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.

To explore how H5N1-VN1203 mediates ISG control, we next focused on viral proteins linked to H5N1-VN1203 virulence and IFN manipulation: NS1, PB1-F2, and PB2 (29) (30) (31) (32) . To test the contributions of these viral proteins, Calu3 cells were infected with H5N1-VN1203 mutants containing a C-terminal truncation of NS1 (NS1trunc124), a deletion of PB1-F2 (PB1-F2del), or PB2 encoding an amino acid substitution (PB2-K627E) that significantly reduces polymerase activity. For each, viral replication and ISG induction was compared to those of the wild type (WT) and demonstrated attenuated replication, noting that each mutant virus replicated to equivalent titers at 18 to 24 h (see Fig. S5A in the supplemental material). However, ISG induction kinetics varied between both the WT virus and each mutant (Fig. 5A) . For the PB1-F2del and PB2-K627E mutants, a significant subset of ISGs (48.5% for PB1-F2del and 38.8% for PB2-K627E; data not shown) had augmented expression (Ͼ1.5 log 2 FC) compared to the WT at 24 hpi; however, 95% of genes downregulated in WT infections also remained downregulated (24%, ϽϪ0.75 log 2 FC expression) or uninduced (74%, Ͻ1.5 log 2 FC expression) by both of these mutants. In contrast, compared to WT H5N1-VN1203, the NS1trunc124 mutant induced a larger subset and augmented expression (71%) of ISGs (Ͼ1.5 log 2 FC) (data not shown). Importantly, of the 41 consensus ISGs downregulated during H5N1 infection, 38 were no longer downregulated and over half (51%) were upregulated (Ͼ1.5 log 2 FC expression) following infection with the mutant NS1 virus. Proteomics analysis validated these results, demonstrating increased protein expression of 11 ISGs in H5N1-NS1trunc124 infection, including MDA5, MX1, and IFIT1, which were not detectable in WT infection (see Fig. S3B ). Together, these results suggest that the NS1 C terminus plays a role in selective ISG downregulation and makes a strong contribution to global ISG control following H5N1-VN1203 infection.