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Revertants of rats cells transformed by avian erythroblastosis virus.

Morphological revertants of the avian erythroblastosis virus (AEV)-transformed rat cell line ATla were isolated and characterised. The revertants are similar to the uninfected parental rat cell line in that they have regained an organized cytoskeleton and they are no longer capable of anchorage-independent growth. The pattern of integrated viral DNA in the revertants is indistinguishable from that of the transformed parent. However, the revertants do not express the integrated viral genome at either the mRNA or protein level. Phenotypic reversion thus is probably .due to reduced transcription of the AEV-transforming gene below a threshold necessary to induce morphological transformation.

Alpharetrovirus↗

Site-specific mutagenesis of avian erythroblastosis virus: v-erb-A is not required for transformation of fibroblasts.

Avian erythroblastosis virus (AEV) is an acutely transforming retrovirus whose putative oncogenes (v-erb-A and v-erb-B) encode the proteins P74gag-erb-A and P61-68erb-B. The existence of these two gene products has prompted the question of whether one or both proteins are required in the transformation of erythroblasts and fibroblasts by AEV. In the accompanying manuscript, we describe the use of site-specific mutagenesis to generate mutants of AEV unable to synthesize P61-68erb-B. Here we present our analysis of the oncogenic potential of an AEV mutant unable to synthesize P74gag-erb-A due to a large deletion encompassing both gag and v-erb-A sequences. The erb-A-mutant retrovirus propagated quite poorly on fibroblasts in culture; however, fibroblasts harboring the erb-A mutant genome were transformed in the absence of P74gag-erb-A expression. The mutant virus failed to induce erythroleukemias in chickens, but the validity of this finding is compromised by the poor replicative capacity of the mutant. The results presented in this and the preceding manuscript indicate that P61-68erb-B is both necessary and sufficient for neoplastic transformation of fibroblasts by AEV; by contrast, a role for p74gag-erb-A in leukemogenesis by AEV has not yet been rigorously excluded.

Alpharetrovirus↗

Subcellular localization of the v-erb-B protein, the product of a transforming gene of avian erythroblastosis virus.

Avian erythroblastosis virus (AEV) is an oncogenic retrovirus capable of transforming both fibroblasts and immature erythroid cells. The v-erb-B locus within the AEV genome encodes a glycosylated protein, expression of which is required for oncogenic transformation of either cell type. Subcellular localization of the v-erb-B glycoprotein in AEV-transformed cells is reported here. Results indicate that the v-erb-B protein is synthesized on dense membrane fractions and appears to possess the properties of an integral membrane protein. The bulk of the v-erb-B protein remains with dense membranes after synthesis, although a small quantity may slowly become associated with the plasma membrane. The biogenesis and subcellular location of the v-erb-B protein are thus quite different from those of the transforming proteins that display protein kinase activity. These differences are especially provocative because the amino acid sequences of the v-erb-B protein and the protein kinases are closely related to one another.

Alpharetrovirus↗

Embryonic erythroid cells transformed by avian erythroblastosis virus may proliferate and differentiate.

Embryonic chick cells from the primitive streak stage to later stages of the developing embryo were infected with avian erythroblastosis virus (AEV). The data indicate that the greatest number of target cells for AEV was observed in the 12-somite blastoderm and gradually decreased in hemopoietic tissues with the development of the embryo. The target cell for AEV is not in the BFU-E compartment, as it is in the adult bone marrow, but is probably recruited within the CFU-M compartment which precedes the BFU-E compartment. Our studies also show that a significant number of transformed colonies derived from embryonic hemopoietic tissues undergo hemoglobinization in contrast with what is observed in transformed colonies of bone marrow. A complete characterization of the embryonic and adult hemoglobin is at present under study.

Alpharetrovirus↗

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.

Alpharetrovirus↗

Identification of two additional v-sea-encoded proteins in avian erythroblastosis virus, S13-infected fibroblasts.

