Murine Leukemia Virus Gp70 Research Articles

Origins of a Vaccine-Induced, Human Anti-HIV-1 Antibody

Origins of a Vaccine-Induced, Human Anti-HIV-1 Antibody

Human monoclonal antibodies specific for conformation-sensitive epitopes of V3 neutralize human immunodeficiency virus type 1 primary isolates from various clades.

The epitopes of the V3 domain of the human immunodeficiency virus type 1 (HIV-1) gp120 glycoprotein have complex structures consisting of linear and conformational antigenic determinants. Anti-V3 antibodies (Abs) recognize both types of elements, but Abs which preferentially react to the conformational aspect of the epitopes may have more potent neutralizing activity against HIV-1, as recently suggested. To test this hypothesis, human anti-V3 monoclonal Abs (MAbs) were selected using a V3 fusion protein (V3-FP) which retains the conformation of the third variable region. The V3-FP consists of the V3(JR-CSF) sequence inserted into a truncated form of murine leukemia virus gp70. Six human MAbs which recognize epitopes at the crown of the V3 loop were selected with the V3-FP. They were found to react more strongly with molecules displaying conformationally intact V3 than with linear V3 peptides. In a virus capture assay, these MAbs showed cross-clade binding to native, intact virions of clades A, B, C, D, and F. No binding was found to isolates from subtype E. The neutralizing activity of MAbs against primary isolates was determined in three assays: the GHOST cell assay, a phytohemagglutinin-stimulated peripheral blood mononuclear cell assay, and a luciferase assay. While these new MAbs displayed various degrees of activity, the pattern of cross-clade neutralization of clades A, B, and F was most pronounced. The neutralization of clades C and D viruses was weak and sporadic, and neutralization of clade E by these MAbs was not detected. Analysis by linear regression showed a highly significant correlation (P < 0.0001) between the strength of binding of these anti-V3 MAbs to intact virions and the percent neutralization. These studies demonstrate that human MAbs to conformation-sensitive epitopes of V3 display cross-clade reactivity in both binding to native, intact virions and neutralization of primary isolates.

Enhanced T cell engraftment after retroviral delivery of an antiviral gene in HIV-infected individuals.

Intracellular expression of gene products that inhibit viral replication have the potential to complement current antiviral approaches to the treatment of AIDS. We previously have shown that a mutant inhibitory form of an essential viral protein, Rev M10, prolongs the survival of T cells transduced with a nonviral vector in HIV-infected individuals. Because these gene-modified cells were not observed in patients beyond 8 weeks, efforts were made to improve the duration of engraftment. In this study, we used retroviral vector delivery of Rev M10 to CD4(+) cells and analyzed relevant immune responses in a pilot study of three HIV-seropositive patients. DNA and RNA PCR analyses revealed that cells transduced with Rev M10 retroviral vectors survived and expressed the recombinant gene for significantly longer time periods than those transduced with a negative control vector in all three patients. Immune responses were not detected either to Rev M10 or to Moloney murine leukemia virus gp70 envelope protein. Rev M10-transduced cells were detected for an average of 6 months after retroviral gene transfer compared with approximately 3 weeks for the previously reported nonviral vector delivery. These findings suggest that retroviral delivery of an antiviral gene may potentially contribute to immune reconstitution in AIDS and could provide a more effective vector to prolong survival of CD4(+) cells in HIV infection.

Inhibition of B6RV2 leukemia growth by immunization with purified unique antigen.

Monoclonal antibody (mAb) NU7‐99 reacted with only B6RV2 cells, not with 28 other leukemia cell lines, fibroblasts or normal tissues. Biochemical analyses of the unique antigen on B6RV2 cells that reacted with NU7–99 mAb indicated its relationship to xenotropic murine leukemia virus gp70. The antigen that reacted with NU7‐99 mAb was extracted from the surface of B6RV2 cells with n‐butanol and purified by ion‐exchange chromatography and affinity chromatography. Growth of B6RV2 tumors in semi‐syngeneic mice was inhibited by immunization of the mice with a purified preparation of this unique antigen.

