Abraham Pinter

Monoclonal antibodies against murine leukemia viruses: Identification of six antigenic determinants on the p15(E) and gp70 envelope proteins

Abstract Hybrid cells that produced monoclonal antibodies against the envelope proteins of murine leukemia virus (MuLV) were prepared by the polyethylene glycol-mediated fusion of a mouse myeloma cell line with lymphocytes from mice immunized with allogeneic MuLV-producing leukemia cells. Twenty-three independent cell lines were cloned and inoculated into syngeneic mice for the production of ascites fluids that contained high-titered (20–75 mg/ml) monoclonal antibodies. Six serologically distinct specificities were detected when these ascites fluids were tested on a broad panel of MuLV and non-murine retra iruses. Prototype cell lines producing monoclonal antibodies that were representative of each pattern of reaction were selected for further study. In immune precipitation assays each of the prototype antibodies reacted with viral envelope proteins; three of these identified antigenic determinants on p15(E), while three others identified antigenic determinants on gp70. The p15(E) antigenic determinants were shared by a diverse panel of MuLV. One of these p15(E) antigenic determinants was also found in feline leukemia virus. The gp70 antigenic determinants, on the other hand, had a more restricted distribution and were found in only selected isolates of MuLV.

Structural domains of endogenous murine leukemia virus gp70s containing specific antigenic determinants defined by monoclonal antibodies

Abstract Eight distinct antigenic determinants, or epitopes (labeled a-h) were identified on endogenous ecotropic murine leukemia virus (MuLV) gp70s using a series of murine and rat monoclonal antibodies. These epitopes were characterized by their distribution patterns in a panel of cloned MuLVs, and by their localization in fragments of gp70 generated either by spontaneous breakdown of purified gp70, or by controlled proteolysis of gp70 in solubilized virions. A major 32K carboxy terminal fragment was formed which contained epitopes b, c, and f. This fragment also possessed the p15(E) disulfide linkage site, and contained approximately four complex (type 1) carbohydrate chains. An amino terminal 35K fragment contained epitopes a, d, g, and h, and possessed two glycosylated sites, including a site which occasionally retained an endoglycosidase H-sensitive oligosaccharide chain. A related 49K fragment was also obtained which included the entire 35K region and contained an additional sequence bearing epitope e. In a series of dual-tropic MCF-type viruses studied, only those epitopes located in the 32K fragment were ever retained, indicating that for these recombinant viruses at least a portion of that domain was derived from the ecotropic parent. A model is presented indicating the likely orientation of these fragments and their structural characteristics.

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.

Studies with inhibitors of oligosaccharide processing indicate a functional role for complex sugars in the transport and proteolysis of friend mink cell focus-inducing murine leukemia virus envelope proteins

The functions of asparagine-linked oligosaccharides on the PrENV protein of Friend mink cell focus-inducing (FrMCF-1) murine leukemia virus were investigated by examining the effect of two inhibitors of different stages of the biosynthetic pathway of these sugar substituents on the synthesis and processing of the viral proteins. Treatment of virus-producing cells with tunicamycin totally inhibited the glycosylation of PrEnv, and resulted in the formation of a nonglycosylated form of the protein of molecular weight 62 kDa. This component was not proteolytically processed inside the cells, and neither it nor any derivative proteins were incorporated into extracellular virions. Treatment of cells with 1-deoxynojirimycin (DNM), which inhibits the cellular glucosidases normally involved in removal of the three glucose residues present on the initially transferred oligosaccharide chains, resulted in the intracellular accumulation of a slightly larger than normal form of PrENV, and decreased levels of cell-associated gp70. Only gp70 was detected on the cell surface. The bulk of the gp70 produced in the presence of the drug was aberrantly glycosylated, and contained decreased levels of complex and increased numbers of high mannose oligosaccharides; almost all of the gp70 molecules however, contained at least one complex sugar chain. Decreased incorporation of both env and gag proteins into extracellular virions was observed, despite the fact that the gag proteins were processed normally intracellularly; in contrast, DNM treatment of Gazdar murine sarcoma virus-infected HTG2 cells, which produce only gag but not env proteins, did not inhibit the release of extracellular virus. Ultrastructural examination of FrMCF-infected cells treated with DNM indicated the presence of large numbers of intracytoplasmic vacuoles, many of which contained viral particles. These studies indicate that the normal maturation process involved in the formation of complex oligosaccharides is necessary to obtain efficient transport to the plasma membrane and proteolysis of PrEnv, and also provide evidence suggesting a role for the env proteins in regulating assembly of gag proteins into virions.

