Richard J. Massey

Topographical analysis of viral epitopes using monoclonal antibodies: Mechanism of virus neutralization

Abstract The mouse mammary tumor virus (MMTV) was used in neutralization studies with monoclonal antibodies (monoclones) to the major external glycoprotein gp52. Monoclones to MMTV gp52 showed that epitopes with different antigenic specificities were targets for virus neutralization. These epitopes were group specific (common to C3H, GR, RIII, and C3Hf MMTVs), class specific (common to C3H and GR MMTVs), and type specific (unique to C3H MMTV). Competition antibody binding assays using a radioiodinated C3H MMTV type-specific neutralizing monoclone revealed that all the monoclones that neutralized virus infectivity also blocked the binding of the type-specific monoclone to C3H MMTV. A topographically distinct site from this determinant was found using other monoclones which did not compete for the binding of the type-specific monoclone and their epitopes did not function as targets for neutralizing antibodies. However, these nonneutralizing monoclones could neutralize virus infectivity in the presence of antimouse IgG sera. Therefore, two topographically distinct sites could be identified on a single protein of the virus. One site functioned as a target for neutralization and the second site bound antibody but did not affect neutralization. The observation that virus bound to cells was no longer susceptible to neutralization by monoclones showed that neutralization affected virus prior to its binding to cell surface receptors. This result further indicated that antibody prevented virus adsorption to cells and that neutralization was mediated by steric hindrance from antibodies binding to epitopes adjacent to those determinants which functioned in virus binding to cell surface receptors.

Gene order of mouse mammary tumor virus precursor polyproteins and their interaction leading to the formation of a virus

Abstract 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 H 2 N-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 H 2 N-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 [ 51 Cr] release with monospecific MMTV antisera, and (iv) membrane immunofluorescence with monospecific MMTV antisera. Analysis of 125 I-labeled MMTV cell surface antigens by immune precipitation with MMTV anti-gp52, gp36, p27, p14, and p10 sera followed by SDS-PAGE revealed only 125 I-gp52. In contrast, cell surface glycoprotein labeling revealed [ 3 H]gp52 and [ 3 H]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.

Generation of a mouse mammary tumor virus (MMTV) pseudotype of Kirsten sarcoma virus and restriction of MMTV gag expression in heterologous infected cells.

Abstract C3H mouse mammary tumor cells producing mouse mammary tumor virus (MMTV) were cocultivated with nonproducer mouse cells (KNIH) transformed by Kirsten sarcoma virus (KiSV). These cocultivated cells were then treated with mitomycin C and overlayed onto human embryonic skin and muscle cells. The virus resulting from this cocultivation could be titrated in a focus-forming assay on Fischer rat embryo (FRE) cells exhibiting one-hit kinetics. Furthermore, focus formation on FRE cells was neutralized specifically by antiserum directed against MMTV and the major MMTV external glycoprotein gp52, but not against a broadly reactive antiserum directed murine leukemia virus (MuLV) gp70 and MMTV gp36, p27, p14, and p10. These results demonstrate the generation of a KiSV(MMTV) pseudotype and further demonstrate that gp52 is a target antigen for neutralization of MMTV. This pseudotype possessed a wide host range, transforming cells of human, rat, mouse, mink, and rabbit origin. MMTV but not MuLV antigen expression was demonstrated in the KiSV(MMTV) pseudotype-infected cells. Analysis of intracellular MMTV protein synthesis in these in vitro -infected cells has indicated that the low yield of extracellular MMTV produced by the transformed cells may be the result of the poor expression of the MMTV gag precursor polyprotein relative to the expression of the env gene polyprotein. These studies thus provide the basis for an in vitro infectivity assay for neutralization and host range studies of MMTV.

Mouse mammary tumor virus and murine leukemia virus cell surface antigens on virus producer and nonproducer mammary epithelial tumor cells

