Functional redundancy in viruses: an alternative E6AP independent mechanism by which high-risk HPV16 E6 facilitates the degradation of p53


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Wilawan Bunjobpol, Ian N. Hampson, Anthony W. Oliver, Xiaotong He, Jie Qi, Henry C. Kitchener, Lynne Hampson

University of Manchester, Gynaecological Oncology Labs, School of Cancer Studies, Manchester, UK

Abstract

Background

High risk types of HPV (16, 18 etc) are a major cause of human cancer. The capacity of these viruses to induce malignant changes depends, in part, on their ability to induce degradation of p53 and it is well known that the viral E6 oncoprotein binds and subverts the function of the E3 ubiquitin ligase (E6AP) to produce this effect.

Method

Yeast and mammalian two hybrid combined with site directed mutagenesis were used to identify, confirm and define the binding site of a novel HPV16 E6 target. Stable transfection, immunoprecipitation, immunohistochemistry and western blotting were used to analyse the expression, location and function of the novel E6 target in mutant and wild type p53 expressing cells.

Results

E6 bound specifically to a canonical E6 binding site in the phospholipid scramblase-1 (PLSCR1) protein and did not induce its degradation. Since PLSCR1 has been previously shown to negatively regulate the ability of onzin (Plac8) to activate MDM2 dependent degradation of p53, we investigated the ability of E6 to suppress this negative regulatory effect. Stable transfecton of E6 up-regulated and induced nuclear localisation of onzin in HPV-ve C33A cells. HPV16+ve SiHa cells express wild type p53 and have low levels of endogenous onzin and E6. Stable transfection of onzin into these cells caused a large reduction in the levels of p53.

Conclusion

Our results indicate that the specific interaction of E6 with PLSCR1 may regulate the ability of onzin to degrade p53 independent of E6AP and we are currently verifying this hypothesis

Background

High risk types of HPV (16, 18 etc) are a major cause of human cancer. The capacity of these viruses to induce malignant changes depends, in part, on their ability to induce degradation of p53 and it is well known that the viral E6 oncoprotein binds and subverts the function of the E3 ubiquitin ligase (E6AP) to produce this effect.

Method

Yeast and mammalian two hybrid combined with site directed mutagenesis were used to identify, confirm and define the binding site of a novel HPV16 E6 target. Stable transfection, immunoprecipitation, immunohistochemistry and western blotting were used to analyse the expression, location and function of the novel E6 target in mutant and wild type p53 expressing cells.

Results

E6 bound specifically to a canonical E6 binding site in the phospholipid scramblase-1 (PLSCR1) protein and did not induce its degradation. Since PLSCR1 has been previously shown to negatively regulate the ability of onzin (Plac8) to activate MDM2 dependent degradation of p53, we investigated the ability of E6 to suppress this negative regulatory effect. Stable transfecton of E6 up-regulated and induced nuclear localisation of onzin in HPV-ve C33A cells. HPV16+ve SiHa cells express wild type p53 and have low levels of endogenous onzin and E6. Stable transfection of onzin into these cells caused a large reduction in the levels of p53.

Conclusion

Our results indicate that the specific interaction of E6 with PLSCR1 may regulate the ability of onzin to degrade p53 independent of E6AP and we are currently verifying this hypothesis