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.     

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.     

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.     

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.     

Characterization of the interaction of reticuloendotheliosis virus with the avian lymphoid system.

Splenic lymphocytes from chickens infected with reticuloendotheliosis virus (REV) are cytostatically impaired in their ability to undergo mitogen-induced blastogenesis ([³H]TdR uptake and proliferation), but are fully capable of eliciting cytotoxic reactions against allogeneic, ⁵¹Chromium-labeled chicken erythrocytes. Spleen cells from birds with reticuloendotheliosis (RE_s) are able to suppress DNA synthesis of normal splenic lymphocytes (N_s), but are unable to inhibit ¹[³H]TdR uptake by chick embryo fibroblasts. The suppression of the N_s mitogenic response is not restricted by major histocompatibility (B-locus) differences between populations of RE_s suppressor and N_s target cells. Moreover, infection of birds with an attenuated form of REV, which replicates in the host but does not cause tumorigenesis, also leads to suppression of phytohemagglutinin-induced, [³H]TdR uptake by host lymphocytes. These results are discussed in terms of the interaction between viral-infected/transformed cells and host defense mechanisms.     

Characterization of reticuloendotheliosis virus strain T DNA and isolation of a novel variant of reticuloendotheliosis virus strain T by molecular cloning.

Reticuloendotheliosis virus strain T (REV-T) is a highly oncogenic avian retrovirus which causes a rapid neoplastic disease of the lymphoreticular system. Upon infection, this virus gives rise to two species of unintegrated linear viral DNA, which are 8.3 and 5.5 kilobase pairs long and represent the helper virus (REV-A) and the oncogenic component (REV-T), respectively. Restriction endonuclease cleavage maps of these two DNA components indicate that REV-T DNA has a large portion of the genome deleted with respect to REV-A DNA and a substitution about 0.8 to 1.5 kilobase pairs long that is unrelated to REV-A DNA. These additional sequences comprise the putative transforming region of REV-T (rel). A chicken spleen cell line transformed by REV-T produced virus which upon infection gives rise to three species of unintegrated linear viral DNA (8.3, 5.5, and 3,3 kilobase pairs). We isolated the proviruses of the 8.3- and 3.3-kilobase pair species from this cell line by cloning in the phage vector Charon 4A. Restriction enzyme mapping showed that the two proviral clones are proviruses of REV-A and a variant of REV-T, respectively. A subclone of the variant REV-T provirus specific for the rel sequences of REV-T was used as a hybridization probe to demonstrate that the rel sequences are different from the putative transforming sequences of Schmidt-Ruppin Rous sarcoma virus strain A, avain myelocytomatosis virus, avian myeloblastosis virus, avian erythroblastosis virus, Abelson murine leukemia virus, and Friend erythroleukemia virus. In addition, the rel-specific hybridization probe was used to identify a specific set of sequences which are present in uninfected avian DNAs digested with several restriction enzymes. The corresponding cell sequences are not arranged like rel in REV-T.     

Avian reticuloendotheliosis virus strain A and spleen necrosis virus do not infect human cells

Spleen necrosis virus (SNV) and Reticuloendotheliosis virus strain A (REV-A) belong to the family of reticuloendotheliosis viruses and are 90% sequence related. SNV-derived retroviral vectors produced by the REV-A-based D17.2G packaging cell line were shown to infect human cells (H.-M. Koo, A. M. C. Brown, Y. Ron, and J. P. Dougherty, J. Virol. 65:4769-4776, 1991), while similar vectors produced by another SNV-based packaging cell line, DSH134G, are not infectious in human cells (reviewed by R. Dornburg, Gene Ther. 2:301-310, 1995). Here we describe a careful reevaluation of the infectivity of vectors produced from the most commonly used REV-A- or SNV-based packaging cells obtained from various sources with, among them, one batch of D17.2G packaging cells obtained from the American Type Culture Collection. None of these packaging cells produced vectors able to infect human cells. Thus, contrary to previously published data, we conclude that REV-based vectors are not infectious in human cells.     

Down-regulation of cytokeratin 14 mRNA in polyoma virus middle T-transformed rat liver epithelial cells.

