Effect of MCMV M35 on global gene expression during PRR signalling in murine fibroblasts
To investigate if the protein M35 of murine cytomegalovirus (MCMV) affects transcription of genes beside IFNbeta, we immortalised murine embryonic fibroblasts (MEF) isolated from wild-type C57BL/6J mice with SV40 Large T antigen to generate iMEF, and transduced these with retrovirus to stably express M35-HAHA or carry a control vector (EV). We stimulated the 2 different cell lines by transfection of Alexa488-labelled immunostimulatory DNA (ISD) for 2, 4 or 6 hours to activate the host transcription factor IRF3. Mock-transfected and untreated cells served as controls. In the last 90 min before harvest, cells were additionally treated with 200µM 4-thiouridine (4sU) for metabolic labelling of nascent transcripts, and total mRNAs were processed accordingly by SLAM-sequencing procedures for gene expression profiling of newly synthesized transcripts. Overall design: Comparative gene expression profiling analysis of total transcripts from SLAM-seq data for differential gene expression in the presence of M35 compared to control cells at different time points of stimulation.