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.     

Avian reticuloendotheliosis virus: characterization of genome structure by heteroduplex mapping

The genome structure of defective, oncogenic avian reticuloendotheliosis virus (REV) was studied by heteroduplex mapping between the full-length complementary DNA of the helper virus REV-T1 and the 30S REV RNA. The REV genome (5.5 kilobases) had a deletion of 3.69 kilobases in the gag-pol region, confirming the genetic defectiveness of REV. In addition, REV lacked the sequences corresponding to the env gene but contained, instead, a contiguous stretch (1.6 to 1.9 kilobases) of the specific sequences presumably related to viral oncogenicity. Unlike those of other avian acute leukemia viruses, the transformation-specific sequences of REV were not contiguous with the gag-pol deletion. Thus, REV has a genome structure similar to that of a defective mink cell focus-inducing virus or a defective murine sarcoma virus. An additional class of heteroduplex molecules containing the gag-pol deletion and two other smaller deletion loops was observed. These molecules probably represented recombinants between the oncogenic REV and its helper virus.     

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: identification of the hematopoietic target cell for transformation

Non-virus-producing hematopoietic cells transformed in vitro by reticuloendotheliosis virus (REV-T) induce lethal "reticuloendotheliosis" when inoculated into histocompatible chickens. This is the first direct demonstration that an in vivo target cell of an avian acute leukemia virus can be transformed in vitro. The tumorigenic, REV-T-transformed non-virus-producing cells fail to express helper-virus-coded proteins. REV-T transformed tumorigenic cells therefore do not require helper-virus functions. Cells transformed in vivo or in vitro by REV-T have lymphoblastoid morphology and express low levels of terminal-deoxynucleotidyl-transferase activity and bursal-cell determinants. One clone synthesized Ig mu. The preferred target cells for REV-T transformation are therefore immature lymphoid cells that express B-cell determinants. We propose that the unique transforming sequence of REV-T be designated rel (lymphoid).     

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.     

Proviruses of avian sarcoma virus are terminally redundant, co-extensive with unintegrated linear DNA and integrated at many sites.

We have analyzed the DNA from 15 clones of avian sarcoma virus (ASV)-transformed rat cells with restriction endonucleases and molecular hybridization techniques to determine the location and structure of proviral DNA. All twenty units of proviral DNA identified in these 15 clones appear to be inserted at different sites in host DNA. In each of the ten cases that could be sufficiently well mapped, entirely different regions of cellular DNA were involved. Thus ASV DNA can be accommodated at many positions in cellular DNA, but the existence of preferred sites has not been excluded. Six of the 15 clones carry only one normal provirus, two contain two normal proviruses, and seven harbor either one or two proviruses that appear anomalous in physical mapping tests. Both ends of at least 18 proviruses, however, were found to contain sequences specific to both the 3' and 5' termini of viral RNA. The organization of these terminally redundant sequences appeared identical to that of the 300 base pair (bp) repeats found at the ends of unintegrated linear DNA (Shank et al., 1978). Proviral DNA is therefore co-extensive, or nearly co-extensive, with unintegrated linear DNA and has a structure we denote as CELL DNA-3'5'----------3'5'-CELL DNA. Three of the four anomalous proviruses which were fully analyzed were deletion mutants lacking 25--65% of the genetic content of ASV; the fourth provirus had a novel site for cleavage by Eco RI but was otherwise normal. Tests for the biological competence of proviral DNA, based upon rescue of transforming virus after fusion with chicken cells, were generally consistent with the physical mapping studies.     

Characterization of avian myeloblastosis-associated virus DNA intermediates.

The major species of unintegrated linear viral DNA identified in chicken embryonic fibroblasts infected with either the avian myeloblastosis-associated viruses (MAV-1, MAV-2) or the standard avian myeloblastosis virus complex (AMV-S) has a mass of 5.3 X 10(6) daltons. An additional minor DNA component observed only in AMV-S-infected cells has a mass of 4.9 X 10(6) daltons. The unintegrated linear viral DNAs and integrated proviruses of MAV-1 and MAV-2 have been analyzed by digestion with the restriction endonucleases EcoRI and HindIII. MAV-2 lacks a HindIII site present in MAV-1. These fragments have been compared to those generated by EcoRI and HindIII digestion of linear viral DNAs of AMV-S. Restriction enzyme digestion of AMV-S viral DNA produced unique fragments not found with either MAV-1 or MAV-2 viral DNAs. The major viral component present in AMV-S stocks has the HindIII restriction pattern of MAV-1. Restriction enzyme analysis of the 5.3 X 10(6)-dalton unintegrated MAV viral DNAs and their integrated proviruses suggests that the DNAs have a direct terminal redundancy of approximately 0.3 megadaltons and integrate colinearly with respect to the unintegrated linear DNA.     

