Fig. 1.

Generation of MuHV-4 mutants. In order to ablate expression, translational stop codons were inserted into the coding sequences of gL, gp70 and gp150 as indicated. Single gL and gp70 mutants have been described previously. (a) For this study, we combined gL and gp70 mutations, and then made a triple mutant by further disrupting gp150. (b) Viral DNA was analysed by Southern blotting, exploiting the novel EcoRI site introduced during mutagenesis. Wild-type (wt) and gp70⁻ viruses provide comparisons. gL⁻gp70⁻.1 and gL⁻gp70⁻.2 are independent mutants. For gL, there is a 3.1 kb invariant band and a 942 bp wt band that changes to 270 bp+595 bp. For gp70, a 12.7 kb wt band changes to 3.4 kb+8.4 kb. The 3.4 kb band was evident by ethidium bromide staining (data not shown), but not by hybridization because it overlaps the probe by only 115 bp. For gp150, a14.9 kb wt band changes to 11.2 kb+3.8 kb. (c) BHK-21 cells were left uninfected (UI) or infected (2 p.f.u. cell⁻¹) with wt (18 h infection), single mutant (gL⁻, gp70⁻, 18 h infections) or double mutant viruses (gL⁻gp70⁻, 66 h infection), then analysed by flow cytometry for surface expression of virion glycoproteins. (d) BHK-21 cells were exposed to BAC⁺ wt (1 p.f.u. cell⁻¹) or gL⁻gp70⁻ (5 p.f.u. cell⁻¹) viruses plus soluble heparin. Both viruses expressed eGFP from an HCMV IE1 promoter, and infection was quantified by flow cytometry after 24 h. Different virus doses were used to ensure comparable eGFP expression. The difference in susceptibility to heparin was much greater than the difference in virus input.