Different localization of the product of the v-rel oncogene in chicken fibroblasts and spleen cells correlates with transformation by REV-T

Reticuloendotheliosis virus strain T (REV-T) is a highly oncogenic avian retrovirus that transforms early lymphoid cells in vivo and in vitro, but REV-T does not transform chicken embryo fibroblasts (CEF). Using antisera to p59v-rel, the v-rel oncogene product of REV-T, we show that p59v-rel is expressed at equal levels and is a phosphoprotein in REV-T infected spleen cells and CEF. Biochemical fractionation and immunofluorescence of REV-T infected nontransformed CEF show that p59v-rel is loosely associated with the nucleus. However, in REV-T transformed spleen cells p59v-rel is primarily a cytoplasmic protein. MSB-1 cells, a Marek's disease virus transformed T cell leukemic line, and E26 virus transformed myeloid cells show nuclear staining of p59v-rel when they are infected by REV-T. Our results indicate that there is a correlation between a cytoplasmic localization of p59v-rel and transformation by REV-T, and they suggest that p59v-rel cannot transform cells in which it assumes solely a nuclear location.     

Activation of oncogenicity of the c-rel proto-oncogene.

Reticuloendotheliosis virus strain T (Rev-T) induces a lethal lymphoma in young birds and transforms avian lymphoid cells in vitro. The transforming gene of Rev-T, v-rel, was derived from the turkey proto-oncogene c-rel. Comparison of the nucleotide sequences of v-rel and c-rel indicates that in addition to several internal amino acid changes relative to c-rel, p59v-rel has amino acid sequences at both ends derived from the reticuloendotheliosis virus strain A-related virus env gene (K. C. Wilhelmsen, K. Eggleton, and H. M. Temin, J. Virol. 52:172-182, 1984). In this report, the v-rel sequences important for transformation were defined by constructing recombinant retroviruses in which c-rel sequences replaced the analogous v-rel sequences. These recombinant viruses expressing chimeric proteins were tested for their ability to transform spleen cells in vitro and to induce tumors in young chickens. Activation of the oncogenicity of c-rel in Rev-T required alteration of the amino terminus and the central region of the protein. Deletion of the noncoding sequences 3' to c-rel and of most of the helper virus-related env sequences was necessary for the formation of Rev-T.     

Serine phosphorylation of the v-rel oncogene product/pp40 complex

The transforming protein encoded by the v-rel oncogene of reticuloendotheliosis virus has been purified from REV-T transformed lymphoid cells by sequential DEAE-Sepharose and immunoaffinity chromatography. The purified preparation consisted of pp59v-rel and the 40 kDa cellular protein which is complexed with the v-rel oncogene product in transformed cells as well as some minor proteins. Incubation of this purified preparation in the presence of Mg2+ and (gamma-32P)ATP resulted in phosphorylation of both pp59v-rel and the 40 kDa protein. This preparation was also able to phosphorylate casein on serine residues. Immunoprecipitates obtained from extracts of REV-T transformed lymphoid cells labeled with 32P-orthophosphate contained 59 and 40 kDa phosphoproteins. Both pp59v-rel and the 40 kDa protein were phosphorylated on serine residues in transformed cells.     

p59v-rel, the transforming protein of reticuloendotheliosis virus, is complexed with at least four other proteins in transformed chicken lymphoid cells

Previous studies have identified the protein product of v-rel, the oncogene carried by reticuloendotheliosis virus (REV), as a 59,000-dalton phosphoprotein located predominantly in the cytosol of transformed chicken lymphoid cells. In immune precipitates of p59v-rel, there is a closely associated protein kinase activity. In chicken lymphoid cells that do not contain REV, p68c-rel is found free in the cytosol not associated with other proteins and not detectably phosphorylated. In this study, we found that immune precipitates of 59v-rel from REV-transformed cells contain at least four other proteins, of approximate molecular weights 124, 115, 68, and 36 kilodaltons (kDa). The 124-, 115-, and 36-kDa proteins are apparently unrelated to p59v-rel in sequence, and their coprecipitation suggests that they are complexed with p59v-rel. The coprecipitating 68-kDa protein was found to be p68c-rel, which, like the other three proteins, precipitates by virtue of its association with p59v-rel. Glycerol gradient analysis suggested the presence of more than one type of complex: one containing p115, p68c-rel, p59v-rel, and p36, and another containing p124, p115, p59v-rel, and possibly p68c-rel. In vitro kinase activity was found in all size classes, coinciding with the distribution of p115 and p59v-rel. The complex(es) was stable under a variety of conditions, including a wide range of ionic strengths, chelators, and detergents, and through multiple cycles of immune precipitation and elution. This suggests a specific and functionally significant interaction among the members that may be of direct relevance to the mechanism of REV-induced transformation.     

