FIG. 4.

Subcellular localization of gB in HF and U373 cells. Confocal images of gB staining of cells infected with RVV gB_wt, RVV gB_Ala, or RVV gB_Asp were obtained. (A to C) Infected HF; (D to F) infected U373 cells. The cells were stained before permeabilization or at 4°C (representing surface gB) with mouse anti-gB (green) and after permeabilization (representing total gB) with mouse anti-gB (red). (A and D) RVV gB_wt infections; (B and E) RVV gB_Ala mutant infections; (C and F) RVV gB_Asp mutant infections. Surface gB staining is a combination of prepermeabilization at 4°C and postpermeabilization (yellow) (A, B, C, and F). (G and H) Confocal microscopy images demonstrating the presence of gB in VV-infected cells. U373 cells were infected with either RVV gB_wt (G) or RVV gB_Ala (H) and subsequently treated with the phosphatase inhibitor tautomycin. The cells were stained with mouse antibody to gB prepermeabilization or at 4°C (green) (surface gB) and with mouse antibody to gB postpermeabilization (red) (total gB). The accumulation of gB trafficking to the surface was observed only in the RVV gB_wt infection (yellow) (G). To demonstrate that transport of gB to the cell surface is not affected by the state of phosphorylation, we coinfected U373 cells with RVV gB_wt (I), RVV gB_Asp (J), or RVV gB_Ala (K) and RVV dyn_K44A. Magnifications, ×303 for panels A to F and I to K and ×473 for panels G and H.