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Biomedical subjects

D Kabat

Publications and source records attributed to D Kabat.

At least 91 records · Page 5Linked to original sources
PubMed1985

Role of a membrane glycoprotein in Friend virus-induced erythroleukemia: studies of mutant and revertant viruses.

We previously reported the isolation and characterization of spontaneous, transmissible mutants of Friend spleen focus-forming virus (SFFV) that are nonpathogenic in adult NIH/Swiss mice and that contain abnormalities in nonoverlapping regions of their envelope glycoprotein (env) genes (M. Ruta, R. Bestwick, C. Machida, and D. Kabat, 1983, Proc. Natl. Acad. Sci. USA 80, 4704-4708). In newborn NIH/Swiss mice, these mutant SFFVs form revertants that are pathogenic in mice of all ages. At least two of three studied revertants contain second site env mutations which affect the sizes and proteolytic fragmentation patterns of their encoded glycoproteins. A variety of structural and genetic evidence suggests that the xenotropic- and ecotropic-related regions of the SFFV glycoprotein fold into separate globular domains that are connected by a flexible proline-rich joint. A glutamyl peptide bond within this joint is exceptionally susceptible to cleavage with Staphylococcus aureus V8 protease. Moreover, disulfide bonds occur within the xenotropic-related domain, but not between the globular domains. These results provide strong additional evidence that the env gene is required for SFFV pathogenesis, and they provide a new system for identifying the features of glycoprotein structure and localization which are essential for its leukemogenic activity.

Animals↗
PubMed1985

Cell surface receptors for murine leukemia viruses: two assays and their implications.

Two assays were developed for identifying individual cells which bear murine leukemia virus receptors: an erythrocyte rosette assay for ecotropic receptors, and an efficient immune cytotoxic assay for cells with ecotropic or amphotropic receptors. Both assays indicate that ecotropic MuLV adsorbed to its cell surface receptor only slowly becomes internalized. Furthermore, attempts to isolate murine fibroblast variants lacking these ecotropic MuLV receptors were unsuccessful, suggesting either that mutations in the receptor gene are rare (less than 10(-7) per generation) or that the receptor is required for cell viability. These assays are rapid and can be used to identify receptor-bearing cells in mixed populations, a prerequisite for molecular genetic studies.

Animals↗
PubMed1985

Cell anchorage determines whether mammary tumor virus glycoproteins are processed for plasma membranes or secretion.

The subcellular localization of mouse mammary tumor virus (MMTV) glycoproteins was analyzed in infected and cloned rat hepatocarcinoma cells cultured with the MMTV transcriptional inducer dexamethasone. When reacted with protein A-coated erythrocytes in the presence of antisera specific for viral glycoproteins or with fluorescent antisera, only some of the cells acquired surface label. This diversity was dependent on cell anchorage to the substratum. In general, the more rounded, less adherent cells contained the MMTV glycoproteins on their surfaces, whereas the flatter, more adherent cells did not. After a change in adherence, a delay preceded complete remodeling of the plasma membranes. Fluorescent antibody studies of fixed cells and analyses of viral glycoprotein synthesis and shedding using L-[35S]methionine indicated that the different expression of MMTV glycoproteins in round versus flat cells is caused by a switch in posttranslational processing. In round cells, the MMTV-encoded precursor glycoprotein is proteolytically cleaved and then transported to plasma membranes as a complex of two subunits, the smaller being the membrane anchor. In flat adherent cells, the smaller subunit is rapidly degraded in an intracellular organelle and the larger is then secreted into the medium. As indicated by labeling of cells with 125I, the concentrations of several host-encoded plasma membrane components are also influenced by cell anchorage. We propose that this switch in cell surfaces and in secretions dependent upon cell-substratum attachments may be a common control mechanism important for embryogenesis, wound healing, and cancer.

Animals↗
PubMed1985

A weakly pathogenic Rauscher spleen focus-forming virus mutant that lacks the carboxyl-terminal membrane anchor of its envelope glycoprotein.

A mutant Rauscher spleen focus-forming virus (mutant 4-3) that causes mild splenic erythroblastosis in mice has a 44-base-pair deletion in the 3' region of its envelope glycoprotein (env) gene. The encoded glycoprotein terminates prematurely, lacks a hydrophobic membrane anchor, and has a shortened intracellular lifespan. An active site for causing erythroblast proliferation may occur in the undamaged amino-terminal domain of the env glycoprotein.

Base Sequence↗
PubMed1985

Roles of helper and defective retroviral genomes in murine erythroleukemia: studies of spleen focus-forming virus in the absence of helper.