Localization of the v-rel protein in reticuloendotheliosis virus strain T-transformed lymphoid cells.

The protein (p59rel) encoded by the transforming gene of reticuloendotheliosis virus strain T (REV-T) has been identified in REV-T-transformed avian lymphoid cells by using antisera raised against synthetic peptides whose sequences were derived from three nonoverlapping regions of v-rel (N. R. Rice, T. D. Copeland, S. Simek, S. Oroszlan, and R. V. Gilden, Virology 149:217-229, 1986). To obtain polyclonal antibodies directed against a larger number of p59rel epitopes, a 262-amino acid segment was expressed in bacteria. Antisera raised against this fusion protein (v-delta-rel) precipitated p59rel from lysates of [35S]methionine-labeled REV-T-transformed cells, thus confirming previous results obtained with the peptide antisera. We used this new antiserum to localize p59rel in REV-T-transformed cells by subcellular fractionation using differential centrifugation and by indirect immune fluorescent staining. After fractionation and immune precipitation, the majority of p59rel was found in the cytosolic fraction. Indirect immunofluorescence experiments also gave results consistent with the cytoplasmic localization of the v-rel protein in transformed lymphoid cells. In previous studies (Rice et al., Virology 149:217-229, 1986) it was shown that immune precipitates formed with one of the three p59rel peptide antisera possessed in vitro protein kinase activity. Immune precipitates formed with the fusion protein antiserum also showed kinase activity in the in vitro assay. Most of this activity was found in the soluble cytoplasmic fraction, indicating that the kinase may be p59rel or a protein closely associated with it.     

Transformation of avian lymphoid cells by reticuloendotheliosis virus

Avian reticuloendotheliosis virus (REV-T) is the most virulent of all retroviruses, inducing an invariably fatal leukemia in chickens with a latent period of 7-10 days. Unlike avian cells transformed by other acutely transforming viruses, lymphoid cells transformed by REV-T are immortalized. Furthermore, in vitro derived, REV-T transformed cells which do not produce virus are tumorigenic and induce lethal reticuloendotheliosis when injected into histocompatible birds. Thus REV-T transforms its target cell both in vitro and in vivo. In addition this transformation is independent of any helper virus functions. Like other acute leukemia viruses, REV-T is replication-defective and must co-replicate with a reticuloendotheliosis associated virus (REV-A). During evolution, a substantial portion of its genome has been deleted and replaced with a host-derived genetic sequence, designated v-rel. Presumably, the v-rel oncogene was transduced from a normal turkey DNA locus, c-rel. There are 9 regions of homology between c-rel and v-rel, however, several differences exist between these genes, suggesting that transformation by REV-T results from the production of an altered v-rel protein. The v-rel sequence is distinct from other known oncogenes and encodes a 57-kDa phosphoprotein. In REV-T transformed cells, this pp57v-rel protein is localized in the cytoplasm. The product of the v-rel oncogene is present at a low level, representing only about 0.003% of total methionine-labelled protein. In addition, pp57v-rel is relatively stable, having an estimated half-life of 4-10 h. The v-rel protein when purified close to homogeneity is complexed with a 40-kDa cellular phosphoprotein in transformed lymphoid cells and possesses serine kinase activity. This review discusses the molecular aspects of transformation by REV-T in the context of other oncogene-encoded proteins.     

Avian reticuloendotheliosis virus-transformed lymphoid cells contain multiple pp59v-rel complexes.

The v-rel oncogene of avian reticuloendotheliosis virus type T (REV-T) encodes a 59-kilodalton (kDa) phosphoprotein located principally in the cytosol of transformed lymphoid cells. All of the detectable pp59v-rel was present in high-molecular-weight complexes containing at least five cellular proteins (p124, p115, p75c-rel, p70hsc, and pp40). Antiserum was developed against the 40-kDa protein, the most abundant cellular protein associated with the complex. The 40-kDa phosphoprotein was complexed with pp59v-rel in REV-T-transformed lymphoid cell lines arrested at different stages of B-cell development as well as in lymphoid tumor cells and in fibrosarcomas. The half-life (8 h) of pp40 in REV-T-transformed lymphoid cells was the same as that of pp59v-rel. Antiserum against pp40 permitted the identification of two pp59v-rel complexes. The most abundant cytoplasmic complex contained approximately 75% of the pp59v-rel and all of the detectable pp40 in REV-T-transformed lymphoid cells. Twenty-five percent of the pp59v-rel was present in a minor complex that contained the majority of p75c-rel along with p115 and p124. In nuclear extracts of REV-T-transformed lymphoid cells, pp59v-rel was complexed with pp40. The two high-molecular-weight proteins (p115 and p124) and p75c-rel were not detected in the nuclear complex. In the cytosolic complexes, pp40 was heavily phosphorylated, whereas the nuclear form was much less extensively phosphorylated.     

