MERS-CoV and H5N1 influenza virus antagonize antigen presentation by altering the epigenetic landscape

Menachery, Vineet D.; Schäfer, Alexandra; Burnum-Johnson, Kristin E.; Mitchell, Hugh D.; Eisfeld, Amie J.; Walters, Kevin B.; Nicora, Carrie D.; Purvine, Samuel O.; Casey, Cameron P.; Monroe, Matthew E.; Weitz, Karl K.; Stratton, Kelly G.; Webb-Robertson, Bobbie-Jo M.; Gralinski, Lisa E.; Metz, Thomas O.; Smith, Richard D.; Waters, Katrina M.; Sims, Amy C.; Kawaoka, Yoshihiro; Baric, Ralph S.

MeDIP-PCR. Briefly, for methylated DNA analysis, Calu3 cells were plated (∼1.5 × 10 6 per well) and infected with MERS-CoV, SARS-CoV, H5N1-VN1203, or H1N1-09 at a MOI of 5 or 3 (H5N1 only). Total genomic DNA was harvested 0, 12, 18, and 24 hpi by using the PureLink Genomic DNA Minikit. Cultures inoculated with PBS alone served as time-matched mock controls. Sonication conditions were chosen to result in the desired size distribution of the sonicated total DNA between 250 and 1,000 bp. Sonicated samples were then used to enrich methylated DNA by using the methyl-CpG binding domain of human MBD2 protein (MethylMiner Methylated DNA Enrichment Kit; Invitrogen). To determine the methylation status of MHC gene promoters, qRT-PCR was performed. For this, primer pairs targeted the CpG islands of target genes. Results were reported as the relative percentage of methylated and unmethylated DNA in each target genomic DNA sequence by using the ΔΔCt method. The fraction methylated of DNA in each sample was calculated by normalizing the total DNA amount to the amount of unmethylated DNA.