THE REGULATORY ELEMENTS OF HPV-16

Abstract

Human Papillomavirus type-16 (HPV-16) is a member of the oncogenic genital I-IPVs postulated to be the main etiological agent of cervical carcinomas. The occurrence of HPV-16 in the majority of cervical carcinomas and benign infections of the human uterine cervix underlies its importance among these oncogenic genital HPVs. The oncogenicity of this HPV is derived from the presence of two tumour genes, E6 and E7. The present study examined how the inducible transcriptional elements in the long control region (LCR) can regulate the E6/E7 promoter, p97, responsible for the transcription of E6 and E7 oncogenes, and other as yet undefined promoters. By primer extension analyses, dexamethasone, a synthetic glucocorticoid, was shown to increase the p97 transcripts from the intergrated HPV-16 in cervical carcinoma cell lines. The HPV-16 GRE, the element responsible for this regulating effect, was determined also to be induced by progesterone but not by androgens. Using several experimental approaches, other genital HPVs, regardless of their oncogenicity were shown to have a GRE in their LCR that is responsive to glucocorticoids and progestagens. In HPV-16, and probably also other genital I-IPVs, the close association of nuclear factor-I binding sites with the GRE acts to enhance the transcriptional activity of the GRE. When cervical carcinoma cell lines were investigated for their ability to support progesterone inducibility, negative results were obtained. Stable or transient transfection of progesterone receptor cDNA expression vectors into these cells could however rescue this negative phenotype. Transient transfection experiments in HeLa cells suggest that the cell-type-specific enhancer of HPV-16 is regulated by tetradecanoyl-phorbol-13-acetate (TPA), a feature shared by other genital HPVs. The two canonical activator protein-I (AP-1) binding sites were shown to be responsible for mediating this effect by mutational analyses, oligonucleotide cloning and band-shift assays and they overlap with two footprints fp4el and fp9e. Mutational analyses also indicated that a single intact AP-1 binding site in the HPV-16 tissue-specific enhancer is sufficient to exert TPA inducibility. In cervical carcinoma cell lines like CaSki, the transcription of the various RNA transcripts from the p97 promoter is also regulated by TPA. On the basis of the general occurrence of the GRE/PRE and AP-1 binding sites in the genital HPVs, these inducible transcriptional cis-elements are suggested to be important for the natural history of this subgroup of HPVs by regulating the early viral gene expression, including E6 and E7 oncogenes. These inducible cis-elements function to increase the transcription of the tumour genes essential for the initiation of malignancy. Once transformation has occurred, these transcriptional cis-elements serve to maintain the continual expression of the HPV tumour genes essential for the transformed phenotype. Thus, the activation of the GRE/PRE activity might explain why the prolonged usage of oral contraceptives by women is a risk factor in cervical carcinoma. Finally, the pertubation of the Protein kinase C pathway by oncogenes and mitogens including carcinogens, by super-induction of the AP-1 activities, can also potentially increase the risk of cervical carcinoma.