Transforming viruses spontaneously arise from nontransforming reticuloendotheliosis virus strain T-derived viruses as a result of increased accumulation of spliced viral RNA.

The highly oncogenic avian retrovirus reticuloendotheliosis virus strain T (Rev-T) contains a substitution of the oncogene v-rel for much of env and a deletion of gag and pol relative to the helper virus Rev-A. Replacement of gag and pol sequences in Rev-T suppresses transformation by reducing the accumulation of spliced viral mRNA and v-rel protein in infected cells (C. K. Miller and H. M. Temin, J. Virol 58:75-80, 1986). After infection of spleen cells with viruses containing gag and pol sequences, revertant viruses that are strongly transforming were found. Approximately three-fourths of the revertant viruses appeared structurally the same as the parental virus, and approximately one-fourth of the revertant viruses had large deletions (similar in size and location to the deletion in Rev-T). Two revertant viruses that appeared structurally the same as the parental virus were molecularly cloned. The regions sufficient to change the parental virus to a strongly transforming virus were determined by construction of recombinant viruses. In one revertant virus, the region sufficient for transformation contained a 327-base-pair insertion 5' of the 3' splice site used by Rev-T. In the other revertant virus, the region sufficient for transformation contained a 1-base-pair transition and a deletion of one copy of a 9-base-pair direct repeat, both 3' of the 3' splice site used by Rev-T. These differences resulted in the accumulation of increased levels of subgenomic v-rel mRNA and protein, ultimately leading to transformation.     

Protein stabilization explains the gag requirement for transformation of lymphoid cells by Abelson murine leukemia virus.

The single protein encoded by Abelson murine leukemia virus is a fusion of sequence from the retroviral gag genes with the v-abl sequence. Deletion of most of the gag region from the transforming protein results in a virus capable of transforming fibroblasts but no longer capable of transforming lymphoid cells. Smaller deletions in gag reveal that p15 gag sequences are responsible for this effect, whereas deletion of p12 sequences had no effect on lymphoid transformation. In transformed fibroblasts, p15-deleted and normal proteins had similar activities and subcellular localization. When the p15-deleted genome was introduced into previously transformed lymphoid lines, its protein product exhibited a marked instability. The tyrosine-specific autophosphorylation activity per cell was less than 1/20th that of the nondeleted protein. Although pulse-Ia-beling showed that the p15-deleted protein was synthesized efficiently, immunoblotting demonstrated that its steady-state level was less than 1/10th that of the nondeleted Abelson protein. The specific instability of the p15-deleted protein in lymphoid cells explains the requirement of these sequences for lymphoid but not fibroblast transformation.     

Transformation of chicken embryo fibroblast cells by avian retroviruses containing the human Fyn gene and its mutated genes.

The transforming activity of the human fyn protein, p59fyn, which is a kinase of the src family, was investigated by testing the effect of recombinant avian retrovirus (Fyn virus) expressing p59fyn on chickens or cultured chicken embryo fibroblast (CEF) cells. The Fyn virus did not induce transformed foci. After several passages of the virus stock on CEF cells, however, a few foci were detected in the presence of dimethyl sulfoxide. Chickens inoculated with Fyn virus at the stage of 12-day-old embryos developed fibrosarcomas 3 to 6 weeks after hatching. The viruses obtained from these foci and from one of the tumor tissues showed high transforming activity in the presence of dimethyl sulfoxide, suggesting that these viruses carry spontaneous mutations of the fyn gene. Four fyn genes from CEF DNAs infected with transforming viruses were molecularly cloned, and their products were confirmed to possess transforming activity. DNA sequence analysis of the fyn genes showed that two of the four mutants have Thr instead of Ile at position 338 in the kinase domain. The other two mutants carry deletions of 78 and 108 base pairs, respectively, which result in complete loss of region C of SH2. The overall level of proteins containing phosphotyrosine was significantly higher in transformed cells than in normal CEF cells. Our data indicate that when expressed at high levels in a retrovirus, normal p59fyn cannot cause cellular transformation, but that mutant p59fyn with either a single amino acid substitution in the kinase domain or a deletion including region C produces a transforming protein, perhaps due to enhanced tyrosine kinase activity. This is the first observation that deletion of region C can unmask the potential transforming activity of a src family kinase.     

