Fig. 2.

gL⁻gp70⁻ MuHV-4 shows a severe deficit in cell binding. (a) BHK-21 or NMuMG cells were exposed to viruses (1 p.f.u. cell⁻¹, 37 °C) for different times, then washed three times with PBS to remove unbound virions. After 24 h, all cells were analysed by flow cytometry for viral eGFP expression. Each point shows 2×10⁴ cells. (b) NMuMG cells were exposed to wt or mutant viruses (2 h, 4 °C, 5 p.f.u. cell⁻¹), then washed three times with PBS and either fixed immediately (4 °C) or first incubated in complete medium (2 h, 37 °C). All the cells were then stained for gN, an abundant component of the virion envelope. New gN expression is not evident until at least 6 h p.i., so this assay detects only input virus. (c) Virus stocks were compared by immunoblot for gB (mAb MG-4D11), ORF17 (mAb 150-7D1) and gN (mAb 3F7). gB-FL, full-length gB; gB-C, C-terminal cleavage product. ORF17 is auto-cleaved and so appears as a doublet. (d) NMuMG cells were exposed to wt or mutant viruses (2 h, 37 °C, 3 p.f.u. cell⁻¹), then washed three times with PBS and analysed for virion binding by fixation, permeabilization and staining for gB or gN. nil, No virus. Each bar shows 2×10⁴ cells. Fixation/permeabilization was used to make virion detection independent of endocytosis.