ABSTRACT
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.
Subject(s)
Human papillomavirus 16 , Oncogene Proteins, Viral/genetics , Oncogene Proteins, Viral/metabolism , Polymorphism, Genetic/genetics , Repressor Proteins/genetics , Repressor Proteins/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Calcium-Binding Proteins/metabolism , Cell Line , Discs Large Homolog 1 Protein , Human papillomavirus 16/genetics , Humans , Membrane Proteins/metabolism , Protein Binding , Transcription, Genetic , Tumor Suppressor Protein p53/metabolism , bcl-2-Associated X Protein/metabolismABSTRACT
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.