Sequences of the A-MuLV protein needed for fibroblast and lymphoid cell transformation

The role of various segments (gag or v-abl) of the Abelson murine leukemia virus (A-MuLV) genome in both lymphoid cell and fibroblast transformation was examined by deletion of areas from cloned, plasmid DNA representations of the genome. The deleted plasmids were tested by transfection into fibroblasts and by infection of bone marrow cells using virus stocks derived from the fibroblast transfectants. Deletion of gag coding sequence from the A-MuLV protein did not affect fibroblast transforming activity but abolished lymphoid transforming activity. The gag- A-MuLV genomes were very unstable in transformed fibroblasts leading to large secondary deletions in v-abl sequences. The gag- A-MuLV proteins also had lower autophosphorylation than their gag+ counterparts although cells transformed by gag- virus had a normal elevation of protein-linked phosphotyrosine. Systematic deletion of v-abl sequences showed that only 45,000 to the 130,000 molecular weight of v-abl sequence in the A-MuLV protein is needed for fibroblast transformation and, at most, slightly more is needed for lymphoid cell transformation.     

Insertion of several different DNAs in reticuloendotheliosis virus strain T suppresses transformation by reducing the amount of subgenomic mRNA.

The highly oncogenic retrovirus reticuloendotheliosis virus (Rev) strain T (Rev-T) has, relative to its helper virus Rev strain A, a substitution of the oncogene v-rel for most of the env gene and a large deletion of gag and pol sequences. When the helper virus sequences that are deleted in Rev-T are replaced, the recombinant virus is nontransforming (I. S. Y. Chen and H. M. Temin, Cell 31: 111-120, 1982). We show that suppression of transformation occurs when several different DNA sequences are inserted in Rev-T and that suppression is correlated with a reduction in the amount of v-rel mRNA and v-rel protein in infected cells. The reduced amount of v-rel protein is insufficient for transformation.     

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.     

Identification of a signal for nuclear targeting in platelet-derived-growth-factor-related molecules.

The v-vis gene encodes p28sis, the transforming protein of simian sarcoma virus. This gene resulted from a fusion of the env gene of simian sarcoma-associated virus and the woolly monkey gene for the B chain of platelet-derived growth factor (PDGF). Previous work has shown that the v-sis gene product undergoes signal sequence cleavage, glycosylation, dimerization, and proteolytic processing to yield a secreted form of the protein. It transport across the endoplasmic reticulum is blocked by the introduction of a charged amino acid residue within the signal sequence, the protein does not dimerize, is not secreted, and is no longer transforming as assayed by focus-forming ability in NIH 3T3 cells. Instead, this mutant protein localizes to the nucleus as demonstrated by both indirect immunofluorescence and cell fractionation. Using a series of deletion mutations, we delimited an amino acid sequence within this protein which is responsible for nuclear localization. This region is completely conserved in the predicted human c-sis protein, although it lies outside of regions required for transformation by the v-sis gene product. This nuclear transport signal is contained within amino acid residues 237 to 255, RVTIRTVRVRRPPKGKHRK. An amino acid sequence containing these residues is capable of directing cytoplasmic v-sis mutant proteins to the nucleus. This sequence is also capable of directing less efficient nuclear transport of a normally cytoplasmic protein, pyruvate kinase. Pulse-chase experiments indicate that the half-lives of nuclear and cytoplasmic v-sis mutant proteins are approximately 35 min. Using the heat-inducible hsp70 promoter from Drosophila melanogaster, we showed that the nuclear v-sis protein accumulates in the nucleus within 30 min of induction. The identification of a nuclear transport signal in the v-sis gene product raises interesting questions regarding the possibility of some function for PDGF or PDGF-related molecules in the nucleus.     

Hormone-regulated v-rel estrogen receptor fusion protein: reversible induction of cell transformation and cellular gene expression.

We describe the construction of a v-rel estrogen receptor fusion protein (v-relER) which allows the regulation of v-rel oncoprotein activity by hormone. In the presence of estrogen, v-relER readily transformed primary chicken fibroblasts and bone marrow cells in vitro. In both cell types, v-rel-specific transformation was critically dependent on the presence of estrogen or the estrogen agonist 4-hydroxytamoxifen (OHT). Withdrawal of estrogen or application of an estrogen antagonist, ICI164,384 (ICI) caused a reversal of the transformed phenotype. We also demonstrate that the v-relER protein binds to NF-kappa B sites in an estrogen-dependent manner, thereby showing that sequence-specific DNA binding of v-relER is critical for the activation of its transforming capacity. In transient transfection experiments, we failed to demonstrate a clear repressor or activator function of the v-rel moiety in v-relER. However, in v-relER-transformed bone marrow cells, estrogen and OHT induced elevated mRNA levels of two cellular genes whose expression is constitutive and high in v-rel-transformed cells. These results suggest that v-rel might exert part of its activity as an activator of rel-responsive genes.