FIG 7.

MeV enters Vero.hSLAM and Vero.hPVRL4 cells through a Rac1- and PAK1-independent pathway. Vero.hSLAM and Vero.hPVRL4 cells were transfected with plasmids expressing wild-type Rac1 (Rac1 WT), the dominant negative form of Rac1 (Rac1 DN), wild-type PAK1 (Myc-PAK1 WT), the dominant negative form of PAK1 (GST-PAK1 DN), or siRNA targeting human PAK1. (A) Expression plasmids for Rac1 or its dominant negative form contained an eGFP reporter gene, and the synthesis of Rac1 WT or Rac1 DN was confirmed by fluorescence microscopy. (B) Vero.hSLAM and Vero.hPVRL4 cells were transfected with Rac1 WT and Rac1 DN expression plasmids and incubated for 2 days. The transfected cells were infected with MeV-Luc at an MOI of 2 for 8 h, and luciferase activity is reported as a percentage relative to the value for the Rac1 WT control. Rac1 DN appeared to have no effect on MeV-Luc entry. (C) Vero.hSLAM and Vero.hPVRL4 cells were transfected with PAK1 siRNA or scrambled control siRNA and incubated for 2 days. Cell lysates were subjected to SDS-PAGE and analyzed by Western immunoblot analysis using antibodies directed against PAK1. Knockdown of PAK1 by PAK1-targeting siRNA was confirmed in both cell lines. (D) Vero.hSLAM and Vero.hPVRL4 cells were transfected with PAK1 siRNA or scrambled control siRNA and incubated for 2 days. The transfected cells were infected with MeV-Luc at an MOI of 2 for 8 h, and luciferase activity is reported as a percentage relative to the values for scrambled control siRNA. PAK1 siRNA failed to inhibit the entry of MeV-Luc in both cell lines. (E) Vero.hSLAM and Vero.hPVRL4 cells were transfected with PAK1 siRNA or scrambled control siRNA and incubated for 2 days. The transfected cells were infected with MeV-eGFP at an MOI of 0.1 and incubated for 24 h. Syncytia were observed by using fluorescence microscopy, and PAK1 siRNA did not appear to affect MeV-eGFP infections. Experiments were repeated 3 times, with similar results. Data from one representative experiment are shown. (F) Vero.hSLAM and Vero.hPVRL4 cells were transfected with expression plasmids for Myc-PAK1 and GST-PAK1 DN and incubated for 2 days. Expression in cell lysates from these cells was confirmed by using Western immunoblot analysis and antibodies directed against the Myc and GST tags. (G) Vero.hSLAM and Vero.hPVRL4 cells were transfected with PAK1 WT and PAK1 DN expression plasmids and incubated for 2 days. These cells were infected with wtMeV-Luc for 8 h, and luciferase activity is reported as a percentage relative to the value for PAK1 WT-transfected cells. The PAK1 DN form had little effect on MeV-Luc entry into Vero.hSLAM and Vero.hPVRL4 cells. (H) Vero.hSLAM and Vero.hPVRL4 cells were transfected with PAK1 WT and PAK1 DN expression plasmids and incubated for 2 days. These cells were incubated with wtMeV-eGFP at an MOI of 2. After 1 h of adsorption, the virus inoculum was removed and replaced with fresh medium containing 200 μM FIP to prevent secondary infection and syncytium formation. Twenty-four hours later, fluorescence due to viral infection was measured by FACS analysis, and data are displayed as a percentage of eGFP-positive cells relative to the value for the PAK1 WT control. All experiments are the means of data from three independent experiments, and error bars indicate standard deviations. Statistically significant differences (P < 0.05 by ANOVA) are indicated by asterisks.