IDENTIFICATION OF ANTIGENIC DIFFERENCES BETWEEN THE PHOSPHORYLATED AND NONPHOSPHORYLATED FORMS OF THE E7 PROTEIN OF HUMAN-PAPILLOMAVIRUS TYPE-16

To analyze the antigenic properties of the human papillomavirus type 1 6 E7 oncoprotein, two monoclonal antibodies, VD6 and IB10, that have d ifferent reactivities to the E7 protein were generated. While the VD6 antibody reacted strongly with E7 protein in CaSki cell extracts, the other antibody, IB10, showed much weaker reactivity with E7. This reac tivity increased in a dose-dependent manner in the presence of the cas ein kinase Ii-specific inhibitor DRB (5,6-dichloro-1-beta-D-ribofurano sylbenzimidazole) Antigenic site estimation and an in vitro phosphoryl ation assay, using bacterially expressed E7 protein, demonstrated that the weak reactivity of IB10 was;elated to the phosphorylation status of the E7 protein. Phosphorylation of E7 reduced considerably the reac tivity of IB10 but did not affect the reactivity of VD6, which reacts with the N-terminal portion of E7. In immunoprecipitation (IF) assays, IB10 precipitated weakly the E7 protein from CaSki cell extracts. Tog ether, these data suggest that unphosphorylated E7 protein shows disti nct antigenic character compared to its phosphorylated form under dena turing conditions; however, under native conditions, the phosphorylate d and nonphosphorylated E7 proteins have some antigenic crossreactivit y. (C) 1998 Wiley-Liss, Inc.