title: The transformation biology of bovine papillomavirus type 4 in established and primary cells
Creators: name: Cairney, Margaret
Publisher: name: University of Glasgow
Year: 1996
Subjects: subject: Virology, papillomaviruses, cattle, infections, cell transformation.

description: Bovine papillomavirus type 4 (BPV-4) induces papillomas in the upper alimentary canal of cattle which can progress to carcinomas in animals feeding on bracken fern. Viral DNA is rarely detected in either naturally occurring or experimentally induced cancers. Similarly, BPV-4 DNA is seldom found in in vitro transformed established cells. These results suggest that presence of BPV-4 is not required for progression to or maintenance of the transformed state. The frequency with which BPV-4 DNA is lost both in vivo and in vitro also suggests that there may be active selection against whole or part of the BPV-4 genome. Several BPV-4-transfected established lines were analysed to examine whether this proposed negative selection was observable at the DNA level. In common with previous in vitro studies, BPV-4 DNA was found to be progressively lost on continued sub-culture. Analysis of cell lines containing BPV-4 DNA showed that there was no overt mutation or rearrangement of viral DNA sequences. The apparent wildtype organisation of viral genes included the E8 open reading frame (ORF). It was originally hypothesised that this viral ORF, which is the second major transforming gene of BPV-4, was an attractive target for negative selection due to the detrimental effect observed on transfecting primary cells with BPV-4 E8 DNA. Previous work also reported amplification and rearrangement of specific host sequences in BPV-4-transformed lines. It was therefore proposed that induction of cellular DNA amplification may be an important aspect of BPV-4 transformation activity. This was investigated in virally-transformed established cells. No BPV-4-mediated DNA amplification was apparent in any of the lines, although involvement of cellular regions of repetitive sequences in manifestation of such amplification may be indicated. The co-factors involved in BPV-4-associated carcinogenesis have been identified as including the mutagens, carcinogens and immunosuppressants present in bracken fern. One of major mutagens present is the flavonoid quercetin. Quercetin has discernible effect on BPV-4 transformation in vitro as it synergises with the virus to fully transform primary bovine fibroblasts (PalF cells). The work described in this thesis confirmed these initial results and extended these findings. Quercetin-treated cells showed a more aggressive transformed morphology than untreated transfectants, whether they had been transfected with whole genome BPV-4 or sub-genomic fragments. However, whereas in non-treated cells the E8 gene was required for anchorage independence, quercetin-treated cells containing the BPV-4 E7 gene alone were found to be capable of anchorage-independent growth. Conversely, and contrary to expectation, quercetin- treated cells transfected with the E8/E7 genes grew very poorly or not at all in semi-solid media. The reasons for the antagonistic action of E8 and quercetin are not yet understood. Independent of quercetin action, results also provided circumstantial evidence that the E8 oncoprotein is responsible for downregulation of gap junctional intercellular communication in BPV-4-transformed cells. BPV-4 does not have an E6 gene and immortalisation of BPV-4-transformed cells is achieved only in the presence of an exogenous E6 gene. Quercetin treatment however conferred immortality on cells transformed by whole genome BPV-4 or by the E7 gene alone. The observed synergism between BPV-4 and quercetin was also found to be dependent on time of treatment. Treatment of cells with quercetin either immediately before or after DNA transfection resulted in an increased degree of cellular transformation compared to that seen on lengthening the interval between treatment with the chemical and transfection with BPV-4 genes. PalF cells treated with quercetin immediately after transfection with the E7 gene were tumourigenic. The tumours grew far more aggressively than when cells were treated with quercetin before transfection with whole genome BPV-4. This indicated that the timing of exposure of viral products and quercetin is crucial and that in certain circumstances only the E7 gene is required for full malignant transformation.