CHARACTERIZATION OF THE INTERACTIONS OF HUMAN-PAPILLOMAVIRUS TYPE-16 E6 WITH P53 AND E6-ASSOCIATED PROTEIN IN INSECT AND HUMAN-CELLS

Human papillomavirus (HPV) 16 E6 induces the degradation of the tumour suppressor protein p53 by the ubiquitin-dependent proteolysis pathway , In vitro, this process involves the formation of a trimolecular comp lex between E6, p53 and a cellular protein E6-associated protein (E6-A P), However, an analysis of their potential interactions in vivo has n ot been carried out, We have established a model for the expression an d analysis of the interactions of these three proteins in insect cells , a eukaryotic system where potentially crucial modifications of the p roteins will occur. In baculovirus-infected cells the degradation of p 53 can occur, However, p53 is only degraded early in the infectious cy cle due to a lack of ATP at later times. Consequently, substantial qua ntities of material can be produced in this system for further analysi s, Evidence is also provided that, in vivo, E6 can interact with p53 i n the absence of EG-AP and that EG-AP can interact with p53 in the abs ence of E6. Furthermore, analysis of the subcellular localization of t he proteins using both biochemical fractionation and indirect immunofl uorescence suggests that the degradation of p53 occurs in the perinucl ear region of the cell.