinants was shown by utilizing different MMTVs grown in the same feline cell line. Interspecies determinants on gp36 were demonstrated by the observations that (i) MC-MTV (a virus isolate from the Asian rodent Mus cervicolor, and morphologically identical to MMTVs) competed, with an altered slope, in the gp36 radioimmunoassay, and (ii) antisera raised against MC-MTV immunopreciptitated 125I-labeled gp36. The detection of gp36 in spontaneous mammary tumors of several strains of mice also facilitates further studies on the replication of MMTVs and the host's immune response to MMTV-mediated oncogenesis.

Antigenic marker of human erythrokaryocytes similar to mouse erythroblast antigen

Identical antigenic determinants are discovered on the surface of human erythrokaryocytes with antibodies against specific antigen of murine erythroblasts (Ag-Ed), previously revealed in study of Rauscher leukemia, in the immunofluorescent and cytotoxic tests. The antigen is present on the membranes of the majority of human embryonic liver and adult bone marrow nuclear erythroid cells, but is not found in fetal thymocytes, newborn kidney cells, adult human hepatic cells and in peripheral blood erythrocytes. Ag-Eb appears to possess an inter-species determinant, shared by mammalian nuclear erythroid cells, and may be used as their specific marker.

A competition immunoassay for characterizing the reverse transcriptase of mammalian RNA tumor viruses

A competition enzyme immunoassay for reverse transcriptase has been developed. This assay is based on competition between the enzymatically active reverse transcriptase probe and a sample tested for the presence of reverse transcriptase antigen on a limited amount of monospecific antibody. The extent of competition is determined from the residual DNA polymerase activity left in the probe. Any reverse transcriptase activity in the test sample that may interfere with the enzymatic assay is irreversibly inhibited by pretreatment with N-ethylmaleimide. Thus, increasing amounts of test antigen neutralize more antibodies and as a result restore more of the reverse transcriptase probe activity. By means of the competition immunoassay we were able to show type, group, and interspecies antigenic determinants in the reverse transcriptase of mammalian RNA tumor viruses. The competition immunoassay has also proved useful for quantitating the amount of reverse transcriptase present in virions. The enzyme was found to comprise 2.5% of the total proteins in Moloney murine leukemia virus.

Type C RNA virus expression in systemic lupus erythematosus. New Zealand mouse model and human disease

An antigen recognized by antisera produced against p30 (core) proteins of the four chief groups of mammalian type C viruses (murine, feline, RD-114 related to endogenous primate, and infectious primate group) is located in an immune-complex pattern in some renal glomeruli of human SLE patients with lupus proliferative glomerulonephritis but is not detected in normal or pathological control human kidneys. This antigen cross-reacts with p30 interspecies determinants shared by the four chief virus groups and cross-reacts with a partially purified antigen extracted from human SLE spleen. The human SLE spleen antigen cross-reacts with p30 group antigen of RD-114 virus but not of feline or murine viruses. Some host immunoglobulins eluted from a human SLE kidney by acid-buffer show antibody-like activity against p30 group antigen of RD-114 virus but not of simian, feline, or murine viruses.

Radioimmunologic characterization of RD-114 reverse transcriptase: Evolutionary relatedness of mammalian type C viral pol gene products

The reverse transcriptase of RD-114, an endogenous mammalian type C virus, has been purified and characterized by radioimmunologic techniques. Using radioimmunoprecipitation assays, specific antibodies to reverse transcriptase were detected in the sera of animals naturally or experimentally infected with mammalian type C viruses. Competition radioimmunoassays using the purified enzyme as a probe detected type-specific antigenic determinants of RD-114 reverse transcriptase, as well as group-specific determinants shared with baboon endogenous virus. The pol gene products of all mammalian type C viruses were shown to share interspecies antigenic determinants not detected among functionally analogous reverse transcriptases of other retroviruses. These findings, together with previous immunologic evidence, strongly favor a common progenitor in the evolution of mammalian type C viruses.

Influence of antisera to oncornavirus glycoprotein (gp71) on infections of cats with feline leukemia virus

Six-month-old kittens that had been inoculated 6 days previously with feline leukemia virus (FeLV) were treated with either normal goat serum, goat antiserum to FeLV, or goat antiserum to the purified Friend leukemia virus (FLV) gp71. All of the kittens treated with normal goat serum became persistently viremic, whereas up to three-fourths of those treated with the antiviral antisera resisted persistent viremia. These results suggest that viral antigens, in particular, their interspecies determinants, can be used as targets for immunotherapeutic regimens for the prevention and control of diseases induced by oncornaviruses.

Deposition of retrovirus associated antigens (p30 and gp70) on cell membranes of feline and murine leukaemia virus infected cells.

