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.

While reduced histone modifications following MERS-CoV infection failed to explain down-regulation of antigen-presentation molecules, it suggested an alternative epigenetic mechanism: DNA methylation. Direct methylation of genomic DNA functionally ablates expression, indirectly leading to reduced access and loss of histone marks. To examine this possibility, we performed methylated DNA-IPs (MeDIPs) with virus-infected cells and then assessed the methylation status of three antigen-presentation-associated gene promoters (HLA-A, B2M, and PDIA3) using PCR (Fig. 5A) . For H1N1-09, infection resulted in modest changes, with increased methylation of HLA-A and reduced methylation of B2M promoter regions. Similarly, SARS-CoV infection generally resulted in modest DNA methylation trending toward reduced methylation relative to mock-infected controls. In contrast, H5N1-VN1203 infection increased methylated DNA levels in the same target genes, and, strikingly, MERS-CoV infection resulted in complete methylation (100%) of all three target genes. To verify the effect observed in MERS-CoV infections, we performed MeDIP-PCR reactions for three additional antigen-presentation gene promoters (CIITA, HLA-E, and PSMB9) and again observed complete promoter methylation for all genes (Fig. S2) . Global methylation analysis of chr. 6 revealed robust methylation across the entire chromosome following MERS-CoV infection (Fig. 5B) . Importantly, while H5N1-VN1203 infection also induced increased methylation across chr. 6, its peaks were less in magnitude than observed in MERS-CoV infection.