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.     

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.     

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.     

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.     

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.     

v-rel oncoproteins in the nucleus and in the cytoplasm transform chicken spleen cells.

The transforming protein encoded by the v-rel oncogene of the highly oncogenic avian retrovirus reticuloendotheliosis virus strain T (Rev-T) is a 59,000-dalton protein, p59v-rel. The mechanism by which p59v-rel induces transformation of early lymphoid cells is unknown. As a step towards understanding the mechanism of v-rel-induced transformation, we sought to establish the subcellular site of action of p59v-rel. In this report, we show that p59v-rel contains sequences that are necessary for its efficient localization in the nucleus of infected chicken embryo fibroblasts. These v-rel sequences when added to the normally cytoplasmic protein, beta-galactosidase, directed that protein to the nucleus. A mutation in the v-rel nuclear-localizing sequence did not affect the transforming function, although it did alter the nuclear-localizing function. The addition of a supplemental nuclear-localizing sequence from simian virus 40 large T-antigen to v-rel resulted in the expression of a transforming rel protein which was located exclusively in the nucleus of transformed spleen cells, in contrast to wild-type p59v-rel, which was largely cytoplasmic in transformed spleen cells. Our results support the hypothesis that v-rel encodes a protein which can act either in the nucleus or in the cytoplasm to transform spleen cells.     

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.     

Evidence that the 90-kDa heat shock protein (HSP90) exists in cytosol in heteromeric complexes containing HSP70 and three other proteins with Mr of 63,000, 56,000, and 50,000

Monoclonal antibody (mAb) 8D3 and 3G3 are unique antibodies capable of precipitating both free 90-kDa heat shock protein (HSP90) and HSP90-protein complexes. Immunoprecipitation of [35S]methionine-labeled Hepa 1c1c7 cytosolic extracts were performed using mAb 8D3 or 3G3. The resulting immunoprecipitates can be dissociated from the mAb with a 500 mM NaCl wash. These washes were subjected to both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. Five major protein spots were detected in addition to HSP90 with the following relative molecular weights: 68,000, 63,000, 56,000, 50,000, and 188,000. On Western blots mAb 3G3 was capable of specifically binding to HSP90. Each of these proteins was localized on two-dimensional gels. Using one-dimensional gel electrophoresis and immunochemical localization on Western blots, the p68 spot was identified as HSP70, and the p56 spot was found to cross-react with polyclonal antibody JP-1 raised against a 59-kDa protein. This 59-kDa protein has been found previously to be associated with several steroid hormone receptors in rabbit uterine cytosol. Immunoprecipitation of [32P]orthophosphate-labeled Hepa 1c1c7 cytosol with mAb 8D3 or 3G3 revealed two major phosphorylated proteins with relative molecular weights of 90,000 and 50,000. The identities of p63 and p188 are currently unknown. This is the first report examining the major proteins that are complexed with HSP90 in mammalian cells.     

I kappa B gamma, a 70 kd protein identical to the C-terminal half of p110 NF-kappa B: a new member of the I kappa B family.

A cDNA corresponding to the 2.6 kb NF-kappa B mRNA species present in a variety of lymphoid cell lines has been molecularly cloned. The deduced 607 amino acid sequence is identical to the sequence of the C-terminal region of 110 kd NF-kappa B protein. A 70 kd protein can be identified in lymphoid cells using antibodies raised against the C-terminal region of p110 NF-kappa B. Comparison of the two-dimensional tryptic peptide maps of the 70 kd protein expressed in cells and the in vitro translated product encoded by the cDNA display extensive homology. The 70 kd protein expressed in bacteria prevents sequence-specific DNA binding of p50-p65 NF-kappa B heterodimer, p50 homodimer, and c-rel. p70 also interferes with transactivation by c-rel and prevents its nuclear translocation. The 70 kd protein, predominantly found in lymphoid cells, is a new member of the I kappa B family of proteins and is referred to as I kappa B gamma.