Absence of missense mutations in activated c-myc genes in avian leukosis virus-induced B-cell lymphomas.

We have determined the nucleotide sequences of two independent DNA clones which contained the activated c-myc genes from avian leukosis virus-induced B-cell lymphomas. Neither of these c-myc genes contained missense mutations. This strongly supports the notion that the c-myc proto-oncogene in avian leukosis virus-induced B-cell lymphomas can be oncogenically activated by altered expression of the gene without a change in the primary structure of the gene product.     

Multiple proto-oncogene activations in avian leukosis virus-induced lymphomas: evidence for stage-specific events.

We have examined avian leukosis virus-induced B-cell lymphomas for multiple, stage-specific oncogene activations. Three targets for viral integration were identified: c-myb, c-myc, and a newly identified locus termed c-bic. The c-myb and c-myc genes were associated with different lymphoma phenotypes. The c-bic locus was a target for integration in one class of lymphomas, usually in conjunction with c-myc activation. The data indicate that c-myc and c-bic may act synergistically during lymphomagenesis and that c-bic is involved in late stages of tumor progression.     

Two unrelated cell-derived sequences in the genome of avian leukemia and carcinoma inducing retrovirus MH2.

Molecularly cloned proviral DNA of avian replication-defective retrovirus Mill Hill No. 2 (MH2) was analyzed. The MH2 provirus measures 5.5 kb including two long terminal repeats (LTR), and contains a partial complement of the structural gene gag, 1.5 kb in size, near the 5' terminus, and a 1.3-kb segment of the v-myc transforming gene near the 3' terminus. These v-myc sequences are closely related to the v-myc transforming gene of avian acute leukemia virus MC29, and to the cellular chicken gene c-myc. The gag and myc domains on the MH2 provirus are separated by unique sequences, 1.3 kb in size and termed v-mil, which are unrelated to v-myc, or to other oncogenes or structural genes of the avian leukemia-sarcoma group of retroviruses. Normal chicken DNA contains sequences closely related to v-mil, termed c-mil. Analyses of chicken c-mil clones isolated from a recombinant DNA library of the chicken genome reveal that c-mil is a single genetic locus with a complex split gene structure. In the MH2 genome, v-mil is expressed via genome-sized mRNA as a gag-related hybrid protein, p100gag-mil, while v-myc is apparently expressed via subgenomic mRNA independently from major coding regions of structural genes. The presence in the MH2 genome of two unrelated cell-derived sequences and their independent expression may be significant for the oncogenic specificities of this virus.     

Infrequent involvement of c-fos in avian leukosis virus-induced nephroblastoma.

To determine whether c-fos is involved in avian leukosis virus-induced nephroblastoma, 28 tumors from chickens were analyzed for novel fos fragments. DNA from 1 of 16 clonal outgrowths (in chicken 6561) contained novel fos-related EcoRI and KpnI fragments which hybridized to both v-fos and viral probes. Oncogenicity tests using filtered 6561 tumor cell homogenates did not reveal a tumor-inducing transduction of c-fos. We conclude that c-fos is only an occasional target for proviral insertions or new transductions in avian leukosis virus-induced nephroblastoma. The results also identify a polymorphism in c-fos in K28 chickens and demonstrate that unintegrated viral DNA is not a general characteristic of avian leukosis virus-induced nephroblastoma.     

Molecular cloning and characterization of gag-, pol-, and env-related gene sequences in the ev- chicken.

Using less stringent hybridization conditions and cloned viral DNA probes representing the avian sarcoma virus gag, pol, env, and long terminal repeat (LTR) gene sequences, we detected related sequences in two avian species purportedly lacking all endogenous avian leukosis viruses, the ev- chicken and the Japanese quail. The blot hybridization patterns obtained with the various probes suggest the presence of between 40 and 100 copies of retrovirus-related sequences in the genomes of these two species. An ev- chicken genomic DNA library was prepared and screened with gag-specific and pol-specific DNA probes. Several different clones were obtained from this library and characterized. Analysis of these clones revealed that the retrovirus-related gene sequences are linked in the order LTR-gag-pol-env-LTR, a structure indicative of a complete provirus. These data indicate the presence of previously unidentified endogenous retrovirus species in avian cells, suggesting that under the appropriate conditions of hybridization additional, more distantly evolved families of endogenous retrovirus genes may be identified in vertebrate species.     

Molecular cloning of proviral DNA and structural analysis of the transduced myc oncogene of avian oncovirus CMII.