Production of monoclonal antibodies reactive with a denatured form of the friend murine leukemia virus gp70 envelope protein: Use in a focal infectivity assay, immunohistochemical studies, electron microscopy and western blotting

Four monoclonal antibodies were selected for their ability to recognize the envelope protein of Friend murine leukemia virus (F-MuLV) in methanol-fixed tissue culture cells. Each of these monoclonal antibodies was found to react only with F-MuLV. By using recombinant retroviruses, it was determined that each of the monoclonal antibodies recognized the C-terminal one-third of the F-MuLV gp70 envelope protein. The monoclonal antibodies were effective in radioimmunoprecipitation of F-MuLV proteins, and one of the antibodies, 720, was also effective in Western blotting. The ability of antibody 720 to react with F-MuLV in methanol-fixed cells facilitated the use of a sensitive immunoperoxidase method with a focal virus infectivity assay. In immunohistochemical studies using light microscopy, antibody 720 could specifically label F-MuLV-infected cells in acetone-fixed tissue sections from F-MuLV-infected animals. Finally, in immuno-gold labelling studies using electron microscopy, antibody 720 could be used to distinguish F-MuLV from amphotropic MuLV.

Turnover and compartmentation of [formula omitted] E in ψ2 cells

The distribution of Moloney murine leukemia virus gp70 p15 E between cell surface and intracellular compartments and the kinetics of transfer between these compartments was examined in ψ 2 cells. A novel biotin derivatization and recovery assay was used to quantitate pulse-labeled protein accessible at 4°C (cell surface), 18°C (cell surface and an intracellular compartment), or inaccessible at any temperature. Cell surface (4°C) gp70 and p15 E turn over more rapidly than intracellular pools of these proteins. The decrease in cell surface gp70 and p15 E after one hour of chase is accounted for by an increase in that which is inaccessible to biotinyl reagent.

Reduction-Modifiable Properties of Moloney Murine Leukemia Virus gp70 as an Indicator of Envelope Glycoprotein Heterogeneity

We have found that preparations of rate-zonal purified Moloney murine leukemia virus originally obtained from the National Cancer Institute Resources Program, when separated by SDS-PAGE in the absence of mercaptoethanol (beta-MSH), exhibited a doublet envelope glycoprotein band of approximately 69/67 kD. When the same samples were run in the presence of beta-MSH, a single band at 70 kD (gp70) was observed. Western blot analysis with polyclonal antiserum identified both the 69- and 67-kD bands as envelope gene products. Tryptic peptide mapping of each of the gp67 and gp69 bands confirmed the serological data, with each showing conserved as well as unique peptides. These results imply that the Moloney murine leukemia virus samples examined above contain two structurally different envelope gene products. Western blot analysis using ecotropic and dualtropic specific sera suggest that gp69 is derived from an ecotropic virus, while gp67 is from a dualtropic virus. This is consistent with the results of an earlier study which showed that the majority of the cysteines (4/5) in dualtropic gp70 are lost by a single deletion relative to the ecotropic gp70 species. This would account for the difference in mobility observed in the SDS-PAGE profile in the absence of beta-MSH. It would indicate that the cysteines play an important role in defining structural differences that separate the ecotropic and dualtropic gp70s.

Monoclonal antibody 45-2D9 recognizes a cell surface glycoprotein on a human c-Ha-ras transformed cell line (45-342) and a shared epitope on human tumors.

A monoclonal antibody (45-2D9) produced after immunization of BALB/c mice with the c-Ha-ras NIH 3T3 tertiary transfectant (45-342) recognized a determinant expressed by the primary, three of three secondary, and one of three tertiary transfectants, but not by NIH 3T3 cells. The determinant was present on the cell surface and was distinct from murine leukemia virus gp70 by absorption studies. Biosynthetic labeling and immunoprecipitation studies with [35S]methionine and [3H]glucosamine demonstrated that 45-2D9 recognizes a 74,000 Mr glycoprotein with minor bands of 90,000 and 180,000 Mr on SDS-PAGE. Pulse chase studies demonstrated a 68,000 Mr precursor molecule that incorporated only [35S]methionine. The distribution of the epitope recognized by 45-2D9 was assessed by immunoperoxidase staining. The antigen was not detected on 10 primary and metastatic murine tumors or 11 transformed murine cell lines. However, a variety of surgically excised human tumors demonstrated intense staining, whereas staining of normal tissues was minimal or not detectable. Thus a human oncogene-transfected cell can express a new cell surface determinant apparently unrelated to the oncogene product, which is also selectively expressed by human tumors.

Characterization of a progressive neurodegenerative disease induced by a temperature-sensitive Moloney murine leukemia virus infection.