Rabbit antibodies prepared against a v-sea-encoded polypeptide expressed in bacteria were used to characterize the v-sea-encoded proteins in cells transformed by the avian erythroblastosis virus, S13. In addition to the two previously described v-sea-encoded proteins, gp155 and gp70, two additional proteins were identified of molecular weights 38,000 and 36,000 Da. Interestingly, these two proteins were found only in fibroblasts infected with the S13 virus and not in S13-transformed erythroid cells. These two proteins were phosphoproteins, but, unlike the two previously characterized v-sea-encoded proteins, they did not appear to be modified by the addition of N-linked sugars. Possible mechanisms for the biosynthesis of these two new proteins are discussed.

Alpharetrovirus↗

Cell-surface glycoprotein synthesis during differentiation of chicken erythroblasts transformed by temperature-sensitive avian erythroblastosis virus.

Chicken erythroblasts transformed by a temperature-sensitive mutant of avian erythroblastosis virus (ts34 AEV) have a greatly increased haemoglobin content (Graf, T., N. Ade and H. Beug: Nature 275, 496-501 (1978)) if allowed to grow for 3-5 days at the non-permissive temperature (41 degrees C), instead of the permissive temperature (35 degrees C) of the virus. Cell-surface molecular changes during this differentiation were investigated by examining the glycoproteins synthesized by a ts34-transformed erythroblast cell line. These cells synthesized a greatly increased amount of a 94,000 molecular weight erythrocyte cell-surface glycoprotein beginning 2-6 h after a shift in growth temperature from 35 degrees to 41 degrees C, consistent with the proposal that such a shift releases these transformed cells from a differentiation block.

Alpharetrovirus↗

Avian myelocytomatosis and erythroblastosis viruses lack the transforming gene src of avian sarcoma viruses.

Using labeled cDNA specific for the detection of the src gene of avian sarcoma viruses, we find that avian myelocytomatosis virus strain MC29 and avian erythroblastosis virus strain ES4 lack nucleotide sequences related to the src gene. Furthermore, chicken fibroblasts as well as hematopoietic cells, infected and transformed with these viruses, show no enhanced level of transcription of the cellular nucleotide sequences related to the src gene of avian sarcoma viruses. These two viruses may thus contain their own transforming gene(s) or induce cellular genes unrelated to the src-like cellular sequences.

Alpharetrovirus↗

Early precursors in the erythroid lineage are the specific target cells of avian erythroblastosis virus in vitro.

In chickens the erythroid differentiation proceeds from stem cells to erythrocytes through several intermediate steps which have been identified in vivo and in vitro. To determine whether Avian erythroblastosis virus (AEV) is able to transform in vitro either one or several types of these precursors, bone marrow cells were separated by physical and immunological methods. It was found that the target cells which could be transformed in vitro by AEV were cells of light density (1.060-1.065 g/cm3), having a modal sedimentation velocity at unit gravity between 4.0 and 6.0 mm/hr, expressing an immature antigen at a low level and a brain-related antigen at a high level. These results indicated that the target cells of neoplastic transformation by AEV were early erythroid precursors, since these precursors shared the same physical and immunological properties with AEV target cells.

Alpharetrovirus↗

Hormone-dependent terminal differentiation in vitro of chicken erythroleukemia cells transformed by ts mutants of avian erythroblastosis virus.

Chicken erythroblast cell strains and a cell line transformed by ts mutants of avian erythroblastosis virus (AEV) terminally differentiate when shifted to the nonpermissive temperature (42 degrees C). The differentiated cells resemble mature erythrocytes with respect to morphology and ultrastructure, expression of differentiation-specific cell-surface antigens, pattern of protein synthesis and hemoglobin content. Terminal differentiation is dependent on conditions favoring the differentiation of normal erythroid progenitor cells, including an erythropoietin-like factor. Colonies of ts AEV cells grown at 42 degrees C semisolid medium resemble erythrocyte colonies derived from normal erythroid progenitor cells. The colonies obtained were comparable in size or slightly larger than the late erythroid precursor (CFU-E) colonies. These results suggest that AEV-transformed cells are blocked at a stage of differentiation that is more advanced than that of the uninfected target cells. ts AEV cells are irreversibly committed to terminal differentiation within 20 to 30 hr after shift to 42 degrees C.

Alpharetrovirus↗

Target cells infected by avian erythroblastosis virus differentiate and become transformed.

