Student Theses and Dissertations
Date of Award
1982
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
William Hayward
Keywords
avian leukosis virus (ALV), c-myc activation, proviral integration, RNA tumor viruses, oncogene recombination, lymphomagenesis
Abstract
The mechanism of oncogenesis by avian leukosis viruses (ALVs) was examined. ALVs are slowly transforming RNA tumor viruses that do not transform cells in tissue culture and that cause neoplastic disease only after a latent period of 4-12 months. ALVs generally induce widely disseminating B cell lymphomas originating in the bursae of Fabricius of infected birds. Occasionally, they cause other neoplasms such as sarcomas, carcinomas and nephroblastomas, as well as a variety of nonneoplastic diseases. Unlike rapidly transforming RNA tumor viruses, which appear to cause neoplastic transformation as a result of high expression (or inappropriate expression) of a "transforming gene" (onc gene) present in the rapidly transforming virus genome, ALVs lack onc genes. Until recently, the mechanism of oncogenesis by these viruses was obscure. Analysis of the virus-related DNA (performed by S. Astrin, Fox Chase Cancer Center) and RNA in ALV-induced lymphomas revealed that the tumors were clonal and thus presumably derived from a single infected cell. Tumor proviruses were often defective and tumors often failed to express normal ALV mRNAs. Thus viral gene expression is not required for maintenance of neoplastic transformation. Most importantly, tumors from different birds had integration sites in common. Tumor cells synthesized high levels of discrete new poly(A) RNAs consisting of viral S' sequences covalently linked to cellular sequences. No virus capable of transforming tissue culture fibroblasts or of inducing tumors in animals was isolated from ALV-induced lymphomas tested. W.S. Hayward (Rockefeller University) found that most ALVinduced lymphomas expressed high levels of RNA that hybridizes to v-rnyc probe. v-myc is the transforming gene of the rapidly transforming virus MC29. The new poly(A) RNAs found in ALVinduced lymphomas contained myc information and ALV proviruses were found integrated adjacent to c-myc (c-myc is the cellular counterpart of v-myc). These results strongly suggest that in lymphoid tumors induced by ALV, an ALV provirus is integrated adjacent to a specific cellular gene, which is usually c-myc. Proviral integration adjacent to c-myc activates transcription of this gene, resulting in neoplasia. Molecular clones of the provirus-host cell junctions (tumor junction fragments) from two independent ALV-induced lymphomas were isolated. The structures of these clones were compared with a clone of the normal c-myc gene. Restriction mapping confirms that the ALV provirus is covalently linked to c-myc in both tumors. Normal proviral integration events had occurred adjacent to the c-myc locus, without gross structural alteration of c-myc. The long terminal repeat (LTR) of an ALV provirus is integrated upstream of the bulk of ALV coding sequences in both tumors, and is oriented such that transcription could initiate within the LTR and continue downstream into c-rnyc. Comparison of the restriction map of a tumor junction fragment clone with the map of the v-rnyc gene shows that, in c-rnyc, there are two regions of v-rnycrelated sequences (which are probably exons), separated by 1 kb of sequences unrelated to v-rnyc (intron). DNA sequence analysis (G. Gasic, Rockefeller University) suggests that integration has occurred in c-myc exons adjacent to splice donor sites. Thus there may be at least 4 exons and 3 introns in c-myc. A model is proposed for the genesis of the tumor-specific RNAs containing viral 5' and c-myc information in ALV-induced lymphomas. In vitro transcription experiments indicate the presence of two potential initiation sites for synthesis by RNA polymerase II in c-myc. Nearly all (>85%) proviral integrations adjacent to c-myc in ALV-induced lymphomas occur downstream from these two sites and upstream of the bulk of c-myc coding sequences. Experiments are in progress to define the initiation site(s) in vivo. The finding by other investigators (Payne et al., 1982) that other proviral orientations can also lead to activation of c-myc and lymphomagenesis is discussed. One ALV-induced fibrosarcoma was obtained. This tumor produced a replication-defective rapidly transforming virus. The particular strain of ALV used was tdl07A, a transformation-defective mutant of subgroup A Schmidt-Ruppin Rous sarcoma virus. The virus isolated from the tumor (16L virus) transforms fibroblasts and causes fatal sarcomas in infected newborn chickens within two weeks. Its genomic RNA is 6.0 kb, and it contains fps sequences [fps is the transforming gene of Fujinami sarcoma virus (FSV) and UR1]. RNase T1 fingerprint analysis shows that the 5' and 3' terminal sequences of 16L virus are identical to (and presumably derived from tdl07A RNA). The central part of 16L viral RNA consists of the fps-related sequences. These oligonucleotides fall into four classes: (i) oligonucleotides common to the putative transforming regions of FSV and UR1 virus; (ii) an oligonucleotide also present in FSV, but not in UR1 (iii) an oligonucleotide also present in UR1, but not in FSV: and (iv) an oligonucleotide not present in either FSV, UR1 or td107A. Cells infected with 16L virus synthesize a protein of molecular weight 142,000 that is a gag fusion protein with protein kinase activity. These results suggest that l6L virus arose by recombination between tdl07A and the c-fps gene. A model for the generation of rapidly transforming viruses by recombination between slowly transforming viruses and cellular genes is presented. Molecular clones of human DNA related to myc and src were obtained by screening a human recombinant bacteriophage library. Preliminary structural analysis by restriction mapping was performed. These studies suggest that coding sequences in both human genes are probably interrupted by introns. Also, the organization of these c-onc genes, especially the human myc-related gene appears to be different in chickens and humans. These clones should be useful for investigating non-viral oncogenesis in humans. The finding that ALVs induce neoplasia by activating the expression of a normal cellular gene suggests that other oncogenic agents might also cause transformation by activating c-onc genes. The generality of oncogene activation in oncogenesis by other viral as well as non-viral agents is discussed.
License and Reuse Information
This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 4.0 International License.
Recommended Citation
Neel, Benjamin G., "Strategies for Oncogenesis by Avian Leukosis Viruses" (1982). Student Theses and Dissertations. 586.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/586
Comments
A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy