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.     

Specificity of avian leukosis virus-induced hyperlipidemia

Rous-associated virus 7 (RAV-7) is a subgroup C avian leukosis virus which does not transform cells in vitro or carry an oncogene. When injected into 1-day-old hatched chicks, RAV-7 causes a low incidence of lymphoid leukosis after a latent period of several months. In contrast, infection of 10-day-old chicken embryos with RAV-7 leads to a disease syndrome characterized by stunting, obesity, atrophy of the bursa and the thymus, high triglyceride and cholesterol levels, reduced thyroxine levels, and increased insulin levels (Carter et al., Infect. Immun. 39:410-422, 1983; J.K. Carter and R.E. Smith, Infect. Immun. 40:795-805, 1983). Histopathological examination of tissues from affected chicks revealed an accumulation of lipid in the liver and an extensive infiltration of the thyroid and pancreas by lymphoblastoid cells. In the present investigation, the subgroup specificity of this syndrome was investigated. Other subgroup C avian leukosis viruses (transformation-defective B77, transformation-defective Prague C strain of Rous sarcoma virus, and RAV-49) caused stunting, infiltration of the thyroid and pancreas, increased liver weights, decreased thyroxine levels, and increased insulin levels, but they did not cause a uniform, profound increase in triglyceride and cholesterol levels. Avian leukosis viruses of subgroup A [myeloblastosis-associated virus 1 causing osteopetrosis [MAV-1(O)] and RAV-1], subgroup B [MAV-2(O), MAV-2 causing nephroblastoma [MAV-2(N)], and RAV-2], subgroup D (RAV-50), and subgroup F (ring-necked pheasant virus and RAV-61) did not cause a syndrome identical to that induced by RAV-7. All of the viruses examined induced some stunting and a reduction in thyroxine levels which correlated with the stunting. The two subgroup F viruses caused an infiltration of the thyroid which may have been secondary to severe lung involvement. We conclude that the RAV-7 syndrome is unique, particularly in the induction of a hyperlipidemia.     

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.     

Chicken leukosis virus genome sequences in DNA from normal chick cells and virus-induced bursal lymphomas.

Genome sequences of two recent field isolates of avian leukosis viruses in the DNA of normal and neoplastic chicken cells were studied by DNA-RNA hybridization under conditions of DNA excess. Comparisons were made between 60-70S RNA from these viruses and that of a chicken endogenous type C virus (RAV-0), and of a series of "laboratory" leukosis and sarcoma viruses, by competitive hybridization analysis. A minimum of 18% of the genome sequences of both ALV isolates detected in DNA from lymphomas they induced were not detected in normal chicken DNA. The vast majority of the fraction of RNA sequences from ALV which do form hybrids with normal chick DNA appear to be reacting with the endogenous provirus of RAV-0. The genomic representation of a variety of avian leukosis and sarcoma viruses in normal chicken cells could not be distinguished by these methods (except that 13% of the RAV-0 genome was not shared with any of the other viruses). In contrast, the portion of the ALV genome exogenous to the normal chicken geome showed significant divergence from that of two sarcoma viruses (Pr RSV-C and B-77). The increased hybridization of ALV RNA with lymphoma DNA was used to detect the appearance of ALV specific sequences in the bursa of Fabricius following infection.increased hybridization was correlated with both the time after infection and the extent of replacement of the bursa by lymphoma. About one half of the increase in hybridization preceded histologic evidence of transformation.     

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.     

Avian bic, a gene isolated from a common retroviral site in avian leukosis virus-induced lymphomas that encodes a noncoding RNA, cooperates with c-myc in lymphomagenesis and erythroleukemogenesis

bic is a novel gene identified at a common retroviral integration site in avian leukosis virus-induced lymphomas and has been implicated as a collaborator with c-myc in B lymphomagenesis. It lacks an extensive open reading frame and is believed to function as an untranslated RNA (W. Tam, Gene 274:157-167, 2001; W. Tam, D. Ben-Yehuda, and W. S. Hayward, Mol. Cell. Biol. 17:1490-1502, 1997). The oncogenic potential of bic, particularly its ability to cooperate with c-myc in oncogenesis, was tested directly by expressing c-myc and bic, either singly or in pairwise combination, in cultured chicken embryo fibroblasts (CEFs) and in chickens using replication-competent retrovirus vectors. Coexpression of c-myc and bic in CEFs caused growth enhancement of cells. Most importantly, chick oncogenicity assays demonstrated that bic can cooperate with c-myc in lymphomagenesis and erythroleukemogenesis. The present study provides direct evidence for the involvement of untranslated RNAs in oncogenesis and provides further support for the role of noncoding RNAs as riboregulators.     

Immunological phenotype of lymphomas induced by avian leukosis viruses.

The production of immunoglobulin by six cell lines derived from bursal tumors induced by avian leukosis virus follows two general patterns: (i) three cell lines that have been extensively passaged in culture synthesize and secrete light chains only; (ii) three cell lines that are recently isolated produce and secrete monomeric immunoglobulin M in addition to free light chains. All six cell lines synthesize and secrete both glycosylated and unglycosylated forms of light chain. We conclude that the cell lines established from lymphomas induced by avian leukosis virus represent relatively mature, but possibly abnormal, stages in the development of chicken B-lymphocytes. The immunoglobulin M produced by the cell lines failed to form detectable immune complexes with avian leukosis virus. It therefore appears that the immunoglobulin M is not directed against viral antigens and that autogenous antigenic stimulus cannot account for the sustained growth of the neoplastic B-lymphocytes.     

Viral DNA in bursal lymphomas induced by avian leukosis viruses.

