Transfection by DNAs of avian erythroblastosis virus and avian myelocytomatosis virus strain MC29.

Chicken embryo fibroblasts and NIH 3T3 mouse cells were transformable by DNAs of chicken cells infected with avian myelocytomatosis virus strain MC29 or with avian erythroblastosis virus. Transfection of chicken cells appeared to require replication of MC29 or avian erythroblastosis virus in the presence of a nontransforming helper virus. In contrast, NIH 3T3 cells transformed by MC29 or avian erythroblastosis virus DNA contained only replication-defective transforming virus genomes.     

Transformation of avian myogenic cultures with myelocytomatosis virus strain 29

Summary Primary cultures of proliferating chick presumptive myoblasts were exposed of either to two RNA tumor viruses and shortly thereafter treated with 5-bromodeoxyuridine (BUdR) to suppress differentiation. The effect of a Rous sarcoma virus which was temperature-sensitive for transformation (tsRSV) has been characterized previously and was used as a reference for evaluating the effect of a myelocytomatosis virus (MC29) and its helper. Two subcultures following exposure, both infected cultures were extensively transformed as indicated by cell morphology. Relaxation of the BUdR block at this time resulted in cultures which still appeared transformed and did not contain myoblast or myotube-like cells or two of their molecular markers. In contrast, uninfected controls and tsRSV-infected cultures which were shifted-up to the nonpermissive temperature produced numerous spontaneously contracting myotubes. The results confirm previous evidence that infection of presumptive myoblasts by tsRSV at the premissive temperature preserves the extant state of differentiation of presumptive myoblasts and suggest, by analogy, that MC29-infection renders a similar effect.     

Generation and characterization of a recombinant Moloney murine leukemia virus containing the v-myc oncogene of avian MC29 virus: in vitro transformation and in vivo pathogenesis.

A new retrovirus consisting of the v-myc oncogene sequences of avian MC29 virus inserted into the genome of Moloney murine leukemia virus (M-MuLV) was generated. This was accomplished by constructing a recombinant DNA clone containing the desired organization, introducing the recombinant DNA into mouse NIH 3T3 cells, and superinfecting the cells with replication-competent M-MuLV. The construction was designed so that an M-MuLV gag-myc fusion protein would be produced. The resulting virus, M-MuLV(myc), morphologically transformed uninfected NIH 3T3 cells. Stocks of M-MuLV(myc)-M-MuLV were infected into secondary mouse embryo cultures. M-MuLV(myc) induced striking growth and proliferation of hematopoietic cells. These cells were of the myeloid lineage by morphology, phagocytic properties, and surface staining with Mac-1 and Mac-2 monoclonal antibodies. They resembled mature macrophages, although they displayed minor properties of immaturity. The myeloid cells were transformed in comparison with uninfected myeloid cells since they were less adherent and had unlimited proliferative capacity and reduced growth factor requirements. The transformed myeloid cells with proliferative potential were actually myeloid progenitors which apparently underwent terminal differentiation to macrophages. It was possible to derive a permanent line of factor-independent macrophages from M-MuLV(myc)-transformed myeloid cells. M-MuLV(myc) also immortalized and morphologically transformed mouse embryo fibroblasts. These in vitro properties closely resembled the biological activity of MC29 virus in avian cells and suggested that the nature of the v-myc oncogene was an important determinant in transformation specificity. Neonatal NIH Swiss mice inoculated intraperitoneally with M-MuLV(myc)-M-MuLV only developed lymphoblastic lymphoma characteristic of the M-MuLV helper alone, and no acute fibrosarcomas or myeloid tumors resulted. In light of the strong myeloid transformation observed in vitro, the absence of acute in vivo myeloid disease was noteworthy. Interestingly, when a derivative of M-MuLV(myc) carried by a nonpathogenic amphotropic MuLV helper was inoculated, T lymphomas developed with long latency. Molecular hybridization confirmed that these tumors contained M-MuLV(myc).     

Structure and dimorphism of c-rel (turkey), the cellular homolog to the oncogene of reticuloendotheliosis virus strain T.

