Fig 6.

Inhibition of the E1-p80 interaction by N40. (A) Schematic representation of HPV31 E1 and of the N40 peptide. E1 is subdivided into its three functional domains: the N-terminal regulatory domain (white), the OBD (light gray), and the helicase domain (Helicase; dark gray). N40 encompasses amino acids 1 to 40 of HPV31 E1, which includes the p80-binding motif (hatched box), and is fused C terminally to the Venus YFP. (B) Coimmunoprecipitation of RFP-p80 with WT or p80-binding-defective N40 (WF, VE, and VI) fused to YFP (N40-YFP). C33A cells were cotransfected with the indicated N40-YFP and RFP-p80 expression vectors and harvested 48 h posttransfection, and whole-cell extracts were subjected to immunoprecipitation using anti-GFP antibodies that also recognize YFP. (C) Inhibitory effect of N40-YFP on the coimmunoprecipitation of RFP-p80 with full-length WT 3F-E1. C33A cells were cotransfected with the indicated E1 and p80 expression vectors, as well as with increasing amounts of WT N40 expression plasmid, and harvested 48 h posttransfection. The WF, VE, and VI mutant peptides were also tested as controls at the highest level of expression vector. Whole-cell extracts were subjected to immunoprecipitation using an anti-Flag antibody.