In vitro assays of substrate degradation induced by high-risk HPV E6 oncoproteins.

The high-risk mucosal human papillomavirus E6 proteins were the first viral proteins that were shown to use the ubiquitin proteasome pathway for the inactivation of their cellular target proteins. The first substrate to be identified was the p53 tumor suppressor protein, and since then many other substrates for E6-induced degradation have been described. All of these require the presence of high-risk mucosal E6 together with the E1, E2, and E3 enzymes of the ubiquitin pathway. This activity of E6, although complex, is nonetheless amenable to in vitro analysis. Many different protocols have been described over the years for performing these assays. In this chapter we describe the most easily used and robust procedure that is routinely used in our laboratory.