A locus has been identified in turkey DNA that contains nucleotide sequences homologous to the oncogene (v-rel) in the avian retrovirus, reticuloendotheliosis virus strain T. This locus, c-rel, has been molecularly cloned from an apparently heterozygous turkey. c-rel is approximately 23 kilobase pairs in length, with at least seven apparent introns, and contains sequences sufficient to account for all of v-rel. Nucleic acid sequence differences exist between v-rel and homologous regions of c-rel. We examined a population of turkeys to determine whether these sequence differences are the result of polymorphism in the population. Within the turkey population, c-rel is dimorphic in apparent introns and 3' flanking sequences, but polymorphism has not been detected within the regions of the c-rel locus that are homologous to v-rel. Additionally, no nucleic acid sequence differences have been detected between the regions of c-rel in turkeys that are homologous to v-rel and the sequences related to v-rel of a homologous locus in chickens (Chen et al., J. Virol. 245:104-113, 1983). The general organization of introns and flanking sequences is conserved for both c-rel in turkeys and this locus in chickens, indicating that c-rel, like other proto-oncogenes, may have an important development or metabolic function.     

Isolation and biochemical characterization of partially transformation-defective mutants of avian myelocytomatosis virus strain MC29: localization of the mutation to the myc domain of the 110,000-dalton gag-myc polyprotein

Recently, we isolated three mutants of MC29 virus which, although able to transform fibroblasts with the same efficiency as wild-type MC29, were 100-fold less efficient at transforming macrophages. In this study we found that MC29-transformed quail producer cell line Q10 was able to generate these partially transformation defective mutants at a high frequency. Using tryptic peptide mapping, we determined that the smaller gag-myc polyproteins encoded by the transformation-defective viruses had lost myc-specific tryptic peptides. This suggested that the mutations which resulted in the transformation-defective viruses being inefficient at transforming macrophages were located in the v-myc sequence and thus directly implicated v-myc and the gag-myc polyprotein in transformation by MC29.     

Characterization of the oncogene (erb) of avian erythroblastosis virus and its cellular progenitor.

Avian erythroblastosis virus (AEV) induces primarily erythroblastosis when injected intravenously into susceptible chickens. In vitro, the hematopoietic target cells for transformation are the erythroblasts. Occasional sarcomas are also induced by intramuscular injection, and chicken or quail fibroblasts can be transformed in vitro. The transforming capacity of AEV was shown to be associated with the presence of a unique nucleotide sequence denoted erb in its genomic RNA. Using a simplified procedure, we prepared radioactive complementary DNA (cDNAaev) representative of the erb sequence at a high yield. Using a cDNAaev excess liquid hybridization technique adapted to defective retroviruses, we determined the complexity of the erb sequence to be 3,700 +/- 370 nucleotides. AEV-transformed erythroblasts, as well as fibroblasts, contained two polyadenylated viral mRNA species of 30 and 23S in similar high abundance (50 to 500 copies per cell). Both species were efficiently packaged into the virions. AEV-transformed erythroblasts contained additional high-molecular-weight mRNA species hybridizing with cDNAaev and cDNA5' but not with cDNA made to the helper leukosis virus used (cDNArep). The nature and the role, if any, of these bands remain unclear. The erb sequence had its counterpart in normal cellular DNA of all higher vertebrate species tested, including humans and fish (1 to 2 copies per haploid genome in the nonrepetitive fraction of the DNA). These cellular sequences (c-erb) were transcribed at low levels (1 to 2 RNA copies per cell) in chicken and quail fibroblasts, in which the two alleged domains of AEV-specific sequences corresponding to the 75,000- and 40,000-molecular-weight proteins seemed to be conserved phylogenetically and transcribed at similar low rates.     

Nucleic acid sequences of the oncogene v-rel in reticuloendotheliosis virus strain T and its cellular homolog, the proto-oncogene c-rel.

Reticuloendotheliosis virus strain T (Rev-T) is a highly oncogenic replication-defective retrovirus which contains the oncogene v-rel. It is thought that Rev-T arose when a virus similar to Rev-A, the helper virus of Rev-T, infected a turkey and recombined with c-rel from that turkey. There is one large c-rel locus in the turkey genome which contains all of the sequences homologous to v-rel (K. C. Wilhelmsen and H. M. Temin, J. Virol. 49:521-529, 1984). We have sequenced v-rel and its flanking sequences, each of the regions of the c-rel locus from turkey that are homologous to v-rel and their flanking sequences, and the coding sequence for env and part of pol of Rev-A. The v-rel coding sequences can be translated into a 503-amino acid env-v-rel-out-of-frame-env fusion polypeptide. We have not detected any sequences in the Los Alamos or University of California-San Diego data bases that are more significantly related to the amino acid or nucleic acid sequence of v-rel than to the randomized sequence of v-rel. Comparison of Rev-A, Rev-T, and c-rel indicates that the v-rel sequences may have been transduced from the c-rel (turkey) locus by a novel mechanism. There are sequences in Rev-A and c-rel that are similar to splicing signals, indicating that the 5' virus-rel junction of Rev-T may have been formed by cellular RNA splicing machinery. Eight presumed introns have presumably been spliced out of c-rel to generate v-rel. There are also short imperfect regions of homology between sequences at the boundaries of v-rel and sequences in Rev-A and c-rel (turkey), indicating that c-rel may have been transduced by homologous recombination. There are many differences between the amino acid sequences of the predicted translational products of v-rel and c-rel which may account for their difference in transformation potential. These sequence differences between v-rel and c-rel include 10 missense transitions, four missense transversions, and three places where Rev-T has a small in-frame deletion of sequences relative to c-rel. Most of the coding sequence differences between c-rel and v-rel are nonconservative amino acid changes.     

