Abnormal glycosylation of the env-sea oncogene product inhibits its proteolytic cleavage and blocks its transforming ability.

Cells transformed by the avian erythroblastosis virus S13 contain three proteins derived from the v-sea oncogene, gp155, gp70 (a cleavage product of gp155), and p38. It is not clear whether only one or all three of these proteins are required for transformation by S13. S13 transformed erythroblasts and fibroblasts revert to a normal morphology in the presence of the alpha glucosidase-1 inhibitor castanospermine, whereas cells transformed by the v-src or v-erbB oncogenes are unaffected by this drug.

Temperature-sensitive v-sea transformed erythroblasts: a model system to study gene expression during erythroid differentiation.

The isolation and characterization of a temperature-sensitive mutant (ts1 S13) of the avian erythroblastosis virus, S13, is described. The temperature-sensitive lesion in ts1 S13 was identified as affecting the tyrosine kinase activity but not the plasma membrane localization of the ts1 S13 v-sea gene product. Erythroblasts transformed by ts1 S13 can be induced to synchronously differentiate into erythrocytes in an erythropoietin (EPO)-dependent fashion.

v-erbA specifically suppresses transcription of the avian erythrocyte anion transporter (band 3) gene.

Previous work has established that the v-erbA oncogene inhibits the temperature-induced differentiation of chick erythroblasts transformed with temperature-sensitive oncogene mutants. Here we demonstrate that v-erbA in differentiating erythroblasts specifically arrests expression of the erythrocyte anion transporter (band 3) gene at the transcriptional level. The v-erbA-induced differentiation block can be overcome by inducing cells to differentiate at alkaline pH.

Common site of mutation in the erbB gene of avian erythroblastosis virus mutants that are temperature sensitive for transformation.

The genome of the avian erythroblastosis virus temperature sensitive mutant ts34 was cloned from a cell line that was shown to contain a single integrated copy of the virus. The mutation was localized to the v-erbB gene by making chimeric viruses between the mutant genome and that of wildtype. Sequencing of the mutant v-erbB gene revealed a single amino acid change of a histidine to an aspartate residue at a position equivalent to amino-acid 826 of the human epidermal growth factor receptor.

Control of erythroid differentiation: possible role of the transferrin cycle.

A monoclonal antibody to the chicken transferrin receptor (JS-8) blocked temperature-induced and spontaneous differentiation of avian erythroid cells transformed by ts- and wt-retroviral oncogenes. In cells committed to differentiate, JS-8 caused an arrest at the erythroblast or early reticulocyte stage, followed by premature cell death, whereas proliferation of noncommitted erythroid cells or other hematopoietic cells remained unaffected. JS-8 had no effect on transferrin binding or internalization, but blocked subsequent receptor-recycling resulting in reduced iron uptake.

Primitive series embryonic chick erythrocytes express the transferrin receptor.

A monoclonal antibody specific for the chicken transferrin receptor was used to study receptor expression on circulating red cells from chick embryos of different ages. The use of indirect immunofluorescence with this antibody showed that all circulating immature reticulocytes and primitive series erythrocytes--but not erythrocytes from the definitive series--expressed the receptor. In all cells, the protein was synthesized as a 90-95-kD form.

Analysis of the autophosphorylation activity of transformation defective mutants of avian erythroblastosis virus.

The v-erb B protein of avian erythroblastosis virus (AEV) possesses an associated protein kinase activity in vitro. Analysis of temperature-sensitive mutants, and nonconditional host range mutants of AEV demonstrated that there was no simple correlation between this autophosphorylation activity and the transformation ability of the various AEV mutants. These data suggest that although this kinase activity may be central to transformation by AEV it is in itself insufficient.

Rous-associated virus 1-induced erythroleukemic cells exhibit a weakly transformed phenotype in vitro and release c-erbB-containing retroviruses unable to transform fibroblasts.

Avian leukosis viruses induce erythroblastosis in chicks by integrating into the c-erbB gene and thus activating c-erbB transcription. We characterized Rous-associated virus 1-induced leukemic erythroblasts in vitro and showed that they mostly resemble erythropoietin-independent but otherwise normal erythroid progenitors. Some leukemic cells, however, were able to both differentiate and proliferate extensively in vitro.

Monoclonal antibodies to novel erythroid differentiation antigens reveal specific effects of oncogenes on the leukaemic cell phenotype.

A panel of new monoclonal antibodies to antigens on the surface of chick erythroid progenitor cells is described. These are characterised with respect to their binding to different classes of normal haemopoietic cells of both the erythroid and myeloid lineages. Using these antibodies, we have examined the phenotype of avian leukaemic cells transformed by retroviruses carrying defined oncogenes.

Two new retroviral onc genes, sea and jun.

Avian leukemia virus S13 induces erythroblastosis, granulocytic leukemia, fibrosarcoma, anemia, and endothelial neoplasia. It transforms chick embryo fibroblasts and primitive erythroid cells in culture and is defective in replication. Its onc gene, sea, is expressed as transformation specific env-sea fusion glycoprotein of 155 kDa.

Identification and characterization of the chicken transferrin receptor.

Among a panel of monoclonal antibodies that recognize chicken haematopoietic-differentiation antigens, one, JS 8, was found to immunoprecipitate a 95000-Mr cell-surface protein from chicken erythroblasts transformed with avian-erythroblastosis virus. This protein was shown, by affinity chromatography on immobilized chicken transferrin (conalbumin), to be the chicken transferrin receptor. Although immunologically unrelated to the human transferrin receptor, biochemical comparison of the chicken transferrin receptor to the human receptor showed similarities with respect to the pattern of biosynthesis, degree of glycosylation, dimerization in the absence of reducing agents and subcellular localization.

