Genetic studies have revealed natural amino acid variations within the human papillomavirus (HPV) type 16 E6 oncoprotein. To address the functional significance of E6 polymorphisms, 10 HPV16 E6 variants isolated from cervical lesions of Swedish women were evaluated for their activities in different in vitro and in vivo assays relevant to the carcinogenic potential of E6. Small differences between E6 prototype and variants, and among variants, were observed in transient expression assays that assessed p53 degradation, Bax degradation, and inhibition of p53 transactivation. More variable levels of activities were exhibited by the E6 proteins in assays that evaluated binding to the E6-binding protein (E6BP) or the human discs large protein (hDlg). The E6 prototype expressed moderate to high activity in the above assays. The L83V polymorphism, previously associated with risk for cancer progression in some populations, expressed similar levels of activity as that of the E6 prototype in most functional assays. On the other hand, L83V displayed more efficient degradation of Bax and binding to E6BP, but lower binding to hDlg. Results of this study indicate that naturally occurring amino acid variations in HPV16 E6 can alter activities of the protein important for its carcinogenic potential.

Previous studies have shown that human papillomavirus (HPV) 16 E6 inhibits apoptosis induced during terminal differentiation of primary human keratinocytes (PHKs) triggered by serum and calcium. E6 inhibition of apoptosis was accompanied with prolonged expression of Bcl-2 and reduced elevation of Bax levels. In the present study, the effect of E6 on Bax mRNA expression and protein stability was investigated. These studies indicate that stable E6 expression in differentiating keratinocytes reduced the steady-state levels of Bax mRNA and shortened the half-life of Bax protein. These results were confirmed in transiently transfected 293T cells where E6 degraded Bax in a dose-dependent manner. Bax degradation was also exhibited in Saos-2 cells that lack p53, indicating its p53 independence. E6 did not form complexes with Bax and did not induce Bax degradation in vitro under experimental conditions where p53 was degraded. Finally, E6 aa 120-132 were shown to be necessary for Bax destabilization and, more importantly, for abrogating the ability of Bax to induce cellular apoptosis, highlighting the functional consequences of the E6-induced alterations in Bax expression. INTRODUCTIONInfection of the anogenital mucosa with papillomavirus types referred to as 'high-risk' or oncogenic such as human papillomavirus (HPV) 16 and HPV18 is a major factor for the subsequent development of cancer (for review see Zur-Hausen 1996Burd, 2003). HPV infection of the basal epithelium perturbs cell differentiation and enhances cell proliferation. Two oncoproteins are encoded by the oncogenic HPVs, E6 and E7, which are consistently expressed in HPV-positive cervical cancers and derived cell lines (Zur-Hausen, 1996). E6 and E7 contribute to the oncogenic process, at least in part, through their ability to interact with and inactivate key cellular regulatory proteins. E7 associates with tumour suppressor Rb and other cell-cycle regulatory proteins that control cell-cycle progression (Zwerschke & Jansen-Durr, 2000;Munger & Howley, 2002), whereas the E6 oncoprotein interacts with a variety of cellular proteins involved in different signalling pathways, (for review see Mantovani & Banks, 2001;Munger & Howley, 2002), of which the best known is p53. HPV E6 binds to p53 via the E6AP protein, a ubiquitin ligase, and induces p53 degradation through the ubiquitin pathway (Scheffner et al., 1990;Huibregtse et al., 1991Huibregtse et al., , 1993. Other targets of E6 include proteins involved in the regulation of transcription and DNA replication, such as p300/CREB-binding protein (Patel et al., 1999;Zimmermann et al., 1999), the transcriptional coactivator ADA3 (Kumar et al., 2002), interferon regulatory factor-3 (Ronco et al., 1998), Gps2 (Degenhardt & Silverstein, 2001, multicopy maintenance protein 7 (hMcm7) (Kuhne & Banks, 1998;Kukimoto et al., 1998) and the DNA repair protein 0 6 -methylguanine-DNA methyltransferase (MGMT) (Srivenugopal & Ali-Osman, 2002); signalling components and enzymes such as PKN and E6 TP1, (Gao et al., 1999); proteins in...