Molecularly cloned proviral DNA of avian oncogenic retrovirus CMII was isolated by screening a genomic library of a CMII-transformed quail cell line with a myc-specific probe. On a 10.4-kilobase EcoRI fragment, the cloned DNA contained 4.4 kilobases of CMII proviral sequences extending from the 5' long terminal repeat to the EcoRI site within the partial (delta) complement of the env gene. The gene order of CMII proviral DNA is 5'-delta gag-v-myc-delta pol-delta env-3'. All three structural genes are partially deleted: the gag gene at the 3' end, the env gene at the 5' end, and the pol gene at both ends. The delta gag (0.83 kilobases)-v-myc (1.50 kilobases) sequences encode the p90gag-myc transforming protein of CMII. In comparison with the p110gag-myc protein of acute leukemia virus MC29, p90gag-myc lacks amino acids corresponding to additional 516 bases of gag sequences and 12 bases of 5' v-myc sequences present in the MC29 genome. Nucleotide sequence analysis of CMII proviral DNA at the delta gag-v-myc and the v-myc-delta pol junctions revealed significant homologies between avian retroviral structural genes and the cellular oncogene c-myc precisely at the positions corresponding to the gene junctions in CMII. Furthermore, the delta gag-v-myc junction in CMII corresponds to sequence elements in gag and C-myc that are possible splicing signals. The data suggest that transduction of cellular oncogenes may involve RNA splicing and recombination with homologous sequences on retroviral vectors. Different sequence elements of both the retroviral vectors and the c-myc gene recombined during genesis of highly oncogenic retroviruses CMII, MC29, or MH2.     

Avian leukosis virus-induced tumors have common proviral integration sites and synthesize discrete new RNAs: oncogenesis by promoter insertion

Unlike other RNA tumor viruses, avian leukosis viruses (which cause lymphomas and occasionally other neoplasms) lack discrete "transforming genes". We have analyzed the virus-related DNA and RNA of avian leukosis virus (ALV)-induced tumors in an attempt to gain insight into the mechanism of ALV oncogenesis. Our results show that viral gene products are not required for maintenance of neoplastic transformation. Primary and metastatic tumors are clonal and thus presumably derived from a single infected cell. Most importantly, tumors from different birds have integration sites in common. Tumor cells synthesize discrete new poly(A) RNAs consisting of viral sequences covalently linked to cellular sequences. These RNA species are expressed at high levels in tumor cells. Our results suggest that in lymphoid tumors, an ALV provirus is integrated adjacent to a specific cellular gene, and the insertion of the viral promoter adjacent to this gene results in its enhanced expression, leading to neoplasia. These results have potentially important implications for the mechanism of non-viral carcinogenesis.     

Retroviral insertional activation of the c-myb proto-oncogene in a Marek's disease T-lymphoma cell line.

Marek's disease virus (MDV) is an avian herpesvirus that causes, in chickens, a lymphoproliferative disease characterized by malignant transformation of T lymphocytes. The rapid onset of polyclonal tumors indicates the existence of MDV-encoded oncogenic products. However, the molecular basis of MDV-induced lymphoproliferative disease and latency remains largely unclear. Several lines of evidence suggest that MDV and Rous-associated virus (RAV) might cooperate in the development of B-cell lymphomas induced by RAV. Our present results indicate for the first time that MDV and RAV might also act synergistically in the development of T-cell lymphomas. We report an example of an MDV-transformed T-lymphoblastoid cell line (T9) expressing high levels of a truncated C-MYB protein as a result of RAV integration within one c-myb allele. The chimeric RAV-c-myb mRNA species initiated in the 5' long terminal repeat of RAV are deprived of sequences corresponding to c-myb exons 1 to 3. The attenuation of MDV oncogenicity has been strongly related to structural changes in the MDV BamHI-D and BamHI-H DNA fragments. We have established that both DNA restriction fragments are rearranged in the T9 MDV-transformed cells. Our results suggest that retroviral insertional activation of the c-myb proto-oncogene is a critical factor involved in the maintenance of the transformed phenotype and the tumorigenic potential of this T-lymphoma cell line.     

Avian acute leukemia virus OK 10: analysis of its myc oncogene by molecular cloning.

