Functional consequences of the E1–KEAP1 interaction. Functional consequences of the E1–KEAP1 interaction. A, Subnetwork of interaction between HPV E1 and KEAP1. Blue arrows denote the main contribution of differential mutation scores to KEAP1 through network propagation. MiST score of virus–human interaction in C33A cells is indicated (0.81). The mechanism of interaction is represented by edge shapes, with +Ub indicating ubiquitination. B–E, Western blot analysis of the virus–host interaction by IP using streptavidin-coated beads to bind the Strep-tag (B, D, E) or an antibody against endogenous KEAP1 (C). Proteins indicated on the right of each blot from IP and input samples were detected using the antibodies indicated. Bands were cropped from the same original membrane. Distinction between different strains of HPV is made using strain number preceding the respective viral bait (HPV-31 = 31, HPV-16 = 16; D). F, Cartoons depicting the truncation/deletion mutants of HPV-31 E1. Scale is provided in amino acid numbers of the full-length protein at the top. G, Luciferase reporter assay for the antioxidant response element (ARE). Relative luciferase light units were normalized based on transfection control. The mean ± standard deviation of technical triplicates are depicted. H, Model depicting how E1 influences the KEAP1–NRF2 pathway. Manon Eckhardt et al. Cancer Discov 2018;8:1474-1489 ©2018 by American Association for Cancer Research