We have previously shown that the E5a gene of human papilloma virus type 11 (HPV-11/HPV-6c) is a transforming oncogene. In order to dissect the biological consequences of E5a gene expression we utilized the operator/repressor system to manipulate E5a gene expression. Cells were cotransfected with the repressor gene and the E5a gene that had been inserted downstream of a simian virus 40 (SV40) promoter containing the operator sequence. The expression of E5a gene could therefore be repressed by binding of repressor to the operator sequence in proximity to this SV40 regulatory region. The transfected cells were cultured in the presence of the inducer IPTG and under G418 selection. IPTG derepressed E5a gene expression by binding to the repressor and reducing its affinity for the operator sequence. In these studies, we found that E5a-transformed cells still maintained the transformed phenotype as judged by growth density, cell morphology and anchorage-independent growth when E5a gene expression was repressed. We also found that c- expression was induced 3 h after E5a expression was induced by IPTG and c- expression was not shut down after repression of E5a expression. This is the first demonstration that the E5a gene of HPV-11 initiates transformation of NIH 3T3 cells but is dispensable for maintenance of the transformed phenotype.


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