Abstract

The multifunctional non-structural protein 1 (NS1) of influenza A viruses (IAVs) inhibits IFN production and can interact with the regulatory subunit of PI3K, p85s, through an SH2-binding domain. Given the contributions of NS1 to immune evasion, we undertook studies to examine the effects of NS1 in the context of the type I IFN signaling response. We provide evidence that expression of avian H5N1 NS1 in cells reduces IFN-inducible STAT phosphorylation and that this is in part a consequence of NS1-mediated inhibition of expression of the IFN receptor subunit, IFNAR1. Using in silico modeling we postulate that residue Y84, located in the SH2-binding domain of H5N1 NS1, might contribute to interactions with p85s and other host proteins. Accordingly, we introduced a mutation into NS1: Y84F. Examination of the effects of this mutation in H5N1 NS1 on an IFN response revealed a reversal of the NS1-mediated inhibitory effects on IFN-inducible STAT activation and IFNAR1 expression. Employing mass spectrometry and protein–protein interaction network analysis, we identify that NS1 expression may abrogate the host IFN response through multiple interactions with IFN-associated effectors. To further characterize the importance of the NS1 SH2-binding domain in the context of IAV infection, we generated recombinant H1N1 A/WSN/33 viruses (rIAVs) expressing either H5N1 NS1 or H5N1 NS1-Y84F, using reverse genetics, and infected human A549 lung epithelial cells and C57Bl/6 mice in time course studies, and in the presence or absence of IFN treatment. In A549 cells, we show that the rIAV expressing the mutant NS1-Y84F replicates to significantly lower titers and is more sensitive to the antiviral effects of IFN than the H5N1 wt NS1 virus. Moreover, in vivo, the rIAV expressing NS1-Y84F also replicates to lower titers in the lungs of infected mice. Viewed altogether, our data reveal that residue Y84 within the SH2-binding domain of NS1 may permit interactions with IFN-inducible signaling effectors, thereby interfering with an IFN-inducible antiviral response and affecting IAV virulence.

Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call