A progressive neurodegenerative disease occurred following infection of mice with a temperature-sensitive (ts) isolate of Moloney (Mo) murine leukemia virus (MuLV), ts Mo BA-1 MuLV. This NB-tropic ecotropic MuLV, which was ts for a late function, induced a syndrome of tremor, weakness of the hind limbs, and spasticity following infection of several strains of laboratory neonatal mice, including NFS, C3H/He, CBA, SJL, and BALB/c. The latent period of 8 to 16 weeks was considerably longer than that observed for the acute paralytic diseases observed following neonatal infection with other ts Mo-MuLV, rat-passaged Friend MuLV, and some wild mouse ecotropic MuLVs. Spongiform pathology without inflammation and degeneration of neurons devoid of budding virions occurred in the cerebellar grey matter, brain stem, and upper spinal cord; but lower spinal cord anterior horn cells were less obviously affected than in other MuLV-associated neuroparalytic syndromes. ts Mo BA-1 MuLV differed from other ts Mo-MuLV mutants that are capable of inducing a neuroparalytic syndrome in that while infected nervous system tissue contained high levels of MuLV p30 and gp70, no evidence of precursor accumulation or abnormal processing of MuLV p30 or gp70 could be demonstrated. The localization of virus within the nervous system suggests that direct neuronal infection may not be the etiologic mechanism in this MuLV-induced neurodegenerative disease.

Carbohydrate dramatically influences immune reactivity of antisera to viral glycoprotein antigens.

Analysis of the ability of heteroantisera, monoclonal antibodies, and antibodies to synthetic peptides to react with viral glycoproteins deglycosylated with endoglycosidase F revealed that the reactivities of most of the antibodies to these glycoprotein antigens were influenced by the attached carbohydrate moieties. All heteroantisera prepared in rabbits or goats to either fully glycosylated retroviruses or influenza virus were virtually unreactive toward the viral glycoproteins after carbohydrate removal. Analyses with a panel of monoclonal antibodies to purified Rauscher murine leukemia virus gp70 indicated that the reactivity of most of these antibodies improved while the reactivity of others decreased or remain unchanged after carbohydrate removal. Most of the antibodies to synthetic peptide sequences in the influenza virus hemagglutinin also improved in reactivity after carbohydrate removal. These data indicate that carbohydrate side chains on viral glycoproteins influence the immune response to these antigens, and the more native the glycoprotein immunogen, the more dramatic the carbohydrate influence. Thus the immune response to these glycoproteins is not simply a function of the immunogenicity of certain domains over others but rather is a direct measure of carbohydrate influences on the host's perception of the foreign antigen.

Transformation of NIH 3T3 cells by DNA of the MCF-7 human mammary carcinoma cell line induces expression of an endogenous murine leukemia provirus.

Previous studies identified two glycoproteins of 86 (gp86) and 72 (gp72) kilodaltons and two nonglycosylated proteins of 70 (p70) and 19 (p19) kilodaltons which were specifically expressed in NIH cells transformed by DNA of the MCF-7 human mammary carcinoma cell line. Pulse-chase experiments and the use of tunicamycin to inhibit glycosylation suggested that gp86, gp72, and p19 were related as precursor products. Characteristics of the four transformation-associated proteins resembled those of murine leukemia virus (MuLV) proteins. Sera raised against disrupted MuLV immunoprecipitated the same four proteins in extracts of NIH(MCF-7) cells and MuLV-infected NIH 3T3 cells. In addition, a monoclonal antibody against MuLV gp70 immunoprecipitated proteins gp86 and gp72, whereas a monoclonal antibody against MuLV p15(E) immunoprecipitated gp86 and p19. These results indicate that proteins gp86, gp72, and p19 expressed in NIH(MCF-7) transformants correspond to MuLV envelope proteins gp80env, gp70, and p15(E), respectively. The transformation-associated protein p70 appears to be a non-envelope MuLV protein, most likely p65gag. Northern blot analysis confirmed that transformation of NIH cells by MCF-7 mammary carcinoma DNA led to the induction of an endogenous MuLV provirus.

Possible DNA-RNA tumor virus interaction in human lymphomas: expression of retroviral proteins in Ramos lymphoma lines is enhanced after conversion with Epstein-Barr virus.