Selective neutralization of ecotropic murine leukemia virus by monoclonal antibodies: Localization of a site on the gp70 protein associated with ecotropism☆

Abstract A panel of nine monoclonal antibodies against the gp70 and p15(E) envelope proteins of MuLV were tested in parallel for their ability to bind to virus and neutralize infectivity. Although each of the antibodies bound to N-ecotropic MuLV, only antibody against the gp70 f epitope neutralized infectivity. These results were interpreted as evidence for a specific site on the gp70 protein of ecotropic MuLV, in proximity of the gp70 f epitope, which was responsible for the ecotropic-specific binding of virus to the surface of cells.

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.

Protein composition of a defective murine sarcoma virus particle possessing the enveloped type-A morphology

Abstract The protein composition of a noninfectious murine sarcoma virus (Gazdar MSV) isolated from the HTG2 cell line was characterized. Electron microscopy of thin sections of the infected cells demonstrated that the virus particles were assembled via the budding mechanism at the plasma membrane, and the released virions possessed almost exclusively the enveloped type-A morphology characteristic of the immature form of retroviral particle. The protein composition of the virions was shown by SDS-PAGE analysis under reducing conditions to consist of a nonglycosylated protein of approximately 65,000 daltons, p65. When analyzed under nonreducing conditions this band was resolved into at least two components, and higher molecular weight forms of p65 were observed as well. p65 could be immunoprecipitated with monospecific antisera against the individual “gag” gene-coded MuLV proteins p30, p15, and p12 but not with antisera against the MuLV envelope proteins, reverse transcriptase, or p10, indicating that the MSV p65 is related to but not identical with the Pr65 gag of MuLV. The processed MuLV core proteins were not observed in purified HTG2 virions or in HTG2 cell extracts, indicating that proteolytic cleavage of the precursor is not required for the assembly or release of the MSV particles. Immunoprecipitation of lysed virions and HTG2 cell lysates with antisera to the MuLV envelope proteins gp70 and p15(E) demonstrated that detectable amounts of these and immunologically related components are not present in the virions and are not expressed in the infected cells. This suggests that the envelope proteins are not essential for the assembly and release of budding retroviral particles.

Interferon-mediated inhibition of production of Gazdar murine sarcoma virus, a retrovirus lacking env proteins and containing an uncleaved gag precursor.

HTG2 cells are murine sarcoma virus-transformed hamster cells. These cells continuously produce Gazdar sarcoma virus particles which are devoid of viral envelope proteins and which contain the uncleaved gag precursor polyprotein, Pr65, as their major protein constituent. Human interferon-alpha elicited an antiviral response in these cells as shown by the inhibition of replication of vesicular stomatitis virus in interferon-treated cells. Extracellular production of the retroviral particles by these cells was also inhibited by interferon in a dose-dependent manner and this inhibition was abolished by a specific antiserum to interferon. The intracellular level of Pr65 was not lowered in the interferon-treated cells, indicating that inhibition of viral protein synthesis was not responsible for inhibition of virus production. The present study suggests that interferon-mediated inhibition of retrovirus production, in general, is not a consequence of either a defective interaction between viral nucleoprotein cores and viral envelope proteins or a defect in the proteolytic processing of the gag polyprotein, since neither of these processes occurs during the morphogenesis of Gazdar particles and their production is nonetheless inhibited by interferon.

Relation of gp70 to spontaneous cytolytic activity of mouse spleen cells.

In comparing spleen cells of inbred and congenic mice for spontaneous capacity to lyse cells of the BALB/c leukemia RLc♂1 in vitro, we found that the activity of 129 spleen cells was more than double that of 129-Gix− spleen cells. The only known difference between these two strains is that 129-Gix− mice express no known demonstrable gp70 or p30, whereas 129 mice express both these MuLV-related components as mendelian traits not associated with the production of virions. We infer that MuLV-related components at the cell surface are concerned in effector-target interactions leading to cytolysis under the conditions described. — Although the congenic strains B6 (Gix−) and B6-Gix+ differ likewise in expression of the type-variant Gix-gp70, both strains express a second type-variant of gp70. The lytic activity of spleen cells of these two strains for RL♂1 cells was equally high, suggesting that involvement in lytic effector-target interactions is common to gp70 molecules in general. — When used as targets rather than as effectors 129 spleen cells were more sensitive to lysis than 129-Gix− spleen cells. Pre-exposure to gp70, purified from R-MuLV, rendered splenic effector cells less lytic. Pre-exposure to gp70 also rendered RL♂1 target cells less sensitive to lysis. One explanation of these findings is that both target cells and effector cells express gp70 and also receptors for gp70 and that this is the basis of mutual cellular recognition leading to lysis in the circumstances described.