Dissociation of transforming and trans-activation functions for bovine papillomavirus type 1

Abstract

It has been shown that genetic information encoded by the 3' open reading frames (ORFs), E2, E3, E4 and E5, of bovine papillomavirus type 1 (BPV-1), is sufficient to induce cellular transformation of certain mouse cells. The product of the E2 ORF has further been shown to be responsible for the trans-activation of a transcriptional regulatory element located in the noncoding region (NCR) of the BPV-1 genome. To examine whether or not the E2 trans-activation function is encoded by the same gene that encodes the 3' ORF viral transformation function, we have now analysed the expression of the trans-activation function in series of mouse C127 cells transformed by BPV-1 deletion mutants. In addition, using mutated complementary DNA clones generated by the insertion of a premature translational termination linker into different sites of a BPV-1 cDNA clone containing the 3' ORFs intact, we demonstrate that transformation and transcriptional trans-activation functions can be dissociated and that they map respectively to the E5 and E2 ORFs.

Phosphorylation of the Bovine Papillomavirus E2 Protein on Tyrosine Regulates Its Transcription and Replication Functions

“…Oncogenically transformed colonies were then obtained from puromycin-selected C127 cell lines by using a soft-agar assay and grown as monolayers. Previous studies demonstrated that BPV genomes carrying E1 or E2 mutations will integrate into and efficiently transform C127 cells (45,46). Total DNA was isolated from multiple cell lines and subjected to Southern blot analysis.…”

Synthesis and assembly of infectious bovine papillomavirus particles in vitro

“…The molecular basis of PV-induced epithelial cell transformation is at least partially understood (Gissmann, 1992;Pater et al, 1988;Yang et al, 1985), but the mechanism of infection of epithelial cells by PV virions and the basis of host resistance to PV infection remain unknown. This is largely because there is no in vitro system permissive for vegetative PV infection, although partial demonstration of the reproductive cycles of human papillomavirus types 11 and 33 (HPV-11 and -33) has now been achieved in in vitro raft culture (Meyers et al, 1992;Dollard et al, 1992).…”

The bovine papillomavirus genome and its uses as a eukaryotic vector

“…This view appeared to be supported by studies based on the expression of subgenomic fragments or cDNAs from the 3' end of the transforming region [19,23]. However, more detailed analysis, either by the introduction of nonsense mutants specifically into the E2 ORF [24] or insertional mutagenesis of the corresponding cDNA clone [25], leads to the conclusion that the E2 product is not a transforming protein but a transactivator of the BPV regulatory unit [26]. Thus some of the effects reportedly caused by mutations in the E2 gene, such as decreased efficiency of transformation, may now be accounted for by a lack of transactivation of the real transforming genes.…”

“…More detailed analysis indicated two transforming functions within this region, one at the 3' end and one at the 5' end, which when linked to a LTR were individually capable of transforming C127 cells [19,31]. Mutational analysis defined these transforming regions as the E5 (3' segment) and E6 (5' segment) ORFs [25,28,29]. The protein products of these genes have now been identified and their cellular localization indicated by the use of specific antisera [30,32, and see above].…”

“…Mutational analysis indicated that this region of the genome has a transforming function [20]. Furthermore, expression of the E5 ORF alone, from heterologous viral promoters, is sufficient to cause transformation of C127 and NIH 3T3 cells, which strongly suggests that E5 is indeed a transforming protein [25,28,29]. Interestingly, the E5 gene product is small, hydrophobic and only 44 amino acids long (15 of which are leucine residues) [30].…”