FIG 4.

HCMV pp65 inhibits cGAS activity. (A) HFFs were infected with the wild type, v65Rev, or v65Stop at an MOI of 1 for 24 h. Extracts from the infected cells were prepared, DNase and heat treated, and incubated with permeabilized HFFs for 6 h. IFN-β RNA induction was analyzed by RT-qPCR and normalized to that of GAPDH and is shown as the mean fold change plus SD following HCMV versus mock infection (**, P < 0.01; one-way ANOVA followed by Bonferroni's posttest). (B) HFFs were electroporated with pp65 Halo-WT or left untransfected and then infected with v65Stop at an MOI of 1 for 24 h. cGAMP was harvested at 24 hpi and assayed on HFFs. IFN-β mRNA induction was measured in HFFs at 6 hpi by RT-qPCR (***, P < 0.001; one-way ANOVA followed by Bonferroni's posttest for comparison of v65Stop-HaloTag-transfected cells versus v65Stop-untransfected cells). (C) Cells were transduced as described for panel B and 24 h later transfected with poly(dA-dT) (4 μg/ml) for 24 h and assayed on HFFs. IFN-β mRNA induction was measured in HFFs at 6 h by RT-qPCR [***, P < 0.001; one-way ANOVA followed by Bonferroni's posttest for comparison of poly(dA-dT)-HaloTag-transfected cells versus poly(dA-dT)-untransfected cells]. (D) WT, cGAS KO, STING KO, and IFI16 KO HFFs were infected with the wild type, v65Rev, or v65Stop at an MOI of 1. cGAMP was harvested at 24 hpi and assayed on HFFs. IFN-β mRNA induction was measured in HFFs at 6 hpi by RT-qPCR (***, P < 0.001; two-way ANOVA followed by Bonferroni's posttest). (E) HFFs were transfected with synthetic 2′3′-cGAMP or 2′3′-cGAMP control (2 μg/ml) or HFFs were electroporated with pp65 Halo-WT and then transfected with 2′3′-cGAMP. IFN-β mRNA induction was analyzed by RT-qPCR at the time points indicated and normalized to that of GAPDH. The experiment was repeated six times, and no statistically significant differences by unpaired t test analysis were observed between cells transfected with cGAMP alone versus cGAMP plus pp65 Halo-WT.