Figure 5.

Glycoprotein D is endocytosed from the plasma membrane to HSV1 wrapping membranes. HFFF-2 cells grown on coverslips were infected with (A) the GFP-VP26 virus or (B, C) Wt HSV1 at a multiplicity of 2. Ten hours after infection, gD antibody uptake experiments were carried out by incubating cells for 30 min on ice with anti-gD monoclonal antibody followed by 30 min at 37°C to allow uptake. Cells were fixed and permeabilised then labelled with (B) anti-EEA1 (green), or (C) anti-TGN46 (green), and counterstained with Alexa 568 anti-mouse secondary antibody to detect anti-gD antibody (red) and DAPI to detect nuclei (blue). (D) HFFF-2 cells infected with the GFP-VP26 virus were fixed at 10 h and labelled with anti-EEA1 (red) and DAPI to detect nuclei (blue). (E) HFFF-2 cells were infected with HSV1 and at 10 h gD antibody uptake assay carried out as described above with the inclusion of a 30-min incubation on ice with an HRP-tagged F(ab′)₂ fragment of anti-mouse IgG prior to incubation at 37°C. Cells were fixed and stained for HRP prior to processing for EM. (F) Uninfected HFFF-2 cells were labelled with anti-transferrin receptor (red). (G) HFFF-2 cells infected with GFP-VP26 virus (green) were fixed 10 h after infection and labelled with anti-transferrin receptor antibody (red). All immunofluorescence images were acquired with a Zeiss LSM510 Meta confocal microscope. In (A–D, F–G), scale bar=10 μm. In (E), scale bar=200 nm.