Genome of Reticuloendotheliosis Virus: Characterization by Use of Cloned Proviral DNA

Reticuloendotheliosis virus is an avian type C retrovirus that is capable of transforming fibroblasts and hematopoietic cells both in vivo and in vitro. This virus is highly related to the three other members of the reticuloendotheliosis virus group, including spleen necrosis virus, but it is apparently unrelated to the avian leukosis-sarcoma virus family. Previous studies have shown that it consists of a replication-competent helper virus (designated REV-A) and a defective component (designated REV) that is responsible for transformation. In this study we used restriction endonuclease mapping and heteroduplex analysis to characterize the proviral DNAs of REV-A and REV. Both producer and nonproducer transformed chicken spleen cells were used as sources of REV proviral DNA; this genome was mapped in detail, and fragments of it were cloned in lambdagtWES.lambdaB. The infected canine thymus line Cf2Th(REV-A) was used as a source of REV-A proviral DNA. The restriction maps and heteroduplexes of the REV and REV-A genomes showed that (proceeding from 5' to 3') (i) REV contains a large fraction of the REV-A gag gene (assuming a gene order of gag-pol-env and gene sizes similar to those of other type C viruses), for the two genomes are very similar over a distance of 2.1 kilobases beginning at their 5' termini; (ii) most or all of REV-A pol is deleted in REV; (iii) REV contains a 1.1 kilobase segment derived from the 3' end of REV-A pol or the 5' end of env or both; (iv) this env region in REV is followed by a 1.9-kilobase segment which is unrelated to REV-A; and (v) the helper-unrelated segment of REV extends essentially all of the way to the beginning of the 3' long terminal repeat. Therefore, like avian myeloblastosis virus but unlike the other avian acute leukemia viruses and most mammalian and avian sarcoma viruses, REV appears to be an env gene recombinant. We also found that the REV-specific segment is derived from avian DNA, for a cloned REV fragment was able to hybridize with the DNA from an uninfected chicken. Therefore, like the other acute transforming viruses, REV appears to be the product of recombination between a replication-competent virus and host DNA. Two other defective genomes in virus-producing chicken cells were also cloned and characterized. One was very similar to REV in its presumptive gag and env segments, but instead of a host-derived insertion it contained additional env sequences. The second was similar (but not identical) to the first in its gag and env regions and appeared to contain an additional 1-kilobase inversion of REV-A sequences.     

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.     

Molecular cloning of the Harvey sarcoma virus circular DNA intermediates. II. Further structural analyses.

Three species of unintegrated supercoiled Harvey sarcoma virus DNA (6.6, 6.0, and 5.4 kilobase pairs) have been molecularly cloned from Harvey sarcoma virus-infected cells. On the basis of restriction enzyme analyses, the 6.6- and 6.0-kilobase pair viral DNAs contain two and one copies, respectively, of a 650-base pair DNA segment which contains sequences present at the 3' and 5' termini of the viral genome. R-loop structures formed between Moloney leukemia virus RNA and the cloned Harvey sarcoma virus DNA indicated that about 500 base pairs of the 650-base pair repeating segment was complementary to the 3' end of the viral RNA. During amplification in the Escherichia coli host, some recombinants containing the 6.6- or the 6.0-kilobase pair Harvey sarcoma virus DNA insert acquired or lost the complete 650-base pair DNA segment. These changes occurred in both recA+ and recA- E. coli.     

Structure of the provirus within NIH 3T3 cells transfected with Harvey sarcoma virus DNA.

