FIG 8.

The growth deficit of the MuHV-4 ORF63 STOP mutant strain is not associated with an increased cell death or with the activation of the inflammasome. (A) Kinetic of ORF63 expression. BHK-21 cells were infected with MuHV-4 (MOI of 0.5 PFU/cell). At the indicated time postinfection, expression of ORF63 was studied by a Sybr green qRT-PCR approach as described in Materials and Methods. Time zero represents uninfected cells. The data are averages from triplicate measurements ± SEMs and were analyzed by 1-way ANOVA and Bonferroni posttests. ***, P < 0.001. (B) BHK-21 cells were mock infected or infected with WT BAC⁺, ORF63 STOP BAC⁺, and ORF63 Rev BAC⁺ MuHV-4 strains at an MOI of 0.5 PFU/cell. Twenty-four hours after infection, cell viability was assessed by annexin V-APC and propidium iodide labeling and flow cytometry analysis. Percentages of doubly positive cells were measured in eGFP⁻ and eGFP⁺ populations. The data are averages from triplicates ± SEMs and were analyzed by 2-way ANOVA and Bonferroni posttests. (C and D) Effect of Z-VAD (C) or glyburide (D) on growth of MuHV-4 in vitro. BHK-21 cells were infected with WT, ORF63 STOP, and ORF63 Rev MuHV-4 strains in 6-well cluster dishes at an MOI of 0.01 PFU per cell in the presence of the pan-caspase inhibitor Z-VAD (20 μM) or the NLRP3 inflammasome inhibitor glyburide (25 μg/ml). Supernatant and infected cells were harvested at different times after infection, and the amounts of infectious virus were determined by plaque assay on BHK-21 cells. The data are averages from triplicate measurements ± SEMs and were analyzed by 2-way ANOVA and Bonferroni posttests. **, P < 0.01; ***, P < 0.001. At time zero p.i., the inocula were retitrated to ensure that similar amounts of virus were put on the cells. (E) Effect of infection supernatant on the growth of MuHV-4 in vitro. BHK-21 cells were infected with WT, ORF63 STOP, and ORF63 Rev MuHV-4 strains in 24-well cluster dishes at an MOI of 0.01 PFU per cell in the presence of supernatant of BHK-21 cells previously infected with WT, ORF63 STOP, and ORF63 Rev MuHV-4 strains (500 μl/well; 50% final concentration). Supernatant and infected cells were harvested at different times after infection and the amounts of infectious virus were determined by plaque assay on BHK-21 cells. The data are averages from triplicate measurements ± SEMs and were analyzed by 2-way ANOVA and Bonferroni posttests. **, P < 0.01; ***, P < 0.001. At time zero p.i., the inocula were retitrated to ensure that similar amounts of virus were put on the cells. To allow comparisons between graphs, a dashed line has been added across the graphs at the mean maximal value measured for WT and ORF63 Rev strains.