Figure 5. RhoA is involved in BV internalization and transgene expression.

(A) BV internalization (wt, MOI 200) in 293 cells transfected for 24 h with wt, CA or DN RhoA-EGFP. The virus uptake (2 h) was detected by BV capsid antibody vp39 MAb (red) and A555-conjugated secondary antibody. Scale bars 10 µm. (B) Ratio of internalized vs. surface BV after RhoA wt and CA construct transfections was calculated from confocal sections after 30 min p.t. using a differential labeling of BV before and after cellular permeabilization (see Materials and Methods sections). (C) The effect of control (scrbl), RhoA or Rac1 siRNA treatments on BV nuclear entry was monitored in 293 cells (6 h p.t., wt, MOI 200). After immunolabeling, the proportion of SiGlo-positive nuclei positive for BV was measured from three separate samples (50–100 cells/each) by confocal microscopy. SiRNA knock down effects were monitored by western blotting with specific antibodies against RhoA (24 kDa) and Rac1 (26 kDa). In all quantitative images, mean values and standard errors are shown. Statistical significance was determined by using the unpaired Student's t test with a two-tailed P value, *P<0.05.