SV40 triggers reversible changes in phospho-Ezrin, dependent on PDK1 function.

Abstract

<p>(A) Cortical actin alterations occur in A431 cells following incubation with SV40 for 10 min, as visualized with phalloidin staining in fixed cells. (B) Phosphorylated ERM proteins become inactive shortly after SV40 treatment. A431 cells were treated with SV40 for the indicated time points, fixed and immunostained with an antibody against phosphorylated T567 of ERM proteins. p-ERM disappears as soon as 5 min after incubation with SV40 and it resumes after 1 h. (C) A phosphomimetic mutant form of Ezrin, T567D, fails to fall off the membrane following SV40 treatment. A431 cells were transfected with EzrinT567D-GFP, treated with SV40 for 15 min, and subsequently fixed and tested for the GFP signal localization. (D) Expression of both a phosphomimetic and an inactive Ezrin mutant form negatively correlates with SV40 infection. Large populations of A431 cells were transfected with EzrinT567D, EzrinT567A or wild-type Ezrin GFP constructs and subjected to SV40. Cells that were both transfected (GFP signal) and infected (T-antigen signal) were scored and compared with the expected number emerging from a random occurrence of the two signals. Negative log2 ratio values represent an anti-correlation, which demonstrates inhibition of infection by the transfected construct (p-value 6.8×10⁻³ and 0.05 for the T567D and T567A scores, respectively). (E, F) Inhibition of PDK1 function leads to increased levels of p-ERM. The PDK1 inhibitor was applied onto A431 cells for 1.5 h before the addition of SV40 for another 15 min. Levels of p-ERM were assessed using either immunofluorescence in fixed cells (E) or immunoblotting (F). Quantification of two different blots was performed using the ImageJ software. (G) Inhibition of PDK1 function blocks increased levels of infection as caused by Ezrin knockdown. A431 cells were subjected to siRNA against Ezrin or control siRNA, and subsequently treated with the PDK1 inhibitor 1.5 h prior to SV40 infection, or left untreated. Percentage of infection was calculated from a large number of cells. p-values: 1×10⁻⁴ (PDK1 inhibitor), 5.4×10⁻³ (Ezrin siRNA), 5.8×10⁻⁴ (Ezrin siRNA, PDK1 inhibitor).</p