Transformation parameters in chicken fibroblasts transformed by AEV and MC29 avian leukemia viruses.

Infection of chicken fibroblasts with avian erythroblastosis virus (AEV) strain ES4 or with avian myelocytomatosis virus strain MC29 leads to a rapid morphological transformation of most cells. AEV-transformed fibroblasts are similar to Rous sarcoma virus (RSV)-transformed fibroblasts in that they exhibit microvilli at their surface, show a disappearance of actin cables, are agglutinable by lectins, and show a decrease in LETS protein and an increase in the rate of hexose uptake. They also elicit slightly increased levels of cell-associated proteolytic activity, but show no increase in the fibrinolytic activity of the harvest fluids. In addition, as shown previously, they are capable of anchorage-independent growth and of sarcoma induction.In contrast, MC29-transformed fibroblasts express a different pattern of transformation parameters. They are similar to both RSV- and AEV-transformed fibroblasts in that they are morphologically transformed, show a disappearance of actin cables and are agglutinable by lectins. They also elicit surface alterations which consist of bleb-like protrusions rather than of microvilli, and are capable of anchorage-independent growth. They are strikingly different from RSV- and AEV-transformed cells, however, in that they express normal levels of LETS protein and elicit no increase in the rate of hexose uptake or in proteolytic activity. They are not sarcomagenic although they show an accelerated growth rate in culture.In conjunction with the finding that MC29 and AEV do not contain sequences related to the fibroblast-transforming src gene of RSV, these results raise the possibility that MC29 and perhaps also AEV transform fibroblasts by a mechanism different from RSV.     

Response of hemopoietic cells to avian acute leukemia viruses: effects on the differentiation of the target cells.

Chicken bone marrow cells were infected with three avian acute leukemia viruses (ALV)--avian myeloblastosis virus (AMV), myelocytomatosis virus strain MC29 and Mill Hill 2 virus (MH2)--and then cultured in agar in the presence of conditioned medium. Under these conditions, it was found that very few cells served as target cells for these three viruses. Density gradient separation showed that ALV target cells were found primarily in the light density fractions and might be represented by cells committed to the mononuclear phagocyte pathway. Separation of bone marrow cells on the basis of their sedimentation velocity at unit gravity suggested that MC29 and AMV did not share the same target cells. In addition, the analysis of surface receptors and functional markers characteristic of macrophages (Fc and complement receptors, phagocytosis and immune phagocytosis) indicated that the ALV-transformed cells were blocked during their differentiation. These results indicate that the transforming ability of ALV interferes with the differentiation of their target cells.     

Oligoribonucleotide map and protein of CMII: detection of conserved and nonconserved genetic elements in avian acute leukemia viruses CMII, MC29, and MH2.

RNA and protein of the defective avian acute leukemia virus CMII, which causes myelocytomas in chickens, and of CMII-associated helper virus (CMIIAV) were investigated. The RNA of CMII measured 6 kilobases (kb) and that of CMIIAV measured 8.5 kb. By comparing more than 20 mapped oligonucleotides of CMII RNA with mapped and nonmapped oligonucleotides of acute leukemia viruses MC29 and MH2 and with mapped oligonucleotides of CMIIAV and other nondefective avian tumor viruses, three segments were distinguished in the oligonucleotide map of CMII RNA: (i) a 5' group-specific segment of 1.5 kb which was conserved among CMII, MC29, and MH2 and also homologous with gag-related oligonucleotides of CMIIAV and other helper viruses (hence, group specific); (ii) an internal segment of 2 kb which was conserved specifically among CMII, MC29, and MH2 and whose presence in CMII lends new support to the view that this class of genetic elements is essential for oncogenicity, because it was absent from an otherwise isogenic, nontransforming helper, CMIIAV; and (iii) a 3' group-specific segment of 2.5 kb which shared 13 of 14 oligonucleotides with CMIIAV and included env oligonucleotides of other nondefective viruses of the avian tumor virus group (hence, group specific). This segment and analogous map segments of MC29 and MH2 were not conserved at the level of shared oligonucleotides. CMII-transformed cells contained a nonstructural, gag gene-related protein of 90,000 daltons, distinguished by its size from 110,000-daltom MC29 and 100,000-dalton MH2 counterparts. The gag relatedness and similarity to the 110,000-dalton MC29 counterpart indicated that the 90,000-dalton CMII protein is translated from the 5' and internal segments of CMII RNA. The existence of conserved 5' and internal RNA segments and conserved nonstructural protein products in CMII, MC29, and MH2 indicates that these viruses belong to a related group, termed here the MC29 group. Viruses of the MC29 group differ from one another mainly in their 3' RNA segments and in minor variations of their conserved RNA segments as well as by strain-specific size markers of their gag-related proteins. Because (i) the conserved 5' gag-related and internal RNA segments and their gag-related, nonvirion protein products correlate with the conserved oncogenic spectra of the MC29 group of viruses and because (ii) the internal RNA sequences and nonvirion proteins are not found in nondefective viruses, we propose that the conserved RNA and protein elements are necessary for oncogenicity and probably are the onc gene products of the MC29 group of viruses.     

