Erwin Fleissner

The presence of disulfide-linked gp70-pl5(E) complexes in AKR murine leukemia virus

Abstract Analysis of 14 C-labeled amino acid- and [ 3 H]glucosamine-labeled AKR virions on SDS-polyacrylamide gels under nonreducing conditions demonstrates the presence of a glycoprotein of 90,000 molecular weight, [gp90]. Characterization of [gp90] by gel analysis under reducing conditions, and by immunoprecipitation with specific antisera against purified viral proteins, indicates that it is composed of gp70 linked to p15(E) by one or more labile disulfide bonds.

Specificities of human immunoglobulins reactive with antigens in preparations of several mammalian type-C viruses.

Abstract Human serological reactivity with type-C RNA tumor viruses was assessed. Probes consisted of radioactively labeled, gradient purified Woolly monkey-derived simian sarcoma virus-simian sarcoma-associated virus (SSV-SSAV), Rauscher murine leukemia virus (R-MuLV), feline leukemia virus (FeLV-AB), and Rous-associated virus (RAV-2). Most human sera, surveyed for precipitating reactivity against intact virus (1 μg protein) by means of a sensitive radioimmuno-precipitation (RIP) assay, were found to precipitate type-C viruses of simian, murine, and feline, but not avian, origin with the reactivity primarily due to immunoglobulin G. No clear differences in reactivity between sera from normal individuals and from those with various neoplasms were found in preliminary surveys. Specificity tests were performed primarily with SSV-SSAV. Absorption studies with sheep and human red blood cells and blood-typing sera excluded involvement of known blood-group substances or Forssman-like antigens in assays with SSV-SSAV. Quantitative absorption tests with fetal calf serum proteins showed some removal of reactivity from several human sera, but only to maximum levels of 20–45% competition. Reactivity of human serum with SSV-SSAV was completely removed by preabsorbing with 2–4 × 106 tissue culture cells producing SSV-SSAV or SSAV while normal cells and SSV- or Kirsten murine sarcoma virus-transformed nonproducer cells showed no absorption capacity at 10-fold higher concentrations. Complete absorption of reactivity could also be achieved with a 70,000 molecular weight fraction from purified virus while fractions containing p30, p14, or p12 had negligible effects. Direct titering of human sera for reactivity with purified viral gp70 in radioimmunoassays, however, proved negative. The relationship between the latter results and those of the competition experiments are discussed.

A core polyprotein of murine leukemia virus on the surface of mouse leukemia cells

A polypeptide of molecular weight approximately 75,000 daltons, p(75), was identified on the surface of AKR spontaneous leukemia cells by lactoperoxidase-catalyzed radio-iodination. This protein was shown by immunoprecipitation to have antigenic determinants of MuLV p30, p15, and p10, but not gp70, suggesting that p(75) represents a polyprotein composed of virion core components. As evidenced by studies on incorporation of radioactive glucosamine, p(75) is probably glycosylated. No p(75) was found on 2 month old AKR thymocytes, and only a small amount of p(75) was detectable of thymocytes from 4 month old animals. However, substantial quantities of p(75) could be found on thymocytes from 6 month old, yet still preleukemic mice.

The GIX antigen of murine leukemia virus: an analysis with monoclonal antibodies.

Abstract The G IX antigen of murine retrovirus gp70 is a marker for virus replication in various cell types, as well as for T-cell differentiation and leukemogenesis in particular mouse strains. The serological definition of G IX depends on the cytotoxicity of a particular rat antiserum for thymocytes from strain 129 mice. We have found that two rat monoclonal antibodies, elicited by immunization with AKR ecotropic virus or AKR leukemia cells, co-type closely with G IX in several assay systems. Like G IX , the epitopes identified by the monoclonal antibodies are amplified on thymocytes during leukemogenesis in AKR mice. These antibodies are cytotoxic for G IX + leukemia cells and for fibroblasts infected with G IX + viral serotypes. Though absorbed by G IX + thymocytes of various strains, the antibodies are not cytotoxic for these cells. We ascribe the latter result to lower representation of antigenic sites on normal thymocytes, and postulate that more than one epitope may participate in classical G IX -mediated cytotoxicity. The monoclonal antibodies do not react with viral env products synthesized in the presence of the inhibitor of glycosylation, tunicamycin. Reactivity with the antibodies appears to require a stable configurational change in the env precursor protein coinciding with glycosylation, rather than direct participation of carbohydrate in the antigenic site. Thus subsequent enzymatic removal of carbohydrate chains in vitro does not alter reactivity with the antibodies.

Properties of the PC-1 molecule

The technique of cell-surface iodination, followed by immuno-precipitation withPC-1.1 antiserum, was applied to normal spleen cells and to MOPC-70A BALB myeloma cells. The results indicate that the cell-surface component bearing PC-1 alloantigen has a molecular weight of from 105,000 to 110,000 daltons and does not resemble any constituent of plasma membranes or MuLV-type virus so far categorized by similar methods. The PC-1 specificity of the molecule was confirmed by comparison of the spleen cells of two mouse strains with spleen cells of their respective PC-congenic partner strains. MOPC-70A myeloma cells, but not spleen cells, yield a fainter band in the PC-1 position in controls in which antiserum is omitted, but peptide maps show that this similarly placed band has no relation to the PC-1 molecule.