A quantitative estimation of retrovirus associated cell membrane antigens of murine and feline cells infected with their respective type C leukosis virus is presented. Using a radio-immune assay with three broadly reactive antisera, the minimum estimated number of retrovirus associated antigenic determinants on YAC [Moloney leukaemia virus (MuLV) infected murine] and FL-74 [feline leukaemia virus (FeLV) infected feline] cells was 1.3 x 10(6) and 1.6 x10(6) determinants per cell respectively. The virus structural proteins p27-30 and gp70 were detected by three component specific antisera on murine and feline cell surfaces in amounts which varied between cell isolates. MuLV infected cells produced as many as 1.9 x 10(5) p30 antigenic determinants and 7.5 x 10(5) gp70 determinants on infected cells. FeLV infected cells (FL-74) expressed 5.6 x 10(5) p27 and 7.5 x 10(5) gp70 antigenic determinants per single cell surface. The major core protein (p27-30) and the major envelope glycoprotein (gp70) antigens are sufficiently physically separated on cell surfaces so that binding of either of the membrane antigens with component specific antibodies does not interfere with binding of antibodies specific for the other. Despite the expression of interspecies determinants for p30, gp70, and other retrovirus associated antigens detected by antibody procedures, interspecies determinants of cell mediated immunity could not be demonstrated in immune mice bearing Moloney sarcoma virus (MSV) induced tumours. Furthermore, xenogeneic immunization of mice with FL-74 cells failed to protect mice against the growth of MSV induced lymphoma or sarcoma.

Primate retroviruses: immunological cross-reactivity between major structural proteins of new and old world primate virus isolates.

The major 35,000-molecular-weight internal antigen (p35) of the squirrel monkey retrovirus (SMRV) was isolated and partially characterized. Immunological analysis of SMRV p35 led to the demonstration of antigenic determinants common to SMRV and the Mason-Pfizer monkey virus (MPMV). A broadly reactive competition immunoassay was developed utilizing antiserum to MPMV to precipitate 125I-labeled SMRV p35. Although the major structural proteins of MPMV and SMRV competed with equal efficiency in this assay, type B and type C oncornavirus proteins lacked detectable reactivity. Antibodies reactive with the major structural proteins of both MPMV and SMRV were observed in sera of several normal rhesus monkeys with known prior exposure to MPMV-infected animals. These findings demonstrate the ability of sera from naturally immunized primates to recognize broadly reactive interspecies antigenic determinants shared by the major structural proteins of type D oncornaviruses, and they suggest possible horizontal transmission of MPMV among rhesus monkeys. Although sera from a number of squirrel monkeys contained antibody to SMRV p35, the possibility that this latter reactivity was due to endogenous virus activation rather than horizontal transmission cannot be ruled out.

Endogenous New World primate type C viruses isolated from owl monkey (Aotus trivirgatus) kidney cell line.

A type C virus (OMC-1) detected in a culture of owl monkey kidney cells resembled typical type C viruses morphologically, but was slightly larger than previously characterized mammalian type C viruses. OMC-1 can be transmitted to bat lung cells and cat embryo fibroblasts. The virions band at a density of 1.16 g/ml in isopycnic sucrose density gradients and contain reverse transcriptase and a 60-65S RNA genome composed of approximately 32S subunits. The reverse transcriptase is immunologically and biochemically distinct from the polymerases of othe retroviruses. Radioimmunoassays directed to the interspecies antigenic determinants of the major structure proteins of other type C viruses do not detect a related antigen in OMC-1. Nucleic acid hybridization experiments using labeled viral genomic RNA or proviral cDNA transcripts to normal cellular DNA of different species show that OMC-1 is an endogenous virus with multiple virogene copies (20-50 per haploid genome) present in normal owl monkey cells and is distinct from previously isolated type C and D viruses. Sequences related to the OMC-1 genome can be detected in other New World monkeys. Thus, similar to the Old World primates (e.g., baboons as a prototype), the New World monkeys contain endogenous type C viral genes that appear to have been transmitted in the primate germ line.

Translational Products of Type-C RNA Tumor Viruses

RNA tumor viruses, commonly called “oncornavirus,” are essentially categorized as type-A, type-B, type-C, and type-D, primarily on the basis of morphologic criteria. Type-C RNA tumor viruses are regarded as the most extensively studied class of oncornaviruses. This group of viruses is characterized by their centrally located nucleoid and a pattern of virion assembly that occurs as a budding process at the plasma membrane. This chapter focuses on type-C viruses and their translational products. It also presents RNA tumor viruses, essentially known as “RNA sarcoma viruses.” Type-C oncornaviruses can be distinguished from either type-B or type-D viruses on the basis of both morphologic criteria and the divalent cation preference of their RNA-dependent DNA-polymerase. A number of structural proteins of all type-C oncornavirus isolates essentially share cross-reactive interspecies antigenic determinants. The type-C virus genome consists of two identical 35 S RNA subunits joined near their 5′ terminus in a hydrogen-bonded dimer linkage structure. The genome is terminally redundant containing short identical segments at its 5′ and 3′ ends that provide a means of forming circular structures during replication. The mammalian type-C viral genome appears to be arranged in the order 5′ gag-pol-env 3′. In the case of replication-defective mammalian sarcoma viruses, src sequences acquired from the host cell genome by genetic recombination are located at varying positions, frequently extending into the 3′terminus of the gag gene.