Fig. 2.
Transport of neutralized virions. (a) Wild-type (WT) MuHV-4 virions (3 p.f.u. per cell) were left untreated or pre-incubated (2 h, 37 °C) with mAbs 8F10 (anti-gH–gL, IgG2a), T2C12 (anti-gH–gL, IgG2a) or SC-9E8 (anti-gB, IgG2a) (400 µg ml−1) before binding to NMuMG cells (2 h, 4 °C). For comparison, other NMuMG cells were similarly exposed to gL− virions (50 p.f.u. per cell, so as to get equivalent binding). The cells were then washed with PBS and either fixed immediately or first further incubated (2 h, 37 °C) to allow virion endocytosis. The cells were then stained for the ORF75c virion tegument protein with mAb BN-8C3 (IgG1, green) and for the late endosomal marker LAMP-1 (red), and with DAPI (blue). Red/green co-localization appears as yellow. Equivalent data were obtained in a repeat experiment. In this and all subsequent figures, the data shown 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. The numbers give the fraction of green signal co-localizing with red signal. The zoomed images show in more detail the relationship between virions (green) and endosomes (red). (b) As in (a), cells were exposed to virions for 2 h at 4 °C, washed in PBS, then either analysed immediately or first incubated for 1 or 2 h at 37 °C, but antibody binding was detected with an IgG1-specific alkaline phosphatase-conjugated secondary antibody and incubation with p-nitrophenylphosphate substrate, and quantified by measuring A405. For each condition, the A405 was normalized to the value obtained at 4 °C. The bars show mean±sem values from six wells. The ORF75c signal after incubation at 37 °C was significantly higher for non-neutralized WT virions than for gL− or neutralized WT virions (P<0.008 by Student’s t-test). The images show the distribution of ORF75c after virion binding at 4 °C, and after incubation at 37 °C for 1 or 2 h. (c) In a similar experiment to (b), virions were bound to cells for 2 h at 4 °C and detected with the gp150-specific IgG2b mAb BH-6H2 plus an alkaline phosphatase-conjugated IgG2b-specific secondary antibody. The bars show mean±sem A405 values from six wells. The signal with 8F10-neutralized virions was reduced significantly relative to other treatments (P<10−5). Equivalent data were obtained in a repeat experiment.