Epstein-Barr virus (EBV)-genome-negative human lymphoma lines, Ramos and BJAB, can be converted by EBV in vitro into EBV-genome-positive virus nonproducer lines. These cell lines have been used to identify surface antigens unique to EBV, with the expectation that such determinants might be related to the antigenic target responsible for EBV-specific immunosurveillance. Antisera prepared in rabbits immunized with either whole cells or purified plasma membranes were used in immunoblot experiments to analyze antigenic differences resulting from expression of the resident EBV genome. Unexpectedly, an increase in polypeptides of 32 and 70 kilodaltons was consistently observed in the EBV-converted Ramos lines. In contrast, these antigens were not expressed in BJAB or in its EBV-converted lines. These data suggested that p32 and gp70 might be murine leukemia virus (MuLV)-coded antigens because Ramos, but not BJAB, had been passaged in athymic nude mice during establishment of this cell line. This conclusion was confirmed by using antisera specific for MuLV p30 and gp70. Retroviral antigens were expressed constitutively at low levels in Ramos. Quantitative immunoblotting showed that EBV conversion of Ramos amplified the expression of MuLV proteins 3- to 5-fold. The molecular mechanism responsible for the enhanced expression is unknown. The biological relevance of this phenomenon is also not clear, but the interaction between a DNA and a RNA tumor virus in a Burkitt lymphoma line that carries both viruses may have important biological consequences in relation to retrovirus latency and tumor induction. These results also show that caution must be used when ascribing "uniqueness" to EBV-determined antigens, particularly in the Ramos lines. This warning extends also to the use of Ramos cell lines as immunogens, because immunization of rabbits elicited antibodies that recognized proteins of the same size as the retroviral antigens.

The expression of the env gene-related gp66 in mouse cells infected with the helper independent friend leukemia virus is restricted to the myelomonocytic and mastocytic lineages

The presence of a gp66 closely related to the Friend ecotropic murine leukemia virus gp70 (F-MuLV) has recently been reported ( D. Mathieu-Mahul, J. M. Heard, S. Fichelson, S. Gisselbrecht, B. Sola, and C. J. Larsen (1982) Virology 119,59–67). In the present work, characterization of this gp66 was continued. First, immunoprecipitation tests, using cytoplasmic membrane subfractions from one of the myelomonocytic cell lines in which gp66 was first detected, indicated that most of it was associated with rough endoplasmic reticulum. Second, to define the limits of gp66 expression, a variety of hemopoietic cell lines were analyzed for gp66 content. These lines were obtained (a) from various tumors (including erythroleukemias, chloroleukemias, and lymphatic leukemias) induced in susceptible mice by F-MuLV and (b) from long-term bone marrow cultures (LTBMC) infected with F-MuLV. In the latter case, lines of adherent fibroblastoid cells and nonadherent cells with myelomonocytic and mastocytic characteristics were obtained. Although several F-MuLV isolates were used, gp66 was only expressed in myelomonocytic and mastocytic cells. This did not result from in vitro culture conditions as gp66 was also found in fresh cells. These data suggested that a particular processing of the env gene product may exist in both myelomonocytic and mastocytic cell lines. In agreement with this hypothesis, a metabolically unstable gp62 related to MCF gp70 was found in one myelomonocytic cell line expressing MCF virus.

The cytoskeleton of murine leukemia virus (MuLV)-infected mouse fibroblasts as observed under varying conditions of formaldehyde fixation.

Mouse fibroblasts chronically infected with Moloney murine leukemia virus (MuLV) were fixed using variable amounts of formaldehyde, then examined by indirect immunofluorescence light microscopy. Several antisera were employed to detect both external and internal antigens associated with the cells, eg, MuLV gp70, tubulin, vimentin, and actin. Our results indicate that the cell membranes could be partially permeabilized to IgG molecules directed against the three cytoskeletal antigens only after 3.7%, but not 1%, formaldehyde treatment. Complete permeabilization was achieved by subsequent acetone treatment of cells after 3.7% formaldehyde fixation. In such cells, normal-appearing cytoskeletal networks of microtubules and intermediate filaments were observed. Stress fibers were also seen; however, they appeared less numerous and thinner than those of uninfected mouse fibroblasts. Further, a significant amounts of F-actin fluorescence was localized in granules in the cytoplasm of infected cells. Similar observations were made using JLS-V9 mouse cells chronically infected with 334C virus, another MuLV. These results taken together suggest that subtle differences exist in the organization of actin within MuLV-infected and uninfected mouse fibroblasts.

Amplified expression of murine leukemia virus (MuLV)-coded antigens on thymocytes and leukemia cells of AKR mice after infection by dualtropic (MCF) MuLV

