Interaction of p53 with the Adenovirus E1B-55 kDa Protein

The E1B-55 kDa oncoprotein of adenovirus type 5 targets the tumor suppressor protein p53. This includes four distinct activities: (i ) biochemical interaction of E1B-55 kDa with p53; (ii ) inhibition of p53-induced transcription; (iii ) relocalization of p53 from the nucleus to the cytoplasm; and (iv ) in the simultaneous presence of E1B-55 kDa and the adenovirus E4-34 kDa (E4orf6) protein, extensive destabilization of p53. These activities can be observed experimentally, using co-immunoprecipitation of p53 with E1B-55 kDa, luciferase reporter assay of p53 activity, immunofluorescence to localize p53 and E1B-55 kDa, and immunoblot analysis of p53 levels. These experimental systems can be useful when analyzing novel interaction partners and modulators of p53, or in deciding whether adenovirus oncoproteins interact with novel growth regulatory proteins. Protocols describing the four methods are provided in this chapter.