H.L. TURKINGTON¹, B.G. HALE<sup>1

1</sup>Institute of Medical Virology, University of Zürich, Zürich, Switzerland

The influenza A virus (IAV) NS1 protein is a multifunctional virulence factor which primarily acts as an interferon (IFN) antagonist, but also promotes cell survival signalling through activation of the host phosphoinositide-3-kinase pathway (PI3K). Identification in 2012/13 of two novel influenza virus genomes (H17N10 and H18N11) in Central and South American bat species presented two unique NS1 proteins with high sequence divergence from other known NS1 proteins. How these divergent NS1s have evolved to function in the bat host-cell environment and whether they possess strain-specific functions, is unknown. We show that both the H17 and H18 NS1 proteins are capable of antagonising the human IFN response in transfected cells, a property strictly dependent upon their capacity to bind double-stranded (ds)RNA. High-resolution crystal structures of their RNA-binding domains (RBDs) revealed a dimeric fold similar to other classical NS1 RBDs, with conservation of important RNA-binding residues. Strikingly, recombinant chimeric bat IAVs expressing NS1 proteins defective in dsRNA-binding were highly attenuated in IFN-competent cells, but replicated similar to wild-type viruses in IFN-deficient cells. This confirms the highly-conserved functional importance of dsRNA-binding by IAV NS1 proteins for antagonising host innate immunity. Interestingly, we found certain well-described IAV NS1 functions associated with the C-terminal effector domain to be lacking in the H17 and H18 NS1 proteins such as the ability to inhibit general host gene expression. Both bat IAV NS1 proteins also lack the ability to interact with the human p85β subunit of host PI3K, with a subsequent inability to activate downstream signalling molecules in this pathway. We show the NS1:p85β interaction can be restored with a small number of amino acid changes in the H17 and H18 NS1 proteins. Our ongoing work seeks to elucidate whether this interaction may be species-specific and additionally whether the bat IAV NS1 proteins preferentially interact with other intracellular factors in order to modulate the bat host-cell environment.