Figure 3.

Rab6 depletion inhibits HSV1 glycoprotein localization to the plasma membrane in infected cells. A) HeLa cells grown on coverslips were transfected with neg, Rab1 or Rab6 siRNAs, fixed 2 days later and stained for giantin (red), and nuclei stained wih DAPI (blue). B) HeLa cells on coverslips transfected with neg, Rab1 or Rab6 siRNAs were infected 2 days later with HSV1 expressing gD-GFP and fixed 16 h later. C) Neg or Rab6 siRNA transfected HeLa cells were infected after 2 days with Wt HSV1 and stained with a monoclonal antibody against gE. D) HeLa cells on coverslips were transfected with neg, Rab1, Rab6 or Rab24 siRNAs and infected 2 days later with Wt HSV1. Sixteen hours later the cells were incubated on ice for 30 min with gD monoclonal antibody to detect cell surface protein, washed and fixed prior to staining with secondary antibody (red). Nuclei were stained with DAPI (blue). E) Cells infected with HSV1 expressing gD-GFP at a multiplicity of 2 were treated 5 h later with BFA, monensin or left untreated. Cells were fixed at 12 h. F) Total virus yield from infections carried out as for (E), and harvested 18 h after infection. G) HeLa cells on coverslips were transfected with neg or Rab6 siRNA, infected 2 days later with HSV1 expressing the tegument protein VP22 as a GFP fusion protein (GFP-VP22), and images acquired after 16 h. Scale bar = 10 µm.