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. 2007 Aug 8;81(20):11468–11478. doi: 10.1128/JVI.01364-07

FIG. 5.

FIG. 5.

Effect of blocking MVB biogenesis on gB and gH localization and maturation. 293T cells were cotransfected with a construct expressing Vps4E228Q (Vps4EQ) or the empty vector pBJ5, along with the pgBwt-MTS plasmid, expressing wt gB (A and C), or gH-MTS-gL-MTS plasmids encoding gH/gL (B and D). At 48 h after transfection, the cells were stained with MAb H1817 to gB (A and C) or MAb 53S to gH (B, D) and analyzed by confocal microscopy. Nuclei were stained with propidium iodide. (E) 293T cells were cotransfected with a construct expressing Vps4E228Q or the empty vector pBJ5, along with the pgBwt-MTS plasmid. Forty-eight hours after transfection, gB was immunoprecipitated from the cell lysates and treated with endo H (+) or left undigested (−). Samples were analyzed by SDS-PAGE and Western blotting with PAb to the major HSV-1 glycoproteins. Circles identify the gB forms exhibiting the indicated apparent Mrs. Arrows identify the electrophoretic mobilities of endo H-digested immature forms of gB. The electrophoretic mobilities of the molecular mass markers are reported. (F to Q) 293T cells cotransfected with pBJ5-Vps4E228Q along with either pgBwt-MTS (I to K) or gH-MTS-gL-MTS (O to Q) were analyzed by confocal microscopy, with MAb to gB (Virusys) or MAb 53S to gH and a PAb to calreticulin, as indicated. 293T cells cotransfected with the empty vector pBJ5 together with either the pgBwt-MTS plasmid (F to H) or the gH-MTS-gL-MTS plasmids (L to N) were used as a control.