Effects of TLR-agonists go beyond NFκB activity and are independent of tegument proteins.

<p>Comparison of 6 biological replicates measuring <i>Ie1</i>-gene expression of chimaeric MCMV recombinant viruses carrying either a human CMV enhancer (hMCMV) (A) or a human CMV-NFκB/AP1/ATF-motif-disrupted-enhancer (hMCMV-Δ3) (B) after stimulation with indicated TLR-agonists in BMDMs by qPCR. Expression was normalised to <i>Gapdh</i> and is shown relative to mock treated samples and asterisks indicate statistical significant changes over mock for each group as identified by Welch two-sample t-test (* = p-value≤0.05; ** = p-value≤0.01, ns = not significant). C) Statistical comparison (two-sample Welch’s t-test) of differences in fold changes between hMCMV and hMCMV-Δ3 viruses for each TLR-agonist (n = 6). D) TLR stimulation acts independent of delivered tegument proteins. Primary MEF cultures were transfected with MCMV-gLUC DNA (16 h) and subsequently either mock (n = 24) or LPS (n = 24) stimulated. At 6 days post stimulus viral plaque production was quantified and plaques were classified into small, medium and large categories (representative plaques for the categories are shown). Bars depict average plaque numbers (indicated above each bar) with SEM. Statistical significance of observed differences between treatments is indicated by asterisks (Mann Whitney U Test, * = p-value≤0.05; ** = p-value≤0.01; ns = not significant). E) In monocytes NFκB activation and IE1 protein levels are not dependent on M45. Representative western blots detecting proteins IκBa, M45, IE1 and b-Actin in primary MEFs, SVEC4-10 endothelial cells, IC-21 and RAW264.7 macrophage cells infected with either MCMV or MCMV-ΔM45 virus (MOI of 10 TCID₅₀/cell). Cells were lysed at the indicated times post-infection, and protein levels determined by immunoblotting.