Avian leukosis viruses (ALV) induce malignant lymphoma of the bursa of Fabricius. Viral DNA in tumors and normal tissues from infected birds were analyzed by using restriction endonucleases. Viral DNA fragments diagnostic of the exogenous ALV were easily detected in tumors, uninvolved bursal tissue, kidney, and erythrocyte nuclei. Exogenous viral DNA was more difficult to detect in liver. Using a restriction endonuclease (SacI) which cleaves linear unintegrated ALV DNA in a single site to define integration sites in DNA from the various tissues, we were able to detect ALV DNA only in tumor tissue. We concluded that the proviral DNA detected in the various nontumor tissue must be integrated in multiple sites. The appearance of ALV integration sites uniquely in tumors suggests that they are clonal growths. Furthermore, the data suggested the presence of a single exogenous integration site for the ALV provirus in each of six early neoplastic bursal nodules. This provirus appeared to retain the organization of EcoRI and BamHI recognition sequences present in the genome of virus used to infect the birds. The ALV integration site appeared different in each of the tumors studied. In a widespread metastatic lymphoma, multiple ALV integration sites were found as well as structural alterations in at least some copies of the ALV provirus.     

Proteins expressed by the c-myc oncogene in lymphomas of human and avian origin

We have prepared antisera against synthetic peptides corresponding to the C-terminal region of the avian and human myc oncogene coding sequences. Immunoprecipitates from avian and human cells show two major proteins which, by the criteria of hybrid-selected translation, transfection, and peptide-blocking assays, are the c-myc protein products. These proteins are phosphorylated nuclear proteins which are tightly bound to the nuclear matrix-lamin and which have a short half-life. Analysis of avian and human lymphoma cell lines containing rearranged c-myc alleles show significant changes in the ratio of the two proteins although only the avian lymphomas have increased quantities of c-myc protein.     

Activation of c-erbB in avian leukosis virus-induced erythroblastosis leads to the expression of a truncated EGF receptor kinase.

Chicken erythroblastosis caused by avian leukosis virus (ALV) is thought to be mediated by activation of the c-erbB/EGF receptor oncogene by a promoter-insertion mechanism. Here we study the proteins expressed by two ALV-induced leukemias and compare them with the avian EGF receptor and with the oncogene product of avian erythroblastosis virus (v-erbB) which was shown to be a truncated EGF receptor. It appears that the two leukemias express truncated EGF receptors of slightly different sizes with intrinsic tyrosine kinase activity. Hence, acute and chronic retroviruses utilize a common pathway for transformation. Moreover, the proteins expressed in the leukemias are similar to the avian EGF receptor with respect to their phosphopeptide maps, suggesting that they do not carry the C-terminal deletion characteristic of v-erbB.     

The c-myc locus is a common integration site in type B retrovirus-induced T-cell lymphomas

Type B leukemogenic virus (TBLV) induces rapidly appearing T-cell leukemias. TBLV insertions near the c-myc gene were detectable in 2 of 30 tumors tested, whereas 80% of the tumors showed c-myc overexpression. TBLV insertions on chromosome 15 (including a newly identified locus, Pad7) may cause c-myc overexpression by cis-acting effects at a distance.     

Molecular characterization of three erbB transducing viruses generated during avian leukosis virus-induced erythroleukemia: extensive internal deletion near the kinase domain activates the fibrosarcoma- and hemangioma-inducing potentials of erbB.

Three new erbB transducing viruses generated during avian leukosis virus-induced erythroblastosis have been cloned and sequenced, and their transforming abilities have been analyzed. Provirus 9134 E1 expresses an amino-terminally truncated erbB product that is analogous to the proviral insertionally activated c-erbB gag-erbB fusion product. This virus efficiently induces erythroblastosis, but does not transform fibroblasts in vitro or induce sarcomas in vivo. In contrast, virus 9134 S3 expresses an erbB product identical to the erbB product of 9134 E1, with the exception of a large internal deletion located between the kinase domain and the putative autophosphorylation site, P1. Interestingly, this virus is no longer capable of inducing erythroblastosis, but can induce both fibrosarcomas and hemangiomas in vivo. Provirus 9134 F3 has sustained an approximately 23-amino-acid carboxy-terminal truncation and is capable of inducing both erythroblastosis and sarcomagenesis. This virus expresses an erbB product with the shortest carboxy-terminal truncation sufficient to reveal the sarcomagenic potential of this protein. The distinct transforming properties of these viruses indicate that different structural domains of the erbB product confer distinct disease specificities.     

Influence of env and long terminal repeat sequences on the tissue tropism of avian leukosis viruses.

Adsorption and penetration of retroviruses into eucaryotic cells is mediated by retroviral envelope glycoproteins interacting with host receptors. Recombinant avian leukosis viruses (ALVs) differing only in envelope determinants that interact with host receptors for subgroup A or E ALVs have been found to have unexpectedly distinctive patterns of tissue-specific replication. Recombinants of both subgroups were highly expressed in bursal lymphocytes as well as in cultured chicken embryo fibroblasts. In contrast, the subgroup A but not subgroup E host range allowed high levels of expression in skeletal muscle, while subgroup E but not subgroup A envelope glycoproteins permitted efficient replication in the thymus. A subgroup B virus (RAV-2), like the subgroup E viruses, demonstrated a distinct bursal and thymic tropism, further supporting the theory that genes encoding receptors for subgroup B and E viruses are allelic. The source of long terminal repeats (LTRs) or adjacent sequences also influenced tissue-specific replication, with the LTRs from endogenous virus RAV-0 supporting efficient replication in the bursa and thymus but not in skeletal muscle. These results indicate that ALV env and LTR regions are responsible for unexpectedly distinctive tissue tropisms.