Fig 5.

Mature vaccinia virus WR enters MEFs through macropinocytosis. (A) Myosin inhibition and blockage of actin dynamics. HeLa cells and MEFs were pretreated with DMSO, blebbistatin (B), jasplakinolide (J), or blebbistatin along with jasplakinolide (J + B); infected with WR MV at an MOI of 5 PFU/cell for 60 min; and harvested at 2 h p.i. for early gene luciferase expression assays. Luciferase activities obtained from the DMSO-treated cells were used as the 100% control. (B) Small GTPase inhibition of vaccinia virus core uncoating. HeLa cells and MEFs cells were transfected with plasmids for the expression of GFP and the GTP-bound forms (G12V) and GDP-bound forms (T17N) of Rac1 and Cdc42. Cells were infected with WR MV at an MOI of 40 PFU/cell for virus core-uncoating assays. For each group, viral cores from at least 40 cells were counted to obtain an average number of viral cores/cell; viral core numbers obtained from GFP-transfected cells were used as the 100% control. (C) Inhibition of macropinocytosis upon vaccinia virus core uncoating. HeLa cells and MEFs were treated with various concentrations of amiloride (Am) and subsequently infected with WR MV for virus core-uncoating assays as described above for panel B. Viral core numbers obtained from DMSO-treated cells were used as the 100% control. All the experiments were repeated three times, and the standard deviations are shown.