Pathogenic Influenza Viruses and Coronaviruses Utilize Similar and Contrasting Approaches To Control Interferon-Stimulated Gene Responses

Menachery, Vineet D.; Eisfeld, Amie J.; Schäfer, Alexandra; Josset, Laurence; Sims, Amy C.; Proll, Sean; Fan, Shufang; Li, Chengjun; Neumann, Gabriele; Tilton, Susan C.; Chang, Jean; Gralinski, Lisa E.; Long, Casey; Green, Richard; Williams, Christopher M.; Weiss, Jeffrey; Matzke, Melissa M.; Webb-Robertson, Bobbie-Jo; Schepmoes, Athena A.; Shukla, Anil K.; Metz, Thomas O.; Smith, Richard D.; Waters, Katrina M.; Katze, Michael G.; Kawaoka, Yoshihiro; Baric, Ralph S.

While unable to completely block activation, H5N1-VN1203 may dampen the total amount of activated transcriptional factor via antagonists like NS1. With limited availability, transcriptional factor binding preference may drive differential expression. To explore this possibility, we focused on STAT1-dependent/augmented ISGs as determined by the IPA knowledge base; the results identified broad variation in ISG expression despite their similar dependence on STAT1 (Fig. 3A ). To verify that transcriptional factor binding contributed to this observation, we performed chromatin immunoprecipitations (ChIP) targeting STAT1, followed by quantitative real-time PCR (qPCR) to examine target promoter binding. The results indicated that CXCL10 and IFIT1, highly expressed ISGs following H5N1-VN1203 infection ( Fig. 3A) , had a corresponding increase in STAT1 binding in their 5= promoter region at 12 hpi (Fig. 3B ). In contrast, CFHR1 and APOL6, ISGs with decreased expression, had no increase in STAT1 binding despite the presence of activated STAT1 in those cells. For each of these genes, we had identified consensus STAT1 GAS motifs (TTC[N 2-4]GAA) within 1 kb of the translational start site and 200 bp downstream (18) ; the number of these motifs varied by gene (CXCL10 [2] , IFIT1 [1] , CFHR1 [1] , APOL6 [1]) but did not dictate expression of STAT1. Notably, similar variation despite STAT1 dependence was observed following MERS-CoV infection as well as with STAT1 ChIP-PCR results (see Fig. S3 in the supplemental material). Together, the results indicated that differential promoter binding exists between genes activated by STAT1 and possibly contributes to differential ISG expression in H5N1-VN1203 and MERS-CoV infections. Altered histone modification plays a significant role in both H5N1 and MERS-CoV ISG antagonism. A number of factors can impact transcriptional factor binding preference, including the promoter binding sequence, site of phosphorylation, and underlying chromatin structure (19, 20) . We initially chose to focus on chromatin structure based on a number of studies that implicated a role for chromatin remodeling during influenza virus infection (21) (22) (23) and identification of histone mimic (24) . Histone modification can mediate a variety of diverse biological processes, including gene regulation (25, 26) . These changes can result in rapid chromatin remodeling, including activation (opening) or repres-sion (closing) depending on the location, type, and number of modifications. While the NS1 histone mimic motif is absent in both H1N1-09 and H5N1-VN1203, other findings suggest that histone remodeling strategies may be conserved across influenza strains (21) (22) (23) . Coupled with substantial downregulation of ISG subsets during H5N1 infection, these facts encouraged us to investigate histone changes. We performed ChIP using antibodies for either an active form of histone H3 (H3K4me3) or a repressive form (H3K27me3) in the context of H5N1-VN1203 or H1N1-09 infection (25) . We then targeted the 5= untranslated region (UTR) of three ISGs with differential expression between the influenza strains (CFHR1, DDX58 [encodes RIG-I], and SMAD9L) and one with similar expression (IRF9) by quantitative real-time PCR. For H1N1-09, H3K4me3 was strongly associated with the 5=UTR of upregulated ISGs compared to mock (Fig. 4A ). In contrast, these same genes, with downregulated RNA expression in H5N1-VN1203 infection, were also found to have significant