Temperature-sensitive mutants of MH2 avian leukemia virus that map in the v-mil and the v-myc oncogene respectively.

MH2 is an avian retrovirus that contains the v-mil and v-myc oncogenes. In vitro it transforms chick macrophages that are capable of proliferation in the absence of growth factor. Earlier work showed that v-myc induces macrophage transformation and that v-mil induces the production of chicken myelomonocytic growth factor (cMGF), thus generating an autocrine system. We describe the isolation of temperature-sensitive (ts) mutants of MH2 virus. As suggested by marker rescue experiments, one mutant bears a ts lesion in v-mil, whereas the other carries a mutation in v-myc. Ts v-mil MH2-transformed macrophages become factor-dependent at the non-permissive temperature (42 degrees C), while ts-v-myc MH2-transformed macrophages cease growing and acquire a more normal macrophage phenotype at 42 degrees C irrespective of the presence of cMGF. Both phenotypes can be reversed by backshift to the permissive temperature. These results suggest that the gene products of v-mil and v-myc function independently of each other and that v-mil is necessary for the maintenance of autocrine growth, whereas v-myc is required to maintain the transformed phenotype.     

Amplification and expression of a cellular oncogene (c-myc) in human gastric adenocarcinoma cells.

Three of 16 human gastric adenocarcinoma samples, maintained as solid tumors in nude mice, were found to carry amplified c-myc genes. In two samples with a high degree of c-myc DNA amplification (15- to 30-fold), double minute chromosomes were observed in karyotype analysis. The level of c-myc RNA was markedly elevated in a rapidly growing and poorly differentiated tumor, whereas it was only slightly elevated in a slowly growing and more differentiated tumor.     

Comparison between the viral transforming gene (src) of recovered avian sarcoma virus and its cellular homolog.

Recovered avian sarcoma viruses are recombinants between transformation-defective mutants of Rous sarcoma virus and the chicken cellular gene homologous to the src gene of Rous sarcoma virus. We have constructed and analyzed molecular clones of viral deoxyribonucleic acid from recovered avian sarcoma virus and its transformation-competent progenitor, the Schmidt-Ruppin A strain of Rous sarcoma virus. A 2.0-megadalton EcoRI fragment containing the entire src gene from each of these clones was subcloned and characterized. These fragments were also used as probes to isolate recombinant phage clones containing the cellular counterpart of the viral src gene, termed cellular src, from a lambda library of chicken deoxyribonucleic acid. The structure of cellular src was analyzed by restriction endonuclease mapping and electron microscopy. Restriction endonuclease mapping revealed extensive similarity between the src regions of Rous sarcoma virus and recovered avian sarcoma virus, but striking differences between the viral src's and cellular src. Electron microscopic analysis of heteroduplexes between recovered virus src and cellular src revealed a 1.8-kilobase region of homology. In the cellular gene, the homologous region was interrupted by seven nonhomologous regions which we interpret to be intervening sequences. We estimate the minimum length of cellular src to be about 7.2 kilobases. These findings have implications concerning the mechanism of formation of recovered virus src and possibly other cell-derived retrovirus transforming genes.     

Site-directed mutagenesis of the gag-myc gene of avian myelocytomatosis virus 29: biological activity and intracellular localization of structurally altered proteins.

Transfection of chicken embryo cells with pMC29, a plasmid vector containing the sequences for the acute transforming virus MC29, and a cloned transformation-defective helper virus, p delta Mst, resulted in morphological transformation, the synthesis of P110gag-myc (the product of the gag-myc oncogene), and the production of infectious virus. MC29 mutants bearing site-directed deletions within the gag-specific sequences or within the middle portion of the myc sequences efficiently induced transformation of chicken embryo cells in culture. However, variants containing deletions of sequences in the amino-terminal half or carboxy-terminal portion of the myc gene were defective for transformation. The gag-myc proteins encoded by these variants efficiently localized to the cell nucleus. Premature termination mutants were isolated which encoded gag-myc proteins lacking the carboxy-terminal 185 residues; these truncated proteins localized to both the nucleus and the cytoplasm. Deletion of as few as 11 residues within the middle of the myc-specific sequences (residues Ile-239 to Glu-249) significantly reduced the efficiency of chicken hematopoietic cell transformation.     