Abstract Mouse mammary tumor virus (MMTV)- and murine leukemia virus (MuLV)- specific cell surface antigens (CSA) on virus producer and nonproducer mammary epithelial tumor cells were studied using the techniques of lactoperoxidase catalyzed iodination of cell surface proteins followed by radioimmune precipitation with monospecific antisera to the major MMTV proteins gp52, gp36, p27, and p10 and to the major MuLV proteins gp70 and p30. The incorporation of iodinated CSA into extracellular virus was determined by analyzing labeled proteins in purified virus. On cells producing only MMTV both gp52 and gp70 were present on the cell surface. Furthermore, gp52 was the only labeled protein in extracellular MMTV produced by these cells. On cells producing both MMTV and MuLV, both gp52 and gp70 were present on the cell surface, and were the only labeled proteins present in their respective extracellular viruses indicating that gp70 and gp52 are present on mutually exclusive cellular viral budding sites. In addition, MuLV anti-p30 serum precipitated two iodinated proteins with molecular weights of 85,000 and 95,000 daltons, analogous to the Gross cell surface antigen (GCSA). Labeled gp52 and gp70 represent true CSA as demonstrated by the fact that they were also present on the surface of cells producing no virus, but producing large amounts of MMTV glycoproteins and nonglyco-proteins. These results further demonstrate that the precursor to the MMTV glycoproteins (gPr75-MMTV env) is cleaved prior to the appearance of gp52 on the cell surface.

Common surface receptors on both mouse and rat cells distinguish different classes of mouse mammary tumor viruses

Abstract Cell surface receptors which bind mouse mammary tumor virus (MMTV) were detected on mouse and rat cells. Virus binding was quantitated by measuring 125 T-protein A binding to immune complexes composed of a C3H MMTV gp52 type-specific monoclonal antibody and receptor-bound MMTV. C3H MMTV binding to normal mouse mammary epithelial cells (NMuMG) was dose dependent and was ≥50% inhibited by GR MMTV, but not by endogenous C3Hf MMTV or Gross murine leukemia virus. These results were confirmed in [ 3 H]leucine MMTV binding inhibition assays in which GR MMTV and C3H MMTV blocked C3H [ 3 H]MMTV binding while C3Hf MMTV did not block. The affinities of C3H [ 3 H]MMTV binding to receptors on NMuMG, NIH Swiss (SLP), and Fischer rat embryo (FRE) cells were identical by Scatchard analysis. C3Hf [ 3 H]MMTV also bound these cells, but with an affinity approximately 10 times weaker than the C3H [ 3 H]MMTV binding. Interference assays using a Kirsten sarcoma virus (C3H MMTV) pseudotype confirmed the importance of C3H MMTV specific binding to viral penetration and expression. Pretreatment of SLP or FRE cells with 100 μg of C3H MMTV or GR MMTV inhibited focus formation by ≥50% while C3Hf MMTV and RIII MMTV did not inhibit. Therefore, the functional C3H MMTV cell receptors on mouse and rat cells were related and were able to distinguish C3H MMTV and GR MMTV from C3Hf MMTV and RIII MMTV. These comparable receptors may represent evolutionarily conserved surface components of murine cells.

Monoclonal and polyclonal antibody studies of VSV(hrMMTV) pseudotypes

Abstract By phenotypic mixing between the host range (hr) variant of mouse mammary tumor virus (MMTV) and the is-045 mutant of vesicular stomatitis virus (VSV), we were able to produce high titers of “complete” pseudotypes bearing the VSV genome and hrMMTV envelope. Four “strains” of pseudotypes designated VSV(C 3 H), VSV(GR), VSV(RIII), and VSV(C 3 Hf) were generated. To find out whether group-specific and type-specific neutralizing antigenic determinants (epitopes) were expressed on some of these pseudotypes, we utilized several monoclonal antibodies to MMTV-gp52 and sera from mammary tumor-bearing C 3 H/HeN mice in neutralization assays. Four monoclonal antibodies to MMTV-gp52, which were group specific in solid-phase MMTV binding assays, were found to neutralize the infectivity of VSV(C 3 H), VSV(GR), VSV(RIII), and VSV(C 3 Hf), indicating the expression of group-specific “neutralizing epitopes” on these pseudotypes. One monoclonal antibody to MMTV-gp52 that was C3Hf type specific in MMTV binding assays was found to neutralize only the infectivity of VSV(C 3 Hf) and not VSV(C 3 H), VSV(GR), or VSV(RIII). In addition, polyclonal antibodies were found in sera of mammary tumor-bearing C 3 H/HeN mice which neutralized specifically the infectivity of VSV(C 3 H). Such antibodies were not found in the sera of normal C 3 H/HeN mice or normal BALB/c mice. These results indicate that group-specific “neutralizing epitopes” were expressed on VSV(C 3 H), VSV(GR), VSV(RIII), and VSV(C 3 Hf). Furthermore, they suggest that type-specific “neutralizing epitopes” were expressed on VSV(C 3 H) and VSV(C 3 Hf) pseudotypes.