Retroviruses that cause acute oncogenesis are generally complexes of a replication-competent helper virus and a replication-defective component. However, the pure defective components have not been previously available. We prepared the defective spleen focus-forming virus component of Rauscher erythroleukemia virus (R-SFFV) by transfecting a colinear R-SFFV DNA clone into a retroviral packaging cell line (psi 2 cells). The transfected cells released virus (psi 2/SFFV) that was free of helper virus and that induced erythropoietin-dependent erythroid burst formation in bone marrow cultures. When injected into normal adult NIH/Swiss mice in moderate doses, psi 2/SFFV caused a rapid splenic erythroblastosis that regressed. Extensive erythroblastosis could be maintained by repeated injections of psi 2/SFFV into anemic mice or by the addition of a helper virus. We conclude that R-SFFV alone causes proliferation but not immortalization of a population of erythroblasts that is normally replenished from a precursor stem cell pool. Because these precursor cells are inefficiently infected, a single moderate inoculum of psi 2/SFFV causes a wave of erythroblastosis. The properties of the proliferating erythroblasts are substantially determined by the R-SFFV viral component.

Animals↗
PubMed1984

Reduced leukemogenicity caused by mutations in the membrane glycoprotein gene of Rauscher spleen focus-forming virus.

We isolated and characterized two spontaneous, weakly leukemogenic mutants of Rauscher spleen focus-forming virus (R-SFFV) that contain mutations in nonoverlapping regions of the membrane envelope (env) glycoprotein gene. As reported previously (M. Ruta and D. Kabat, J. Virol. 35:844-853, 1980), the replication-defective R-SFFV encodes a membrane glycoprotein with an apparent Mr of 54,000 (gp54) which is structurally and immunologically related to the membrane envelope glycoproteins of dual-tropic murine leukemia viruses. Mutant R-SFFV clones 3-25 and 4-3 encode abnormally sized gp54-related glycoproteins with apparent Mrs of 52,000 (gp52) and 45,000 (gp45), respectively. Northern and Southern blot analyses of the mutant R-SFFV nucleic acids indicated that an insertion has occurred in the 3-25 env gene and that a deletion has occurred in the 4-3 env gene. Furthermore, restriction endonuclease analyses and comparisons of the fragmentation patterns of the wild-type and mutant glycoproteins generated by partial proteolysis with Staphylococcus aureus V8 protease indicated that the mutations affect nonoverlapping domains of the envelope glycoprotein (amino-terminal fragment affected in 3-25 glycoprotein and carboxyl-terminal fragment affected in 4-3 glycoprotein). Glycosylation inhibition studies indicated that the reduced size of gp52 is caused at least partly by loss of an asparagine-linked oligosaccharide. In addition, these mutant viruses have dramatically reduced leukemogenicities compared with wild-type R-SFFV. We conclude that the gp54 structural gene is required for initiation or amplification of the splenic erythroblast hyperplasia which characterizes the preleukemic phase of Rauscher disease.

Animals↗
PubMed1984

Molecular cloning of biologically active Rauscher spleen focus-forming virus and the sequences of its env gene and long terminal repeat.

Rauscher and Friend spleen focus-forming viruses (R- and F-SFFVs) cause similar progressive erythroleukemias dependent upon a virus-encoded membrane glycoprotein. Moreover, these SFFV glycoproteins are immunologically related to each other and to the recombinant-type glycoproteins encoded by the env genes of dual tropic murine leukemia viruses. To better understand these diseases and the viral origins, we isolated a pathogenically active molecular clone of R-SFFV proviral DNA, sequenced its 3'-terminal 2,163-base-pair (bp) region, and compared these sequences with previously determined sequences of F-SFFV. The 516-bp R-SFFV long terminal repeat is highly homologous to those of F-SFFV and Friend murine leukemia virus, although only the latter contains a 65-bp direct repeat in its U3 region. The env gene of R-SFFV encodes a glycoprotein with 408 amino acids that is identical in its basic domain organization to the glycoprotein of F-SFFV. Thus, the junctions between the dual tropic-related and ecotropic sequences occur at the same nucleotide, and both SFFV env genes contain identical 585-bp deletions in their ecotropic domains and single-bp insertions which cause premature terminations at the same amino acid in their ecotropic p15E domains. Consistent with their independent origins, however, the env sequences of R- and F-SFFV are distinctive in both their 5' dual tropic-related and 3' ecotropic-related domains. Furthermore, there are several consistent amino acid differences between the polycythemic F-SFFV sequences and the anemia-inducing R-SFFV sequence. The striking similarities of the independently formed F- and R-SFFV env genes imply that all of the glycoprotein domains arranged in a precise organization may be required for its leukemogenic activity

Amino Acid Sequence↗
PubMed1984

Frequent hereditable shutdown of murine retrovirus gene expression in murine cell lines.