Biological effects of a murine retrovirus carrying an activated N-ras gene of human origin

We have introduced a genomic DNA clone of a mutated human N-ras gene from a T-cell leukemia cell line into a retroviral vector equipped with a neo resistance gene and with SV40 and pBR322 origins of replication. The helper free N-ras virus, which was recovered after transfection of the construction in the psi 2 packaging cell line, contained a correctly spliced N-ras gene. Proviral DNA was amplified in cos cells and subsequently cloned in bacteria. Nucleic acid sequence analysis of the activated N-ras gene revealed a point mutation at codon 12 resulting in a glycine to aspartic acid substitution. The N-ras virus was able to transform mouse fibroblastic cell lines, but failed to fully transform mouse primary embryo fibroblasts. MoMuLV or amphotropic 4070A pseudotypes of the virus were injected intraperitoneally into newborn mice. The MoMuLV pseudotype produced only helper-virus-induced leukemias. The amphotropic pseudotype caused fibrosarcomas after a long latent period. The results of these and other in vivo experiments are discussed in relation to known pathogenic effects of other retroviruses carrying H-ras or K-ras genes.

Biochemical genetics of TL antigens

TL antigens are class I glycoproteins which are expressed on thymocytes and which are coded by the Tla region of the major histocompatibility complex of the mouse. Biochemical analysis of TL molecules from different strains of mice revealed structural variation determined by the Tla region which is detectable by peptide mapping, isoelectric focusing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two-dimensional gels, and by differential reactivity of allelic forms of TL molecules with a panel of anti TL reagents. The quantity of TL expressed on thymocytes is also influenced by the Tla region; three quantitative phenotypes were identified: high (Tlaa, Tlad, Tlac), intermediate (Tlac, Tlaf), and low (Tlab). (Relative amounts: 1000 : 100 : 1.) Some thymic leukemias arising in (Tlab, Tlac, Tlac) mice with genetically determined reduced levels of thymic TL were found to express TL molecules which were structurally indistinguishable from TL isolated from thymocytes but were present in larger amounts. This suggests that TL structural genes are intrinsically capable of full expression in all mice but that the Tla region of mice expressing an intermediate or low quantity of TL is marked by some feature which causes the thymocyte to express less than the full amount of TL possible.

Endogenous murine leukemia virus-encoded proteins in radiation leukemias of BALB/c mice

Abstract To explore the role of endogenous retroviruses in radiation-induced leukemogenesis in the mouse, we have examined virus-encoded proteins in nine BALB/c leukemias by pulse-chase labeling procedures and serological typing with monospecific and monoclonal antibodies. The major gag precursor protein, Pr65 gag , was observed in all cases, but only three leukemias expressed detectable amounts of the glycosylated gag species, gP95 gag , or its precursor, Pr75 gag . No evidence was found for synthesis of gag -host fusion proteins. None of the leukemias released infectious xenotropic or dualtropic virus, but all nine expressed at least one env protein with xenotropic properties. In two instances a monoclonal antibody, 35 56 , which is specific for the MuLV G IX antigen, displayed a distinctive reactivity with this class of env protein, although this antibody is unreactive with replicating xenotropic viruses. An ecotropic/xenotropic recombinant env protein with the same 35 56 phenotype was observed in a leukemia induced by a strongly leukemogenic virus isolated from a BALB/c radiation leukemia.

Oncoviral proteins as cellular antigens.

This review will be concerned with immunological recognition of proteins encoded by oncoviruses and the biological consequences of this recognition. We shall discuss virion structural proteins and variants of these gene products which do not actually reside on virions, as well as proteins specified by cellular genes which have become incorporated into oncoviral genomes and can cause the malignant transformation of host cells. A number of the proteins in question are to be found on the surface of cells. This circumstance is connected with the capacity of the viruses to bud from the plasma membrane, and, in the case of some transformation-related viral proteins, may have to do with the phenotype of transformation. Cell-surface location makes a protein an efficient antigen, provides a point of attack for immune surveillance mechanisms defending against malignancy, and facilitates serological analysis in vitro.

Polymorphism of endogenous murine leukemia viruses revealed by isoelectric focusing in polyacrylamide gels.

Abstract An analysis of structural proteins from a group of endogenous murine type-C viruses has been made by the application of isoelectric focusing in polyacrylamide gels. The gp70 and p12 components focused at rather acidic pIs, while the p10 protein was found at a strongly basic pI (9.6). Significant isoelectric heterogeneity was observed in the major virion core protein p30, for which isoelectric points were found to vary from a pI of 5.8 for the B-tropic isolate from the BALB c mouse strain, WN1802B, to a pI of 6.8 for the Gross leukemia virus.