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-Seq. ChIP analysis for NGS sequencing was performed by using the MAGnify ChIP System (Invitrogen). Briefly, for ChIP analysis, Calu3 cells were plated (∼1.5 × 10 6 per well) and infected with H5N1-VN1203 or H1N1-09 at an MOI of 5 and harvested at 0 and 12 hpi; 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 chromatin between 250 and 1,000 bp. Sonicated samples were then immunoprecipitated with anti-H3K4me3 (Qiagen) and anti-H3K27me3 (Qiagen). To determine genome-wide histone modification, ChIP DNA was subjected to next-generation sequencing (NGS) on a TruSeq ChIP Library Preparation Kit (Illumina) and sequenced on an Illumina HiSeq. NGS data analysis was performed by utilizing the CLC Genomics Workbench. The Histone ChIP-Seq plugin provided analysis tools for a complete histone modification analysis. Briefly, paired end reads were mapped against the human GRCh37/ hg19 reference genome by using a stringent alignment setting (mismatch cost = 2). Peaks were called against the time-matched mock reference reads. To determine specific genomic regions of histone modification enrichment, the maximum P value for peak calling was set to P < 0.05 to detect regions that had a significant fit with the peak shape.