Figure 5.

HPV16 E5 protein binds to EVER1, EVER2, and ZnT-1 and interferes with their activity. (A) Clear lysates (CL) from HaCaT cells expressing FLAG-EVER and/or FLAG-ZnT-1, together with GFP-16 E5 or GFP alone, were immunoprecipitated (IP) with anti-GFP antibody and subsequently immunoblotted (IB) with the indicated antibodies. Immunoprecipitation experiments with ZnT-1 and HPV16 E5 gave a band of unknown origin with a migration pattern similar to that of EVER2. (B) HaCaT cells were transiently transfected with plasmids encoding FLAG-EVERs or ZnT-1 and GFP-16 E5 and stained with anti-FLAG monoclonal antibodies, followed by incubation with CY-3–conjugated secondary antibodies. Colocalization of EVER/ZnT-1 (red) and 16 E5 (green) is presented on merged images (yellow). Bars, 10 μm. (C) HaCaT cells were transiently transfected with pMT1/luc and plasmids expressing EVER or ZnT-1 or control empty plasmid in the absence (E5-) or presence (E5 + ) of a plasmid encoding 16 E5. Cells were cultured for 24 h in medium with 140 μM zinc, and luciferase activity was measured. (D) HaCaT cells stably expressing control pCiNeo plasmid or EVER2 were starvated and transfected with a plasmid encoding 16 E5 (or control plasmid) and with a mixture of plasmids expressing the luciferase reporter gene (pGal4/luc) and chimeric Gal4/c-Jun protein. Cells were incubated for 24 h in a culture medium supplemented with 50 ng/ml EGF, and luciferase activity was determined. (E) Keratinocytes with a wild-type (+/+) or mutant (−/−) EVER2 gene were transiently transfected with pMT1/luc, together with the control empty plasmid (control) or plasmids expressing 16 E5 or truncated 16 E5 corresponding to the N-terminal (M1 to I51) and C-terminal (I51 to T83) parts of the viral protein. Cells were cultured for 24 h in medium with 40 μM zinc, and luciferase activity was measured. *, P < 0.05; #, P < 0.01.