NIH 3T3 cells transformed with unintegrated Harvey sarcoma virus (HSV) linear DNA generally acquired a complete HSV provirus. Infection of these transformed cells with Moloney murine leukemia helper virus was followed by release of infectious particles. The HSV provirus within these transfected cells was convalently joined to nonviral DNA sequences and was termed "cell-linked" HSV DNA. The association of this cell-virus DNA sequence with the chromosomal DNA of a transfected cell was unclear. NIH 3T3 cells could also become transformed by transfection with this cell-linked HSV DNA. In this case, the recipient cells generally acquired a donor DNA fragment containing both the HSV provirus and its flanking nonviral sequences. After cells acquired either unintegrated or cell-linked HSV DNA, the newly established provirus and flanking cellular sequences underwent amplifications to between 5 and 100 copies per diploid cell. NIH 3T3 cells transfected with HSV DNA may acquire deleted proviral DNA lacking at least 1.3 kilobase pairs from the right end of full-length HSV 6-kilobase-pair DNA (corresponding to the 3'-proximal portion of wild-type HSV RNA). Cells bearing such deleted HSV genomes were transformed, indicating that the viral transformation gene lies in the middle or 5'-proximal portion of the HSV RNA genome. However, when these cells were infected with Moloney murine leukemia helper virus, only low levels of biologically active sarcoma virus particles were released. Therefore, the 3' end of full-length HSV RNA was required for efficient transmission of the viral genome.     

McDonough feline sarcoma virus: characterization of the molecularly cloned provirus and its feline oncogene (v-fms).

The genetic structure of the McDonough strain of feline sarcoma virus (SM-FeSV) was deduced by analysis of molecularly cloned, transforming proviral DNA. The 8.2-kilobase pair SM-FeSV provirus is longer than those of other feline sarcoma viruses and contains a transforming gene (v-fms) flanked by sequences derived from feline leukemia virus. The order of genes with respect to viral RNA is 5'-gag-fms-env-3', in which the entire feline leukemia virus env gene and an almost complete gag sequence are represented. Transfection of NIH/3T3 cells with cloned SM-FeSV proviral DNA induced foci of morphologically transformed cells which expressed SM-FeSV gene products and contained rescuable sarcoma viral genomes. Cells transformed by viral infection or after transfection with cloned proviral DNA expressed the polyprotein (P170gag-fms) characteristic of the SM-FeSV strain. Two proteolytic cleavage products (P120fms and pp55gag) were also found in immunoprecipitates from metabolically labeled, transformed cells. An additional polypeptide, detected at comparatively low levels in SM-FeSV transformants, was indistinguishable in size and antigenicity from the envelope precursor (gPr85env) of feline leukemia virus. The complexity of the v-fms gene (3.1 +/- 0.3 kilobase pairs) is approximately twofold greater than the viral oncogene sequences (v-fes) of Snyder-Theilen and Gardner-Arnstein FeSV. By heteroduplex, restriction enzyme, and nucleic acid hybridization analyses, v-fms and v-fes sequences showed no detectable homology to one another. Radiolabeled DNA fragments representing portions of the two viral oncogenes hybridized to different EcoRI and HindIII fragments of normal cat cellular DNA. Cellular sequences related to v-fms (designated c-fms) were much more complex than c-fes and were distributed segmentally over more than 40 kilobase pairs in cat DNA. Comparative structural studies of the molecularly cloned proviruses of Synder-Theilen, Gardner-Arnstein, and SM-FeSV showed that a region of the feline-leukemia virus genome derived from the pol-env junction is represented adjacent to v-onc sequences in each FeSV strain and may have provided sequences preferred for recombination with cellular genes.     

Identification of unintegrated forms of Kirsten murine sarcoma viral DNA and restriction endonuclease cleavage map of linear DNA.

We detected unintegrated linear 7.0-kilobase pair DNA and covalently closed circular DNA species in NIH3T3 cells recently infected with Kirsten murine sarcoma virus. Using the linear DNA, we constructed a restriction endonuclease cleavage map and compared it with the map of Harvey murine sarcoma virus. The restriction endonuclease maps of two segments, one 1.2 kilobase pairs (SmaI site) to 3.7 kilobase pairs (HindIII site) from the right end (corresponding to the viral 3' side) and the other 0.5 kilobase pair (SmaI and KpnI sites) to 0.9 kilobase pair (KpnI site) from the left end, were identical in the two virus types.