Friend spleen focus-forming virus shuts down its gene expression frequently (ca. 10(-3) per generation) in a cis-dominant hereditable fashion in various murine cells but much less frequently in rat cells (less than 10(-6) per generation). Thus, nonexpresser variants were isolated at high frequency from murine cell lines by immunoselection directed against virus-encoded cell surface glycoproteins and also simply by subcloning cells from lines which had been cultured for many generations. Studies of independently infected cell clones indicate that shutdown is a property of the cell line rather than of the specific proviral site. Nucleic acid blot analyses suggest that shutdown correlates with decreased transcription. Moreover, preliminary evidence indicates that other murine retroviruses also shut down frequently in murine but not in rat cells and that shutdown of replication-competent murine leukemia viruses with accompanying loss in interference to superinfection may be the rate-limiting reaction enabling cells to acquire multiple proviruses in their chromosomes. High-frequency shutdown in vivo would have important pathological consequences.

Animals↗
PubMed1983

Loss of leukemogenicity caused by mutations in the membrane glycoprotein structural gene of Friend spleen focus-forming virus.

Friend virus infection of mice causes progressive leukemogenesis--a rapid splenic erythroblastosis that develops weeks later into a disseminating erythroleukemia. Furthermore, the replication-defective Friend spleen focus-forming virus (F-SFFV) encodes a membrane glycoprotein with an apparent Mr of 55,000 (designated gp55), which is structurally and immunologically related to the membrane envelope glycoproteins of dual tropic murine leukemia viruses. We now have isolated three spontaneous F-SFFV mutants that encode abnormally sized gp55-related glycoproteins with apparent Mrs of 40,000, 54,000, and 58,000, respectively. RNA blot and Southern blot analyses indicate that the mutant nucleic acids do not have substantial deletions or insertions in their glycoprotein gene regions. Protein fragmentation patterns indicate that the mutations affect nonoverlapping domains of the glycoprotein. Furthermore, these mutant glycoproteins seem to be defective in their processing to the plasma membranes. Although transmitted efficiently between cultured cells, the mutants have dramatically reduced leukemogenicities compared with the same titers of wild-type F-SFFV. We conclude that the gp55 structural gene is necessary for initiating the erythroblast proliferative phase of Friend disease and that changes in membranes can be primary causes rather than only secondary consequences of tumor progression.

Animals↗
PubMed1982

Glycosylation and intracellular transport of membrane glycoproteins encoded by murine leukemia viruses. Inhibition by amino acid analogues and by tunicamycin.

Addition of asparagine-linked oligosaccharides to nascent murine leukemia virus (MuLV)-encoded membrane glycoproteins was inhibited either completely by tunicamycin or specifically at Asn-X-Thr glycosylation sites by incorporation of the threonine analogue beta-hydroxynorvaline. In conditions of partial analogue substitution, a series of subglycosylated components is formed which are related by a constant apparent Mr difference when assayed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The total number of asparagine-linked oligosaccharides is then estimated by dividing the measured apparent Mr of one oligosaccharide into the total apparent Mr difference between the complete glycoprotein and the polypeptide chain that is synthesized in cells incubated with tunicamycin. Correct results were obtained using glycoproteins with known numbers of oligosaccharides. Our analyses indicate that the gp70 membrane envelope glycoproteins of certain ecotropic MuLVs contain seven oligosaccharides, whereas the GIX+ antigen-containing variant gp70 contains one fewer Asn-X-Thr-linked oligosaccharide. The membrane glycoprotein encoded by the gag gene of Friend MuLV contains only one asparagine-linked oligosaccharide. Similarly, the gp55 membrane glycoprotein encoded by Friend erythroleukemia virus contains four asparagine-linked oligosaccharides. Pulse-chase and cell surface iodination analyses indicate that MuLV membrane envelope glycoprotein processing by partial proteolysis and transport to the cell surface can be efficiently blocked by structural perturbations caused by incorporation of different amino acid analogues or by loss of oligosaccharides. Our data also suggest that loss of oligosaccharides may expose new antigenic sites in viral membrane glycoproteins and increase their susceptibility to intracellular proteolysis.

