CHARACTERIZATION OF THE BOVINE PAPILLOMA VIRUS TYPE-1 GENOME AND TRANSFORMATION OF MAMMALIAN CELLS.

Abstract

The papillomaviruses do not appear to be capable of passage in cultured cell lines despite numerous attempts to identify conditions permissive for their propagation. These viruses induce benign tumors in animals (warts, papillomas) by unknown mechanisms. A broad range of animal species is susceptible to papillomavirus-induced tumorogenesis. The basic molecular mechanisms of papillomavirus replication, transcription, and translation to produce virus-specific products in cells are unknown. Since the viruses do not reproduce in vitro, no conditional-lethal and deletion mutants like those characterized in other systems exist. Thus, progress in understanding the papillomavirus-host cell interactions leading to neoplasia has been severely hindered. This dissertation is concerned with biochemical analysis of bovine papillomavirus type 1 (BPV-1) DNA and RNA in BPV-1 transformed and tumor cells. The specific conditions of infection resulting in stable lines of BPV-1 transformed cells are described. Colonies of BPV-1 transformed cells exhibiting anchorage-independent growth in agarose-containing medium were isolated and cloned cell lines were established. Formation of tumors in a rabbit following inoculation with BPV-1 is reported and represents the first evidence of BPV-induced tumorogenesis in this animal species. The BPV-1 transformed and tumor cells are characterized with respect to the quantity and physical state of the BPV-1 genome in the cells. The BPV-1 DNA is present in high copy numbers in free, non-integrated supercoiled and nicked open-circular forms. No evidence of integrated BPV-1 sequences is noted. This is unusual since all other characterized DNA tumor viruses require covalent integration of at least a portion of the viral DNA in the cellular genome during transformation. A detailed restriction endonuclease cleavage map of the BPV-1 genome is presented, representing a more complete physical characterization of the viral genome. The first evidence of BPV-1 specific transcripts in transformed cells is reported. These results should aid in functional characterizations of the BPV-1 genome, particularly the determination of the specific regions of the BPV-1 DNA transcribed during transformation of cells, analogous to early regions defined in other DNA tumor virus systems, and analysis of post-transcriptional processing mechanisms involved in the synthesis of BPV-1 specific messenger RNA.