Transformation in vitro of bone marrow cells by avian erythroblastosis virus (AEV) gives rise to rapidly growing cells of erythroid nature. Target cells of neoplastic transformation by AEV are recruited among the early progenitors of the erythroid lineage, the burst-forming units-erythroid (BFU-E). They express a brain-related antigen at a high level and an immature antigen at a low level. We show that AEV-transformed cells express low levels of the brain antigen and high levels of the immature antigen. Their response to specific factors regulating the erythroid differentiation indicates that they are very sensitive to erythropoietin. Furthermore, cells transformed by a temperature-sensitive mutant of AEV differentiate into hemoglobin-synthesizing cells 4 days after being shifted to the nonpermissive temperature. All these properties are similar to those of late progenitors of the erythroid lineage, the colony-forming units-erythroid (CFU-E). These results indicate that the AEV-transformed cells are blocked in their differentiation at the CFU-E stage.

Alpharetrovirus↗

Isolation and characterization of chicken DNA homologous to the two putative oncogenes of avian erythroblastosis virus.

The genome of avian erythroblastosis virus contains two independently expressed genetic loci (v-erbA and v-erbB) whose activities are probably responsible for oncogenesis by the virus. Both loci are closely related to nucleotide sequences found in the DNA and RNA of chickens and other vertebrates. We have isolated and characterized chicken DNA homologous to v-erbA and v-erbB. The two viral genes are represented by separate domains within chicken DNA (c-erbA and c-erbB), which are separated by a minimum of 12 kilobases (kb) of DNA and may not be linked at all. The nucleotide sequences shared by the viral and cellular erb loci are colinear, but the cellular loci are interrupted by multiple intervening sequences of various lengths. Polyribosomes prepared from normal chicken embryos contain two polyadenylated RNAs transcribed from c-erbA and two transcribed from c-erbB. The evident coding regions of these RNAs represent an unusually small fraction of the lengths of the RNAs, as if the 3' untranslated domains of the RNAs might be exceptionally large (3-11 kb). These findings indicate that the c-erb loci are normal vertebrate genes rather than genes of cryptic endogenous retroviruses, and that they may have a role in the metabolism of normal cells. It appears that the viral erb genes, like most other retrovirus oncogenes, have been copied from cellular genes. In the viral genome, the two genes are devoid of introns, but they remain independently expressed loci, and they remain colinear with the coding domains of their cellular progenitors.

Alpharetrovirus↗

Transforming capacities of avian erythroblastosis virus mutants deleted in the erbA or erbB oncogenes.

Mutants of avian erythroblastosis virus (AEV) were constructed by deleting large nucleotide segments in each of the viral oncogenes termed v-erbA and v-erbB. Mutants in erbA (erbA -B +) retained the ability to transform fibroblasts in vitro, and these cells exhibited most of the transformation characteristics that typify wild-type AEV-transformed fibroblasts. In addition, the mutants induced small erythroid colonies upon infection of bone marrow cells in culture. Chickens inoculated with erbA -B + virus or with erbA -B +-transformed cells developed sarcomas or atypical erythroid leukemias. The erythroid cells transformed in vivo or in vitro by the erbA -B + viruses appeared not to be as tightly blocked in differentiation as wild-type transformed cells. In contrast, fibroblasts infected with the erbA +B - mutant resembled normal cells in all transformation parameters tested, and no bone marrow cell transformation was observed with the mutant. The results indicate that the main transforming properties of AEV are encoded in erbB and that its effects are enhanced by erbA.

Alpharetrovirus↗

Temperature-sensitive mutants of avian erythroblastosis virus: surface expression of the erbB product correlates with transformation.

The v-erbB gene of avian erythroblastosis virus (AEV) codes for an integral plasma membrane glycoprotein, gp74erbB. Expression of gp74erbB and its intracellular precursors, gp66erbB and gp68erbB, has been studied in cells transformed by two temperature-sensitive mutants of AEV. After shift to 42 degrees C, the processing of gp68erbB is blocked in tsAEV-transformed, but not in wtAEV-transformed, erythroblasts and fibroblasts. In addition, gp74erbB disappears from the surface of tsAEV cells within 12 hr after shift. Thus tsAEV mutants probably bear a lesion in v-erbB that affects the maturation and subcellular localization of gp74erbB. The tsAEV erythroblasts, when "committed" to differentiation by a pulse-shift to 42 degrees C, reexpress gp74erbB during terminal differentiation at 36 degrees C. This suggests that tsAEV erythroblasts become insensitive to the transforming functions of gp74erbB at a certain stage of differentiation.

Alpharetrovirus↗

On the possible presence of a beta 2-microglobulin-like protein in extracts of livers from normal chickens and chickens with erythroblastosis--I. Recognition of a small-molecular weight (mol. wt 11,000) protein.

1. An extract from the livers of both normal chickens (N) and chickens infected with avian erythroblastosis virus (Eb) contains a small molecular weight protein (SMWP, mol. wt 11,000). 2. Double immunodiffusion studies with rabbit antiserum against fowl serum proteins shows a precipitin arc for SMWP in N and Eb extracts, which is continuous with one from one of the normal chicken serum proteins. 3. When treated with 60% saturated ammonium sulphate the SMWP in the liver extracts divides between the precipitate and the supernatant although the specific serological activity of Eb extracts (gag--or COFAL--determined antigenic activity) is restricted to the precipitated SMWP fraction. 4. The COFAL activity of Eb liver extracts could be associated with SMWP by its attachment to this protein, or this phenomenon of "association" could represent the result of changes in synthesis of SMWP or post-synthetic changes.

Alpharetrovirus↗

On the possible presence of a beta 2-microglobulin-like protein in extracts of livers from normal chickens and chickens with erythroblastosis--V. Studies on an immunological relationship of a small molecular weight liver protein (SMWP) to serum albumin.

An extract of the livers of normal chickens (N) and chickens (Eb) infected with avian erythroblastosis virus (EbV) contained a small molecular weight protein (SMWP, mol. wt 11,000). When the extract was not dehaeminized, SMWP in agarose electrophoresis was shown to have peroxidase activity probably due to the presence of haem. When this non-dehaeminized extract was chromatographed on Con-A Sepharose neither SMWP nor an antigen (EbAg) present in Eb livers were retained. The association of EbAg with SMWP is still unexplained. Immunoelectrophoresis shows a reaction of identity between chicken SMWP and serum albumin. Chicken SMWP is thus not a beta 2-microglobulin. The finding of an immunological relationship between SMWP and albumin confirms the biochemical homology of SMWP with serum albumin in terms of amino acid residues. It has been suggested that SMWP may be a precursor or fragment of albumin but the possibility of its being a distinct entity, a microalbumin, should not be discounted.

Animals↗

On the possible presence of a beta 2-microglobulin-like protein in extracts of livers from normal chickens and chickens with erythroblastosis-IV. Homology of small molecular weight (mol.wt 11,000) protein with serum albumin based on the content of amino acid residues.

A small molecular weight (mol.wt 11,000) protein (SMWP) was obtained from the livers of normal chickens and chickens infected with erythroblastosis virus. SMWP, which had been shown to have no biochemical homology with beta 2-microglobulin, is homologous with chicken serum albumin. SMWP shows a similar order of homology with another small molecular weight protein isolated by others from chicken plasma. Like chicken albumin, SMWP is more closely homologous with bovine albumin than with human albumin.

Alpharetrovirus↗

Identification of the binding site for the Shc protein to the avian erythroblastosis virus (AEV-H) v-erbB protein.

Activation of tyrosine kinase growth factor receptors leads to autophosphorylation of specific tyrosine residues within the intracellular region of the receptor. The phosphorylated tyrosines serve as binding sites for various cytoplasmic proteins. The Shc protein is one such protein. Upon activation of the chicken c-erbB protein by ligand Shc binds to the c-erbB protein and becomes phosphorylated on tyrosine. Similarly, Shc is found bound to the constitutively phosphorylated v-erbB protein encoded by the avian erythroblastosis virus strain H, AEV-H. Utilizing various mutant forms of the v-erbB protein, the residue equivalent to tyrosine 1154 in the chicken c-erbB protein was shown to serve as a binding site for the Shc protein to the AEV-H v-erbB protein. However, binding to this site was not essential for transformation since v-erbB oncoproteins which lacked this site still transform both erythroid cells and fibroblasts.

Adaptor Proteins, Signal Transducing↗