IE temporal gate for TLR-triggered signalling to boost IE-gene expression and viral replication.

<p>A) Inhibitory effects of long-term pre-treatment with TLR ligands. BMDMs were incubated for 24 h with indicated TLR ligands and subsequently infected (MCMV-gLuc). Reporter activity was measured at indicated time point p.i. Bars represent mean of normalised activity from n = 64 with error bars showing SEM and asterisks indicating statistical significant changes identified by Wilcoxon two-sample test (* = p-value ≤ 0.05, ** = p-values ≤ 0.01, ns = not significant). B) and C) IE-Time kinetic experiments of TLR-agonist pre-treatment and its effect on MIEP activity. BMDMs were pre-treated for indicated time points and infected with MCMV-gLuc (MOI 0.2). Reporter activity in culture SN was measured at 3 hpi (B, n = 6) or 4 hpi (C, n = 8). Averages of cultures are shown with SEM, normalised to mock activity and asterisks indicate statistical significant changes identified by Welch two-sample t-test (* = p-value ≤ 0.05, ** = p-values ≤ 0.01). D) Linear phases of time-effect curves for TLR4 (black circles) and TLR9 (grey circles) agonists were estimated from plots in Fig 2C. Linear trend-lines were fitted to the respective data points (average with SEM) and were used to determine ET50 values (TLR4 = -5.25 h; TLR9 = -6.15 h). E) Quantification of viral genomes in infected BMDMs (MCMV-gLuc) shows increased numbers of genomes for all used TLR-agonists. Genome copy numbers were absolutely quantified, corrected by <i>Gapdh</i> as cellular control and then normalised to copy numbers from mock-treated samples. Dotted line indicates level in mock samples and thick solid black lines mark the average of the treatment groups (n = 6). Asterisks indicate statistical significant changes over the mock group identified by Wilcoxon two-sample test (** = p-value≤0.01). F) Measurement of viral replication in BMDMs by plaque assay (MCMV-gLuc). Supernatant from n = 6 infected BMDM cultures were harvested at day 3 p.i. and assayed for infectious viral particles on <i>Stat1</i>^-/- MEFs. Plaque numbers were normalised to average of mock samples (2.8x10³ PFU/ml) and asterisks indicate statistical significant changes over mock identified by Welch two-sample t-test (* = p-value≤0.05; ** = p-value≤0.01).