FIG 11.
Binding, endocytosis, and fusion of ORF63 STOP virions. (A and B) WT, ORF63 STOP, and ORF63 Rev virions were bound to BHK-21 cells (3 h, 4°C) with either the same numbers of PFU for the three strains or dilutions of this amount (1/5, 1/10, 1/30) for the ORF63 STOP strain. Cell surface-bound virions were detected by washing, fixing, and staining them for gN with MAb 3F7 (38). Secondary detection was with Alexa 488-conjugated goat anti-mouse IgG pAb. The cells were then analyzed by flow cytometry (A). This experiment was performed in triplicates (B), and the differences in gN detection (mean fluorescence intensity [MFI]) between samples were analyzed by 1-way ANOVA and Bonferroni posttests. ***, P < 0.001 compared to the value for the WT sample. (C) BHK-21 cells were exposed to WT, ORF63 STOP, and ORF63 Rev BAC+ (expressing eGFP) strains (0.5 PFU/cell) for the times indicated and then washed either with PBS (pH 7.4) or with isotonic buffer (pH 3; acid wash). Viral infection was assayed by measuring eGFP expression 24 h p.i. by flow cytometry. The data are average ± SEMs for triplicate measurements. The data were analyzed by 2-way ANOVA and Bonferroni posttests. (D) MuHV-4 WT, ORF63 STOP, and ORF63 Rev virions were bound to BHK-21 cells (particles equivalent to 30 WT PFU/cell, 3 h, 4°C). The cells were then washed with PBS and either fixed immediately or first further incubated (2 h, 37°C) to allow virion endocytosis and membrane fusion. The cells were then stained for the gN envelope glycoprotein (IgG2a [green]) and for the ORF75c virion tegument protein with MAb BN-8C3 (IgG1 [red]), and with DAPI (blue). Red and green colocalization appears as yellow. Equivalent data were obtained in a repeat experiment. The data are fully representative of at least 100 cells examined. The confocal settings were the same for the corresponding images at 4°C and after 2 h at 37°C.