We have recently shown that rat liver nonparenchymal epithelial cells, such as T51B cells, selectively express cytokeratin (CK) 14 as a partner of CK8 in their intermediate filaments, and we proposed CK14 as a unique cell lineage marker of the liver epithelial cell population (R. Blouin, M-J. Blouin, I. Royal, A. Grenier, A. Loranger, D. R. Roop, and N. Marceau, Differentiation, submitted for publication, 1992). In the present study, T51B-261A (spontaneously transformed) and T51B-261B (aflatoxin B1-treated) clones and clones derived from T51B cells transfected with SV40 large T (LT) and polyoma virus middle T (MT) were used to investigate CK gene expression in nontransformed and transformed liver epithelial cells. T51B-261A, T51B-261B, MT-T51B, and LT/MT-T51B clones all grew in calcium-deficient medium and formed colonies in soft agar, whereas LT-T51B clones did not grow at all in either one of these assays. T51B-261A and T51B-261B clones formed small, slow growing tumors when injected into newborn syngenic rats, whereas the MT-T51B and LT/MT-T51B clones produced rapidly forming, large tumors. There was no effect of cell transformation on CK expression, except in the clones expressing MT, where the CK intermediate filaments were completely lost. Analyses of [35S]methionine incorporation into the Triton-resistant cytoskeleton and of total proteins confirmed that CKs were absent. In contrast, vimentin intermediate filaments remained unaffected in all of the clones.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of medium of lowered NaCl concentration on virus release and protein synthesis in cells infected with reticuloendotheliosis virus.

When cultures producing reticuloendotheliosis virus were incubated for 24 h in medium of lowered NaCl concentration, virus production was inhibited. The extent of inhibition increased as the salt concentration of the medium was decreased. The inhibition was rapidly reversed by replacement of low-salt medium with normal medium. During the first hour after the inhibited cultures were returned to normal medium, virus was released at an accelerated rate, making the total amount of virus released by inhibited and control cultures the same. After 1 h in normal medium, the rate of virus production in the previously inhibited cultures was the same as in the control cultures. Incubation of infected cells in low-salt medium resulted in a 60% decrease in the overall rate of protein synthesis. Although returning the cells to normal medium rapidly reversed the inhibition of virus production, it did not rapidly increase the rate of protein synthesis. These results suggest that host cell-directed protein synthesis is preferentially inhibited by the low-ionic-strength medium, whereas that required for virus production continues.     

Interaction of cells of the lymphoid organs in the production of antibodies against the influenza virus. Report 1. Localization of the virus in the cells of the spleen]

A study was made of localization of crude and inactivated influenza virus injected intravenously and intraperitoneally. Macrophages were found to engulf the virus penetrating into the spleen; this was shown by the quantitative and qualitative immunofluorescent method, elcetron microscopy and inoculation to syngenous recipients of homogenates of macrophagal and lymphocytic fractions of splenic cells. No virus was revealed in the lymphocytes.     

Substitution of 5' helper virus sequences into non-rel portion of reticuloendotheliosis virus strain T suppresses transformation of chicken spleen cells

The genome of the highly oncogenic avian retrovirus reticuloendotheliosis virus strain T (REV-T) differs from that of the helper virus reticuloendotheliosis virus strain A by a substitution (rel and a large deletion. Further deletions, constructed in vitro, within the helper-virus-related sequences of REV-T have little effect on the ability of the virus to transform chicken spleen cells in vitro. However, deletions that extend into rel abolish transformation. Substitution of helper-virus-related sequences for the deleted region in the non-rel portion of REV-T also abolishes transformation. Viruses with revertant phenotype were obtained both spontaneously and by construction in vitro from these substituted recombinants. The revertant viruses have various mutations, including deletions and insertions, in the helper-virus-related sequences. Thus the additional helper-virus-related sequences suppress expression of transformation in cis, and the deletion in REV-T seems necessary for expression of the transforming properties of the virus.     

Sequence analysis for the complete proviral genome of reticuloendotheliosis virus Chinese strain HA9901

Reticuloendotheliosis virus (REV), a member of avian retrovirus, can cause tumor, immune suppression and a runting disease syndrome[1,2]. With several clas-sical reference strains, such as strain SNV from ducks, a replication defective oncogenic strain T …