Recently, we have shown that several isolates of recombinant, dualtropic (MCF) murine leukemia virus (MuLV) can induce amplification of MuLV gag and env gene-coded antigens on thymocytes of young AKR mice and, subsequently, can accelerate leukemia in these same animals. With respect to env gene-coded antigens, it is unclear whether antigen amplification represents expression of env gene products of the input virus on the surface of infected thymocytes or whether infection by MCF viruses induces the expression of other endogenous env gene sequences. Infection by antigenically marked viruses has allowed us to decide between these alternatives. We have found that AKR MCF isolates 69L1,13, and 247 can be distinguished serologically by specific patterns of reactivity with mouse and rat monoclonal antibodies that recognize 10 distinct epitopes (single antigenic determinants) of AKR ecotropic MuLV gp70 and p15(E) proteins. Accordingly, 1-monthold AKR mice were injected intrathymically with either MCF 69L1, MCF 13, or MCF 247 viruses and thymocytes of virus-injected mice were then assayed for amplification of specific epitopes at 32—36 days postinjection. Quantitative expression of MuLV antigens was determined by flow microfluorometry using a fluorescence-activated cell sorter. The patterns of reactivity with monoclonal antibodies which were originally determined for each virus in vitro were found to breed true on thymocytes infected in vivo. Moreover, the phenotype of virus recovered in vitro from virus accelerated leukemias was identical to that of the injected virus. Thus, the preleukemic change of MuLV antigen amplification appears to represent expression on thymocytes of env gene products encoded by the infecting MCF viral genome which continues to be expressed in the resulting leukemias without apparent changes in the recombinant env gene.

Cell cycle-dependent expression of surface antigens in murine T-lymphoma cells: II. Murine leukemia virus gp70 increases in S phase

The surface expression of the murine leukemia virus (MuLV) major envelope protein, MuLV gp70, is shown here to be cell cycle-dependent. AKR mouse T-lymphoma cells, BW 5147, synchronized by double thymidine blockade, were collected in S or G1 phase and lactoperoxidaselabeled with 125I and 131I. The iodinated surface proteins were solubilized with 0.05% Triton X-100 and aliquots of each preparation containing equal amounts of protein were mixed and precipitated by goat anti-Rauscher gp70 (70000 D MuLV envelope glycoprotein) serum and Staphylococcus aureus or by rabbit anti AKR gp70 serum and sheep anti-rabbit Ig serum. The precipitates were solubilized, reduced and separated in 9% SDS-PAGE gels. The gels were sliced and analyzed in a gamma-counter programmed for double-labeled samples. The gp70 molecule, precipitated by both antisera had the same quantitative cell cycle variability and the same pI heterogeneity as the 70 K antigen precipitated by an antiserum made against BW 5147 cell membranes at the G1 phase of the cell cycle. The expression of MuLV gp70 on BW 5147 cells is increased 2-fold in the S phase of the cell cycle as compared to the G1 and G2 phases. This increase is associated with one of the two gp70 components which has a lower isoelectric point and a lower apparent molecular weight.

Antigenic modulation as a mechanism for tumor escape from immune destruction: identification of modulation-positive and modulation-negative mouse lymphomas with xenoantisera to murine leukemia virus gp70.

Antigenic modulation as a mechanism for tumor escape from immune destruction: identification of modulation-positive and modulation-negative mouse lymphomas with xenoantisera to murine leukemia virus gp70.

Synthesis, processing, and cell surface expression of friend and xenotropic murine leukemia virus gp70 antigens on friend virus-induced erythroleukemia cell clones.

Synthesis, processing, and cell surface expression of friend and xenotropic murine leukemia virus gp70 antigens on friend virus-induced erythroleukemia cell clones.

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.

Murine leukemia virus proteins expressed on the surface of infected cells in culture.

Infection of JLS-V9 cells in culture with Rauscher murine leukemia virus induced the appearance on the cell surface of two classes of viral proteins: Rauscher murine leukemia virus gp70, and glycoproteins related to the viral core (gag) proteins with apparent molecular weights in sodium dodecyl sulfate polyacrylamide gels of 80 x 10(3) and 95 x 10(3). The latter proteins were identified by lactoperoxidase-catalyzed iodination of the cell surface and by metabolic labeling with [(3)H]mannose followed by immunoprecipitation with an antiserum directed against the major viral core protein, p30. Tryptic peptide maps of chloramine T-iodinated proteins indicated that 80 x 10(3) - and 95 x 10(3)-molecular-weight proteins were closely related. The 95 x 10(3)-molecular-weight protein from Rauscher murine leukemia virus-infected cells had a tyrosine fingerprint which was identical to that of the 95 x 10(3)-molecular-weight gag surface polyprotein of endogenous virus-producing AKR-A cells, suggesting that expression on the cell surface of glycosylated forms of gag precursor polyproteins may not be an exclusive property of leukemic thymocytes, but a more general phenomenon in murine leukemia virus infection. Tryptic fingerprint analysis of iodinated viral and cell-bound gp70's before and after desialylation indicated a lower level of glycosylation in the cell-bound gp70 population than in virions. Analysis of only surface-iodinated gp70 showed a simple pattern of exposed tryptic peptides which was very similar in Rauscher murine leukemia virus-infected cells and in AKR-A cells.