Figure 3.

Overexpression of dynamitin reduces immediate-early viral gene expression. (A) Overexpression of dynamitin (a) or dynamitin-GFP (b) reduced the immediate-early viral gene expression compared with control (a–c) or GFP-transfected cells (c). Immunofluorescence microscopy of PtK₂ cells transfected with dynamitin or dynamitin-GFP and 30 h later infected with HSV1 for 3 h. Cells were fixed with PFA and either double-labeled with anti-myc (a; top panel) to detect transfected cells and anti-ICP4, an immediate-early protein of HSV1 (a–c; bottom panels) or single-labeled with anti-ICP4 (b and c; bottom panels), and the transfected proteins were detected by their intrinsic GFP fluorescence (b and c; top panels). (B) Quantification of viral ICP4 synthesis after transfection. The overexpression of dynamitin reduced immediate-early viral gene expression. Two-sided Student's t test confirmed that ICP4 expression is significantly lower in dynamitin (p = 2.52 × 10⁻³) or dynamitin-GFP (p = 3.57 × 10⁻³) transfected cells compared with GFP-transfected cells. There was no significant difference in ICP4 expression between untransfected and GFP-transfected cells (p = 1). The experiment described in A was quantitated (three independent experiments; altogether >500 cells analyzed for each condition). Cells overexpressing dynamitin, dyna-mitin-GFP, or GFP and untransfected cells were divided into two classes: cells displaying a nuclear ICP4 signal and cells not displaying a nuclear ICP4 signal.