A Temporal Gate for Viral Enhancers to Co-opt Toll-Like-Receptor Transcriptional Activation Pathways upon Acute Infection
Fig 9
Family of IRF proteins represents new candidates for driving IE-gene expression.
A) Sequence analysis of murine and human CMV enhancer regions shows potential IRF binding motifs with (~70% identity with canonical motif) at indicated bp positions (from +1 position of Ie1/3 and Ie1/2 respectively). Structure of the MIEP region in the MCMV-ΔMIEP recombinant is shown for comparison. B) Irf5 is expressed in MEF cells. Relative qPCR data for Irf5 expression, with mock-treated cells as calibrator. Cells were infected with MCMV for 6h before isolation of total RNA (n = 2) for qPCR. Samples were measured in technical replicates (n = 3, SEM). C) Representative ChIP experiment in infected RAW264.7 cells (MCMV, MOI 0.5, 24 hpi) with IgG as unspecific control and antibodies specific (sAB) for IRF3, IRF5, NFκB (p65) and RNA-polymerase II (Pol II), detecting the ActB and the viral MCMV enhancer (Ie1) by SYBRgreen qPCR measured in duplicates. Fold enrichment over IgG is shown and specificity was controlled by infection with the enhancer deletion mutant (ΔMIEP).