Evidence for p15 cleavage site in myc-specific proteins of avian MC29 and OK10 viruses

Myc-related proteins were precipitated from MC29 virus-transformed cells (PR-2) and from OK10 virus-transformed cells (9C) by anti-gag and anti-myc sera. Immunoprecipitates were cleaved with the avian retroviral protease p15 and the cleavage products analyzed in SDS-PAGE. Cleavage fragments of p110gag-myc (product of MC29 virus) and p58myc (product of OK10 virus) showed the presence of a p15 cleavage site within the myc-specific region. The site is missing in deletion mutants of MC29 virus.     

Chicken hematopoietic cells transformed by seven strains of defective avian leukemia viruses display three distinct phenotypes of differentiation.

Chicken hematopoietic cells transformed in vitro and in vivo by seven strains of replication-defective avian leukemia viruses were assayed for the expression of six erythroid and five myeloid differentiation parameters, including differentiation-specific surface antigens as detected by newly developed antisera. The transformed cells were found to display three distinct phenotypes of differentiation. First, cells transformed by AEV resemble erythroblasts. They express heme, globin, carbonic anhydrase and erythrocyte cell surface antigen at low levels, and histone H5 and erythroblast cell surface antigen at high levels. Second, cells transformed by MC29, CMII, OK10 and MH2 viruses have macrophage-like properties. They strongly express Fc receptors, phagocytic capacity and macrophage cell surface antigen, but only weakly express myeloblast cell surface antigen and are negative for ATPase activity. Third, cells transformed by AMV and E26 viruses resemble myeloblasts in that they weakly express Fc receptors, phagocytic capacity and macrophage cell surface antigen but strongly express myeloblast cell surface antigen and ATPase activity. No difference was found between in vitro- and in vivo-transformed cells in the parameters tested. In light of recent genetic and biochemical evidence, we believe that these phenotypes reflect the action of three new types of viral-transforming genes, designated erb (erythroblast), mac (macrophage) and myb (myeloblast).     

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.     

Structural relationship between a normal chicken DNA locus and the transforming gene of the avian acute leukemia virus MC29.

We screened a recombinant chicken DNA/lambda phage library for sequences homologous to the transformation-specific sequences of the avian acute leukemia virus MC29 by hybridization with molecularly cloned MC29 proviral DNA. Three cellular DNA clones were found and compared with each other and with the viral genome by physical mapping with restriction endonucleases and by heteroduplex analysis. These experiments indicated that the three cellular clones overlap and represent a single cellular locus. The RNA genome of MC29 and normal cell DNA share a homologous region of 1.6 kilobases which is interrupted in the cellular DNA by 1.0 kilobase of sequences not present in the viral genome. Hybridization of the cloned cellular DNA to viral RNA and analysis of the protected viral RNA by fingerprinting techniques indicated that there is extensive sequence homology between the helper virus-unrelated mcv sequences of the viral RNA and the cellular DNA, with only minor base differences. The cellular mcv locus, however, lacks all helper virus-related sequences of MC29, including those of the partial viral gag gene which, together with mcv, encodes the probable transforming protein of MC29. We conclude that although the mcv locus of the normal cell does not represent a complete structural homolog to the onc gene of MC29, it is probably the precursor to the onc-specific sequence in the virus.     