Several DNAs representing the genome of the avian acute leukemia virus OK 10 were isolated by molecular cloning from a transformed quail cell line, 9C, which contained at least six OK 10 proviruses. Recombinant lambda phages harboring the OK 10 genome and additional flanking cellular DNA sequences were studied by restriction endonuclease mapping and hybridization to viral cDNA probes. Six of the clones represented complete proviruses with similar, if not identical, viral sequences integrated at different positions in the host DNA. The organization of the OK 10 genome was determined by electron-microscopic analysis of heteroduplexes formed between the cloned OK 10 DNA and DNAs representing the c-myc gene and the genomes of two other avian retroviruses, Rous-associated virus-1 and MC29. The results indicated that the OK 10 proviral DNA is about 7.5 kilobases in size with the following structure: 5'-LTR-gag-delta polmyc-delta env-LTR-3', where LTR indicates a long terminal repeat. The oncogene of OK 10, v-mycOK 10, forms a continuous DNA segment of around 1.7 kilobases between pol and env. It is similar in structure and length to the v-myc gene of MC29, as demonstrated by restriction endonuclease and heteroduplex analyses. Two of the OK 10 proviruses were tested in transfection experiments: both DNAs gave rise to virus with the transforming capacities of OK 10 when Rous-associated virus-1 was used to provide helper virus functions.     

Circles with two tandem long terminal repeats are specifically cleaved by pol gene-associated endonuclease from avian sarcoma and leukosis viruses: nucleotide sequences required for site-specific cleavage.

The avian retroviral pol gene-encoded DNA endonuclease (pol-endo) has been shown to selectively cleave the viral long terminal repeat sequences (LTRs) in single-stranded DNA substrates in a region known to be joined to host DNA during integration (G. Duyk, J. Leis, M. Longiaru, and A.M. Skalka, Proc. Natl. Acad. Sci. USA 80:6745-6749, 1983). The preferred sites of cleavage were mapped to the unique U5/U3 junctions found only in covalently closed circular DNA molecules containing two tandem LTRs. The cuts occurred three nucleotides 5' to the axis of symmetry of the 12-of-15-base-pair nearly perfect inverted repeat which marks the LTR junction. Experiments with double-stranded supercoiled DNA substrates revealed a similar specificity for nicking. Also, the endonuclease associated with the pol cleavage product, pp32, has the same specificity as the alpha beta form. The limits of sequence required for site-selective cleavage near the U5/U3 junction were established with single-stranded DNA substrates. A domain no larger than 44 base pairs allowed site-selective cleavage in each strand in vitro. Recognition of either strand appeared to be independent of the other, and in each case, the critical sequence was asymmetrically distributed with respect to the U5/U3 junction. The predominant contribution was from the U5 domain; this is consistent with its conservation in the LTR sequences of a number of avian sarcoma and leukosis viruses.     

Long terminal repeat (LTR) sequences, env, and a region near the 5'' LTR influence the pathogenic potential of recombinants between Rous-associated virus types 0 and 1.

A series of recombinants between Rous-associated virus type 0 (RAV-0), RAV-1, and a replication-competent avian leukosis virus vector (RCAN) have been tested for disease potential in day-old inoculated K28 chicks. RAV-0 is a benign virus, whereas RAV-1 and RCAN induce lymphoma and a low incidence of a variety of other neoplasms. The results of the oncogenicity tests indicate that (i) the long terminal repeat regions of RAV-1 and RCAN play a major role in disease potential, (ii) subgroup A envelope glycoproteins are associated with a two- to fourfold higher incidence of lymphoma than subgroup E glycoproteins, and (iii) certain combinations of 5' viral and env sequences cause osteopetrosis in a highly context-dependent manner. Long terminal repeat and env sequences appeared to influence lymphomogenic potential by determining the extent of bursal infection within the first 2 to 3 weeks of life. This would suggest that bursal but not postbursal stem cells are targets for avian leukosis virus-induced lymphomogenesis. The induction of neutralizing antibody had no obvious influence on the incidence of lymphoma.     

Nucleotide sequence of a transduced myc gene from a defective feline leukemia provirus.

The nucleotide sequence of a feline v-myc gene and feline leukemia virus (FeLV) flanking regions was determined. Both the nucleotide and predicted amino acid sequences are very similar to the murine and human c-myc genes (ca. 90% identity). The entire c-myc coding sequence is represented in feline v-myc and replaces portions of the gag and env genes and the entire pol gene. The coding sequence is in phase with the gag gene reading frame; v-myc, therefore, appears to be expressed as a gag-myc fusion protein. Viral sequences at the 3' myc-FeLV junction begin with the hexanucleotide CTCCTC, which is also found at the 3' fes-FeLV junction of both Gardner-Arnstein and Snyder-Theilen feline sarcoma viruses. These similarities suggest that some sequence specificity may exist for the transduction of cellular genes by FeLV. Feline v-myc lacks a potential phosphorylation site at amino acid 343 in the putative DNA-binding domain, whereas both human and murine c-myc have such sites. Avian v-myc has lost a potential phosphorylation site which is present in avian c-myc five amino acids from the potential mammalian site. If these sites are actually phosphorylated in normal c-myc proteins, their loss may alter the DNA-binding affinity of v-myc proteins.