Fig. 4: Disrupted gB fusion function did not interfere with VZV immediate early, early or late protein synthesis or with virus particle assembly.

a MeWo cells transfected with pOka-TKGFP (WT), pOka-TKGFP-gB[E526P], pOka-TKGFP-gB[H527P], or pOka-TKGFP-gB[V528P] BACs were imaged by confocal microscopy at 48 hrs post transfection. Immediate-early protein (IE62, red), early protein, thymidine kinase (TK) expressed as a GFP fusion protein (green) and late protein (capsid-ORF23, violet); nuclei stained with Hoechst 33342 (blue). Areas highlighted by a small white box are magnified in the larger white boxes, white arrows indicate cells at the early stage of infection identified by punctate expression of IE62 in nuclei with little to no TKGFP expression. Representative examples from 5 fields of view recorded from n = 2 independent experiments are shown. Scale bar = 20 µm. b Diagram of the flow cytometry-based transmission electron microscopy (FC-TEM) assay. MeWo cells were transfected with VZV BACs expressing TKGFP, harvested at 48 hrs post transfection, and sorted for GFP-positive cells; cells were fixed, embedded in 10% gelatin, infiltrated with resin, heavy metal staining and mounted on the grid. c TEM images of MeWo cells transfected with pOka-TKGFP (WT), pOka-TKGFP-gB[E526P], pOka-TKGFP-gB[H527P], or pOka-TKGFP-gB[V528P] BACs. Panels at the right are magnifications of areas indicated by boxes (N: nuclei; C: cytoplasm). Boxes outlined in red show representative viral capsids, with solid red arrowheads indicating viral DNA containing C-capsid and open red arrowheads indicating empty A-, or scaffold containing B-capsids. Boxes outlined in blue show representative viral particles, with solid blue arrowheads indicating complete virus particles and open blue arrowheads indicating light particles. Assay efficiency is provided in Supplementary Table 4. In the magnified images, scale bar = 0.1 µm.