Amino Acids↗
PubMed1982

Role of partial proteolysis in processing murine leukemia virus membrane envelope glycoproteins to the cell surface. A viral mutant with uncleaved glycoprotein.

We have isolated a mutant Rauscher murine leukemia virus (R-MuLV) with a mutation in its envelope (env) glycoprotein gene. This mutant encodes a membrane glycoprotein with an apparent Mr = 80,000 (gPr80env) that contains both gp70 and p15E antigenic determinants found in the larger wild type R-MuLV env precursor molecule gPr90env. Glycosylation inhibition and peptide mapping analyses indicate that the smaller size of the mutant glycoprotein is caused by a shortening of its polypeptide chain rather than by reduced glycosylation. Unlike gPr90env of wild type R-MuLV which contains Asn-linked high mannose oligosaccharides and is processed by partial proteolysis and by further glycosylation in the Golgi apparatus to produce gp70 plus p15E, the mutant glycoprotein can reach the cell surface without proteolysis. The uncleaved plasma membrane component, which undergoes further glycosylation during its transit through the Golgi apparatus, has an apparent Mr = 85,000. Furthermore, this cell surface glycoprotein is incorporated into released virions which are infectious. However, the mutant envelope glycoproteins on the cell surface do not block the receptors needed for superinfection by wild type MuLV. These results indicate that transport of uncleaved env gene-encoded glycoproteins to the cell surface is not a unique attribute to leukemia-producing recombinant of dual tropic MuLVs (Famulari, N. G., and English, J. K. (1981) J. Virol. 40, 971-976) but can also occur with a mutant of ecotropic MuLV.

Animals↗
PubMed1981

Mutant cells that abnormally process plasma membrane glycoproteins encoded by murine leukemia virus.

Wild-type normal rat kidney fibroblasts infected with the Friend strain of murine leukemia virus (MuLV) contain two virus-encoded glycoproteins on the outer surfaces of their plasma membranes: an envelope glycoprotein with an apparent molecular weight of 70,000 (gp70), and a glycoprotein that reacts with antisera to the major virion internal core proteins p30, p15, p12 and p10 and has an apparent molecular weight of 93,000 (gp93gag). To analyze the functions of these glycoproteins and to develop a model system for studying genetics of membrane synthesis, we used an immunoselection method to isolate variant cell clones defective in processing these glycoproteins into their plasma membranes. Several lines of evidence, including complementation of glycoprotein processing defects by fusion with uninfected wild-type cells, indicate that the immunoselected variants have stably inherited membrane synthesis abnormalities that are encoded by cellular rather than by viral genes. The H-4 cell line, which was selected by use of antiserum to gp70, has metabolic defects that interfere with processing of both gp70 and gp93gag into its plasma membranes. Nevertheless, this cell line releases noninfectious MuLV. Furthermore, two cell lines (2 and 5), which were selected by use of antiserum to the virion core protein p30, specifically lack detectable cell surface or intracellular gp93gag but contain cell surface gp70 and release infectious MuLV. These results suggest that MuLV particles can bud efficiently from cells that lack known virus-encoded plasma membrane constituents.

Animals↗
PubMed1980

Immunoselection of mutants deficient in cell surface glycoproteins encoded by murine erythroleukemia viruses.

We have described a heterogeneously processed glycoprotein with an apparent molecular weight of 55,000 (gp55) that is encoded by the Friend spleen focus-forming virus, an acute erythroleukemia virus [Dresler, S., Ruta, M., Murray, M.J. & Kabat, D. (1976) J. Virol. 30, 564-573]. Several lines of evidence suggest that a small proportion of the gp55 in infected cells is located on the surface membranes. First, different nonproducer cell lines infected with cloned Friend spleen focus-forming virus were efficiently killed in the presence of complement by cytotoxic antisera that react with gp55. Furthermore, a clone of cells selected for resistance to the cytotoxic antibody synthesized an altered intracellular form of gp55. This immunoselection procedure appears to also be useful for isolating glycoprotein mutants of other RNA tumor viruses. Analysis of cell surface proteins labeled with [125I]iodine supported the idea that gp55 occurs on the plasma membranes. However, the cell surface gp55 had more highly processed oligosaccharides than the majority of the gp55, which occurs within the infected cells. In addition, we have found that leukemia cells from mice infected with the erythroleukemic Rauscher virus complex contain a membrane glycoprotein that appears similar to the gp55 encoded by Friend spleen focus-forming virus. The encoding of similar glycoproteins by independently isolated acute erythroleukemia viruses suggests that these glycoproteins may be important in leukemogenesis.

Animals↗