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.

ChIP-PCR. ChIP analysis was performed by using the EpiTect ChIP OneDay Kit (Qiagen). Briefly, infected Calu3 cells were cross-linked, harvested, and frozen at −80°C. Cells were then lysed and chromatin sheared via sonication to generate chromatin fragments between 250 and 1,000 base pairs. Sonicated samples were then immunoprecipitated with anti-STAT1 (clone C-24; Santa Cruz Biotechnology), anti-H3K4me3 (Qiagen), anti-H3K27me3 (Qiagen), or anti-mouse IgG (Qiagen) as a control. To determine the histone modification distribution, quantitative real-time PCR (qPCR) was performed by targeting the 5′ UTR (−750 bp to transcription start site) of select genes; target and primer information are included in Supporting Information. ChIP results were reported as fold difference (or differential occupancy), allowing comparison across multiple samples. For this, each sample was normalized to the input, and then the fold enrichment was calculated by using the ΔΔCt method. The fold difference for each gene was determined by dividing the appropriate time-matched mock by the experimental group. Finally, the fold difference values were converted to log2 and plotted. Data presented are the means ± SEs for triplicate samples.