Activation of c-erbB in avian leukosis virus-induced erythroblastosis leads to the expression of a truncated EGF receptor kinase.

Chicken erythroblastosis caused by avian leukosis virus (ALV) is thought to be mediated by activation of the c-erbB/EGF receptor oncogene by a promoter-insertion mechanism. Here we study the proteins expressed by two ALV-induced leukemias and compare them with the avian EGF receptor and with the oncogene product of avian erythroblastosis virus (v-erbB) which was shown to be a truncated EGF receptor. It appears that the two leukemias express truncated EGF receptors of slightly different sizes with intrinsic tyrosine kinase activity.

The putative transforming protein of S13 avian erythroblastosis virus is a transmembrane glycoprotein with an associated protein kinase activity.

S13 is an avian retrovirus that transforms both fibroblasts and erythroblasts. The gene product responsible for the oncogenic effects of S13 is the env-related glycoprotein gp155. In this report we show that gp155 is a transmembrane protein with a 55-kDa cytoplasmic domain.

Nucleotide sequence of HBI, a novel recombinant MC29 derivative with altered pathogenic properties.

HBI is a recombinant avian retrovirus with novel pathogenic properties that was derived from the myc-containing virus MC29. In contrast to MC29, which causes endotheliomas in chickens, HBI induces lymphoid tumors. The results of molecular cloning and nucleotide sequencing of HBI reported here show that the virus contains sequences derived from both c-myc and ring-neck pheasant virus, in addition to MC29.

Analysis of a deleted MC29 provirus: gag sequences are not required for fibroblast transformation.

Recovered avian myelocytomatosis virus HBI is an MC29-related virus that induces lymphoid tumors in chickens rather than the predominant neoplastic disease induced by wild-type MC29 (namely, endotheliomas). An analysis of the structure of the HBI provirus(es) in the tumors demonstrated that the provirus(es) could be either full size or deleted. One tumor was found to be clonal in that it contained a single provirus which had been partially deleted; this raised a question concerning the role of this provirus in the maintenance of tumor growth.

Molecular analysis of myc gene mutants.

We describe the generation and characterization of a series of deletion mutants of the avian acute leukaemia virus MC29 which allow the study of the function of the myc in transformation of quail embryo fibroblasts in vitro and tumour induction in vivo. These mutants, which are deleted in the 3' portion of the myc gene, fail to transform macrophages in vitro or induce tumours in vivo but are still able to transform morphologically fibroblasts. From one of these mutants a 'recovered' MC29 virus was generated which, like wild type MC29, transformed fibroblasts and macrophages in vitro.

How do retroviral oncogenes induce transformation in avian erythroid cells?

The v-erb B oncogene, as well as other oncogenes of the src-gene family transform immature erythroid cells from chick bone marrow in vivo and in vitro. The erb B-transformed erythroid cells differ from normal late erythroid precursors (CFU-E) in that they have acquired the capacity to undergo self-renewal as well as to differentiate terminally. They also do not require the normal erythroid differentiation hormone, erythropoietin, for either process.

S13, a rapidly oncogenic replication-defective avian retrovirus.

The avian leukemia sarcoma virus S13 transforms chicken and Japanese quail embryo fibroblasts and chicken erythroid cells in tissue culture. S13-induced erythroid transformation requires culture conditions suitable for the growth of normal erythroid precursors (H. Beug and M.

Effects of inhibitors of glycoprotein processing on the synthesis and biological activity of the erb B oncogene.

Three glycoprotein-processing inhibitors were used to resolve whether correct glycosylation was required for the oncogenic activity of erb B. The two glucosidase-I inhibitors, 1-deoxynojirimycin and 2,5-dihydroxymethyl 3,4-dihydroxypyrrolidine, arrested processing of v-erb B at the immature 68-kd form whereas, in the presence of the alpha-mannosidase-II inhibitor (swainsonine), cells synthesised an abnormally processed 70-kd form of v-erb B. Transport of incorrectly processed v-erb B to the cell surface was, however, unaffected, suggesting that correct processing is not a prerequisite for intracellular routing of v-erb B.

Transformation of mammalian fibroblasts and macrophages in vitro by a murine retrovirus encoding an avian v-myc oncogene.

A murine retrovirus which expresses the avain v-myc OK10 oncogene was constructed. The virus, denoted MMCV, readily transforms fibroblasts of established lines, such as mouse NIH/3T3 and rat 208F cells, to anchorage-independent growth in agarose. The virus also transforms primary mouse cells: (i) virus-infected macrophages are induced to form large colonies in semi-solid media, and can easily be expanded into mass cultures; (ii) MMCV-infected fibroblastic cells from mouse limb buds undergo morphological transformation and grow in semi-solid medium.

Identification of a form of the avian erythroblastosis virus erb-B gene product at the cell surface.

Avian erythroblastosis virus (AEV) induces both erythroblastosis and fibrosarcoma in chickens. The viral oncogene responsible for these diseases, erb, is divided into two regions, erb-A and erb-B, although recent evidence suggests that it is primarily the erb-B gene product that is responsible for the transforming activity. The erb-B gene product has been reported previously to be a membrane glycoprotein of 68,000 molecular weight (MW), gp68erb -B.

Comparison of genome structures among three different strains of avian erythroblastosis virus.

The genomic DNAs of two strains of avian erythroblastosis virus (AEV), AEV-R and AEV-H, were molecularly cloned in Escherichia coli using lambda Charon 16A as a vector. Comparison of the restriction maps of the cloned DNAs with that of AEV-ES4 DNA revealed that R strain is identical with ES4 strain, but totally differs from H strain. The erbB gene product of AEV-R and that of AEV-H were shown to be glycoproteins with molecular weights of 68,000 and 72,000, respectively.