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. 2019 Apr 19;15(4):e1007575. doi: 10.1371/journal.ppat.1007575

Fig 1. NHERF1 protein levels are reduced by both high and low-risk E6 proteins.

Fig 1

(A) NHERF1 protein levels are reduced in an E6 and E6AP dependent manner. Plasmids encoding the indicated FLAG_E6AP (1 ug), HA_NHERF1 (0.5 ug), human p53 (0.5 ug), HA_GFP (0.08 ug), and the listed E6 proteins (1 ug) were transiently transfected into E6AP-null 8B9 cells and HA-NHERF1 expression was analyzed by western blot. 2X_FLAG_11E6_WT and 2X_FLAG_18E6_WT constructs were used. Reduction of NHERF1 protein levels by high or low-risk E6 requires ligase active E6AP (E6AP_WT) but does not require the E6 PDZ binding motif (PBM). To disrupt the 16E6 PBM (ΔPBM), we mutated the carboxy terminal PBM from ETQL* to EL*. FLAG_18E6* is a truncated splice isoform of 18E6. E6AP_Ub− denotes an E6AP mutant defective for ubiquitin ligase activity created by mutating the active cysteine residue at position 843 to an alanine (C843A). Quantitation is the result of three independent experiments (N = 3) where NHERF1 levels are normalized to co-transfected HA_GFP. Shown is a single representative blot. Vertical black line in blots represents removal of an irrelevant sample. The means of triplicate independent experiments ± standard error are shown. N = 3. *<0.05, **<0.01 by Student’s t-test. (B) Reduction of NHERF1 protein is not an overexpression artifact. Titrations of the indicated E6 proteins were co-transfected with FLAG_E6AP_WT (1 ug), HA_GFP (0.02 ug), and either HA_NHERF1 (0.5 ug) or p53 (0.5 ug) in murine 8B9 cells. With increased E6 expression, NHERF1 decreased for each E6 protein parallel with p53. As expected, p53 degradation was observed for the high-risk 16E6 proteins (both WT and ΔPBM) but not by low-risk 11E6 protein despite reduction of NHERF1 protein levels by 11E6. The means of triplicate independent experiments ± standard error are shown. (C) NHERF1 protein is reduced in keratinocytes containing episomal HPV16. Vector-transfected keratinocytes and keratinocytes transfected with re-circularized HPV16 genomes were seeded at equal confluency. NHERF1 and p53 protein levels were decreased in cells containing episomal HPV16. Quantitation was normalized to cells lacking the HPV16 episome. The means of triplicate independent experiments ± standard error are shown. N = 3, ***<0.001 by Student’s t-test.