Figure 5.

Role of IRF3 and RelA in the Antiviral Response to HCMV and SeV Particles

(A–D) Wild-type (WT), IRF3 KO, or RelA KO THFs were treated with increasing levels of UV-inactivated HCMV or SeV (A and B), and 16 hr post-treatment supernatants were transferred to naive WT THFs (C and D). All THFs were subsequently challenged with VSV-GFP. Mean GFP fluorescence and standard error from 3 biological replicates were plotted as a percentage of mock treated, VSV-GFP infected cells. IRF3 and RelA KO THFs were compared with wild type by two-way analysis of variance (ANOVA) with Bonferroni post-tests (∗ for p < 0.05, ∗∗ for p < 0.01 and ∗∗∗ for p < 0.001).

(E–G) WT, IRF3, or RelA KO THFs were treated with 84 or 1,344 particles/cell of HCMV-UV or 12 or 200 particles/cell of SeV-UV and transcript levels of IFIT1, ISG15, and CXCL10 were measured by quantitative RT-PCR. Mean fold change relative to mock and standard error from 7 biological replicates was graphed. IRF3 and RelA KO THFs were compared with wild type by two-way ANOVA with Bonferroni post-tests (∗ for p < 0.05, ∗∗ for p < 0.01 and ∗∗∗ for p < 0.001).

(H) WT THFs were treated with 1,344 particles/cell of HCMV-UV or 200 particles/cell of SeV-UV, fixed 4 hr post-treatment and IRF3 and cell nuclei visualized by immunofluorescence microscopy. Scale bars of 100 μm are shown, and IRF3 +ve nuclei in SeV-UV infected THFs are marked.

(I) IRF3-positive nuclei as a percentage of total nuclei were calculated from 3 independent fields of view and mean % IRF3 positive nuclei and standard error from 3 biological replicates were graphed. SeV-UV and HCMV-UV infected THFs were compared by student's t-test (∗∗∗ for p < 0.001).