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.

Proteomic analysis validated ISG antagonism following CoV and influenza virus infection. In parallel to RNA expression experiments, infected Calu3 cells were examined by global proteomics analysis. Numerous factors affect proteomics coverage, including a protein's solubility, hydrophobicity, and location within the cell, thus preventing analysis of major protein subsets, including secreted and membrane-bound peptides. Despite these limitations, significant changes in protein levels provided independent validation of the RNA expression trends. For H1N1-09, the limited control of ISG RNA expression also manifested in robust ISG protein production; 27 ISG proteins were among 530 significantly upregulated host proteins (P or G test value of Ͻ0.05 at any time point; see Fig. S1A in the supplemental material), with production detected as early as 12 hpi and the majority of ISG protein (63%) produced by 24 hpi. In contrast, only 3 of the 382 proteins significantly upregulated by H5N1-VN1203 belonged to the consensus ISG list, validating the RNA expression analysis. While SARS-CoV induces 20 ISG proteins over its 72-h time course, none of these ISG proteins are detected prior to 30 hpi; similarly, MERS-CoV induces five ISG proteins, with only one (STAT1) detected prior to 18 hpi, thus confirming delayed ISG induction in both CoVs.