Characterization of target cells for MCF viruses in AKR mice

The recombinant (MCF) class of murine leukemia virus appears to play an important role in lymphomagenesis in AKR and other mice. Although much effort has been extended in characterizing MCF viruses, relatively little is known about the cells they infect. I examined what cells were targets in AKR mice for both lymphomagenic and nonlymphomagenic MCF viruses. Lymphomagenic MCF viruses of thymic origin (AKR-247 and C58L1) were found to infect and replicate selectively in immature lymphocytes only present in thymic cortex, whereas nonlymphomagenic MCF viruses of splenic origin (C58v-1-C77 and C58v-2-C45) selectively infected and replicated in cells that appeared to B lymphocytes. Virus-binding studies suggested that neither T- nor B-lymphocyte tropisms were determined by selective attachment of virus to the respective cells. These findings demonstrate that in contrast with ecotropic viruses, which can infect many types of cells in the mouse, specific cellular tropisms can exist for MCF viruses, and that MCF infection, and therefore oncogenicity, is closely linked to cellular differentiation.     

Intranuclear degradation of the transformation-inducing protein encoded by avian MC29 virus

The nuclear protein, p110, encoded by the avian MC29 virus degrades with a half-life of 30 to 40 min in virus-transformed cells. Inhibitors of lysosomal proteolysis had no effect on this degradation. When inhibitors of RNA or protein synthesis were added immediately after pulse-labeling the p110 with [35S]methionine, degradation was impeded. Treatment of cells with cycloheximide prior to, and after, the pulse extended the half-life of p110 further than post-treatment alone, and addition of both actinomycin D and cycloheximide to cells pretreated with cycloheximide extended the half-life even further. In cells depleted of cellular ATP using a glucose-deficient medium containing oligomycin, degradation of p110 was only partially inhibited, indicating no direct involvement of ATP in degradation. Isolation of nuclei or nuclear matrices containing labeled p110, with subsequent incubation, resulted in minimal loss of p110 during several hours. These results suggest that p110 is degraded by a protease which is itself labile and freely diffusible from the nucleus, and, in addition, degradation may involve interaction of p110 with newly synthesized RNA.     

Biochemical characterization of transformation-specific proteins of acute avian leukemia and sarcoma viruses

The biological and biochemical properties of the transformation-specific proteins of three avian oncornaviruses with different oncogenic potentials were compared, namely the gag-myc protein of the avian myelocytomatosis virus MC29, the gag-erb A protein of the avian erythroblastosis virus AEV, and the gag-fps protein of Fujinami sarcoma virus FSV. These oncogenes were analyzed in transformed fibroblasts that expressed only the transforming proteins but showed no virus replication. Monoclonal antibodies against the viral structural protein p19, which is the N-terminus of the proteins, were used for indirect immunofluorescence, for immunoprecipitation of the proteins from subcellular fractions, and for immunoaffinity column chromatography. With this last method a 3000-fold purification of the proteins was obtained. By indirect immunofluorescence it was shown that the gag-myc protein was located in the nucleus, and bound to DNA after purification. The gag-erb A protein was not nuclear but probably located in the cytoplasm and did not bind to DNA after purification. Neither of the two proteins exhibited protein kinase activity. In contrast, the gag-fps protein did not bind to DNA but showed protein kinase activity after purification. It was not located in the nucleus either.     

Identification of nucleotide sequences which may encode the oncogenic capacity of avian retrovirus MC29.

