Fig. 1.

Generation of MuHV-4 expressing luciferase. (a) A 2 kb luciferase-polyA cassette was placed downstream of a 500 bp M3 promoter, in a MfeI restriction site between ORFs 57 and 58. Relevant restrictions sites are shown. (b) Viral DNA was digested with EcoRI or HindIII and probed with the 75 338–78 717 BglII clone shown in (a). The luciferase expression cassette changes a 14.9 kb EcoRI band to 5.5 kb+12.0 kb, and a 14.5 kb HindIII band to 6.8 kb+10.1 kb. M3-LUC1.6 and M3-LUC2.1 are independently generated recombinant viruses. (c) BHK-21 cells were left uninfected or infected overnight (1 p.f.u. cell⁻¹), then lysed and assayed for luciferase expression. Each bar shows the mean±sd of triplicate cultures. (d) BHK-21 cells were infected with wild-type or M3-LUC MuHV-4 (0.01 p.f.u. cell⁻¹, 2 h), washed with PBS to remove unbound virions, then incubated at 37 °C. The infectious virus in each culture was measured by plaque assay. (e) BHK-21 cells were infected with wild-type MuHV-4, the M3-LUC2.1 recombinant or its ORF50⁻ derivative. Luciferase expression was assayed 18 h later by luminometry. Each bar shows mean±sd of five replicate infections. The ORF50⁻ cultures contained no replication-competent virus by plaque assay.