Characterization of lymphoid tumors induced by a recombinant murine retrovirus carrying the avian v-myc oncogene. Identification of novel (B-lymphoid) tumors in the thymus

Lymphoid tumors induced by a recombinant murine retrovirus carrying the v-myc oncogene of avian MC29 virus were characterized. The Moloney murine leukemia virus myc oncogene (M-MuLV (myc], carried by an amphotropic MuLV helper, induced tumors in NIH Swiss and NFS/N mice after a relatively long latency (8 to 24 wk). Tumor masses appeared in the thymus, spleen, and lymph nodes. Flow cytometry of the tumor cells indicated that approximately 50% were positive for Thy 1.2. Most of these tumors also expressed one or more other cell surface markers of thymocytes and mature T cells (CD4, CD8). Southern blot hybridization revealed genomic rearrangements for the TCR beta genes. The TCR beta analysis suggested that the M-MuLV(myc)-induced Thy 1.2+ tumors were derived from somewhat less mature cells than tumors induced by M-MuLV, which is a classical non-acute retrovirus lacking an oncogene. The remainder of the M-MuLV(myc)-induced tumors were Thy 1.2-, but they were positive for Ly-5 (B220) and also for MAC-2. The Thy 1.2- tumors were characteristically located in the thymus. However, they were negative for TCR beta gene rearrangements. Some, but not all, of the Thy 1.2- tumors contained rearrangements for Ig genes. Additionally, they typically expressed mRNA specific for B but not for T cells. Thus, these thymic tumors had characteristics of the B cell lineage. Tumor transplantation experiments demonstrated that the Thy 1.2- tumor cells could reestablish in the thymus and spleen of irradiated hosts, and low level expression of the Thy 1 molecule was observed in the thymus but not the spleen on the first passage. After serial passage, one Thy 1- tumor altered its cell surface phenotype to Thy 1low B220-.     

Sequences in human cytomegalovirus which hybridize with the avian retrovirus oncogene v-myc are G + C rich and do not hybridize with the human c-myc gene.

The degree of relatedness between previously identified cross-hybridizing regions within human cytomegalovirus strain AD169 and the avian retrovirus oncogene v-myc were investigated by nucleotide sequence comparison. We found that the homologous regions between the human cytomegalovirus genome and v-myc are limited to short G + C-rich regions in each genome and that the human cytomegalovirus genome shares little or no homology with the human c-myc gene.     

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. MMCV thus transforms both mouse fibroblastic cells and macrophages in vitro, in a fashion similar to the v-myc-containing avian viruses in chicken cells. The possibility of introducing a transforming myc gene into mammalian cells by virus infection provides a novel approach for studying the mechanism of myc transformation in cells from many lineages.     

Association of gag-myc proteins from avian myelocytomatosis virus wild-type and mutants with chromatin.

The localization of the transformation-specific proteins was analyzed in quail embryo fibroblast cell lines transformed by wild-type avian myelocytomatosis virus MC29 and by three of its deletion mutants, Q10A , Q10C , and Q10H , with altered transforming capacities, and in a chicken fibroblast cell line transformed by the avian erythroblastosis virus (AEV). These viruses code for polyproteins consisting of part of the gag gene and of a transformation-specific region, myc for MC29 and erb A for AEV. Analysis by indirect immunofluorescence using monoclonal antibodies against p19, the N-terminal region of the polyprotein, showed that the gag-myc proteins in cells transformed by the wild-type MC29 as well as by the three deletion mutants are located in the nucleus. In contrast, cells transformed by AEV, which express the gag-erb A protein, give rise to cytoplasmic fluorescence. Fractionation of cells into nuclear and cytoplasmic fractions and analysis by immunoprecipitation and gel electrophoresis confirmed these results. About 60% of the gag-myc proteins of wild-type as well as of mutant origin were found in the nucleus, while 90% of the gag-erb A protein was present in the cytoplasm. Also, pulse-chase analysis indicated that the gag-myc protein rapidly accumulates in the nucleus in just 30 min. Further, it was shown that the wild-type and also mutant gag-myc proteins are associated with isolated chromatin. Association to chromatin was also observed for the gag-myc protein from MC29-transformed bone marrow cells, which are believed to be the target cells for MC29 virus in vivo.