The retrovirus strain MC29 induces a variety of tumors in chickens, including myelocytomatosis and carcinomas of the kidney and liver. In addition, the virus can transform cultures of embryonic avian macrophages and fibroblasts. We have characterized the genome of MC29 virus and have identified nucleotide sequences that may encode the oncogenic potential ofthe virus. MC29 virus can replicate only with the assistance of a related helper virus. The defect in replication is apparently a consequence of a deletion in one or more viral genes: the haploid genome of the MC29 virus has a molecular weight of ca. 1.7 X 10(6), whereas the genome of the helper virus MCAV has a molecular weight of ca. 3.1 X 10(6). Although MC29 virus transforms fibroblasts in culture, its genome has no detectable homology with the gene src that is responsible for transformation of fibroblasts by avian sarcoma viruses. We prepared radioactive single-stranded DNA complementary to nucleotide sequences present in the genome of MC29 virus but not in the genome of MCAV (cDNA(MC29)). If they are contiguous, these sequences (ca. 1,500 nucleotides) are sufficiently complex to encode at least one protein. Homologous sequences were not detectable in several strains of avian sarcoma viruses or in an endogenous virus of chickens. Our findings confirm and extend recent reports from other laboratories and lead to the conclusion that MC29 virus may contain a previously unidentified gene(s) that is capable of transforming several distinct target cells. The evolutionary origins of this putative gene and its location on the viral genome can be explored with cDNA(MC29).     

Restriction enzyme analysis of partially transformation-defective mutants of acute leukemia virus MC29

Restriction enzyme mapping and limited sequence analysis have been used to study the generation and genome structure of three partial-transformation mutants of avian acute leukemia virus MC29. The three mutants, td10A, td10C, and td10H, could be shown to have sustained overlapping deletions of 200, 400, and 600 base pairs, respectively, in their genomes. The precise location of the deletions was mapped within the v-myc gene of the mutants by limited sequence analysis of cloned MC29 DNA. The data obtained are discussed in terms of the effect of these deletions on the mechanism of transformation by MC29.     

Phosphorylation of the nonstructural proteins encoded by three avian acute leukemia viruses and by avian fujinami sarcoma virus.

The gag gene-related, nonstructural proteins of three avian acute leukemia viruses (namely, myelocytomatosis viruses MC29 and CMII and avian erythroblastosis virus) and of avian Fujinami sarcoma virus (FSV) isolated by immunoprecipitation from cellular lysates with anti-gag serum were shown to be phosphoproteins in vivo. The specific 32P radioactivity of the nonstructural proteins of MC29, CMII, and FSV was significantly higher than that of helper viral, intracellular gag proteins. Two of these proteins, i.e., the 140,000-dalton FSV and the 110,000-dalton MC29 proteins, were also phosphorylated in vitro by a kinase activity associated with immunocomplexes. This kinase activity is either separated from these proteins or inactivated by incubation of cellular lysates with normal serum followed by adsorption to staphylococcal protein A or sedimentation at 100,000 x g or both. It remains to be resolved whether the 110,000-dalton MC29 and 140,000-dalton FV proteins, in addition to being substrates for phosphorylation, also have intrinsic kinase activity.     

Primary structure of p19 species of avian sarcoma and leukemia viruses.

The internal structural proteins of avian sarcoma and leukemia viruses are derived from a precursor polypeptide that is the product of the viral gag gene. The N-terminal domain of the precursor gives rise to p19, a protein that interacts with the lipid envelope of the virus and that may also interact with viral RNA. The C terminus of p19 from the Prague C strain of Rous sarcoma virus was previously assigned to a tyrosine residue 175 amino acids from the N terminus. We have used metabolic labeling and carboxypeptidase digestion to show that the C terminus of p19 is actually tyrosine 155. This implies the existence of a sixth gag protein 22 amino acids in length and located between p19 and p10 on the gag precursor. The p19 species of some recombinant avian sarcoma viruses and of the defective endogenous virus derived from the ev-1 locus migrate on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as if they were about 4,000 daltons smaller than p19. We have elucidated the structure of these forms, called p19 beta, by analysis of the proteins and determination of the DNA sequence of the p19 region of the gag gene from ev-1 and ev-2. Esterification of carboxyl groups completely suppressed the differences in migration of p19 and p19 beta. Peptide mapping showed the altered mobility to be determined by sequences in the C-terminal cyanogen bromide fragment of the proteins. We conclude from the DNA sequence that a single glutamate-lysine alteration is responsible for the altered electrophoretic mobility.