Structural analyses reveal the mechanism of inhibition of influenza virus NS1 by two antiviral compounds

Abstract

The influenza virus is a significant public health concern causing 250,000-500,000 deaths worldwide each year. Its ability to change quickly results in the potential for rapid generation of pandemic strains for which most individuals would have no antibody protection. This pandemic potential highlights the need for the continuous development of new drugs against influenza virus. As an essential component and well established virulence determinant, NS1 (nonstructural protein 1) of influenza virus is a highly prioritized target for the development of anti-influenza compounds. Here, we used NMR to determine that the NS1 effector domain (NS1) derived from the A/Brevig Mission/1/1918 (H1N1) strain of influenza (1918) binds to two previously described anti-influenza compounds A9 (JJ3297) and A22. We then used X-ray crystallography to determine the three-dimensional structure of the 1918 NS1 Furthermore, we mapped the A9/A22-binding site onto our 1918 NS1 structure and determined that A9 and A22 interact with the NS1 in the hydrophobic pocket known to facilitate binding to the 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30), suggesting that the two compounds likely attenuate influenza replication by inhibiting the NS1-CPSF30 interaction. Finally, our structure revealed that NS1 could dimerize via an interface that we termed the α-α dimer. Taken together, the findings presented here provide strong evidence for the mechanism of action of two anti-influenza compounds that target NS1 and contribute significant structural insights into NS1 that we hope will promote and inform the development and optimization of influenza therapies based on A9/A22.

Molecular recognition of a host protein by NS1 of pandemic and seasonal influenza A viruses

“…Recently, structures of free 1918 NS1 ED W187A and W187R mutants were reported (10,40). This allowed us to compare the structures of 1918 NS1 ED between the free and p85β-bound states.…”

“…Nonstructural protein 1 (NS1) of IAV has attracted considerable attention because of its role as a multifunctional virulence factor during the viral infection cycle (5)(6)(7)(8). Thus, it is generally considered a potential drug target for the treatment of IAV infections (9)(10)(11). The primary functions of NS1 are to antagonize host innate immune responses (12), such as the expression of type I interferon (IFN) (13)(14)(15), and to increase viral replication (7,16,17).…”

The Central Role of Non-Structural Protein 1 (NS1) in Influenza Biology and Infection

“…In this study, A9 and A22, small molecules that are derivatives of NSC125044, were the most promising molecules, A22 being 10 times more potent than A9. Recently, Kleinpeter and coworkers [140] continued Jablonski's research [139]. Both molecules bind to NS1-ED and, by mapping the interaction region through X-ray crystallography, it was shown that they share the same binding pocket as CPSF30, meaning that these compounds can act as inhibitors of the NS1-CPSF30 interaction.…”

New therapeutic targets for the prevention of infectious acute exacerbations of COPD: role of epithelial adhesion molecules and inflammatory pathways

“…Inhibition of IFN pre-mRNA was found to be mediated by the binding of viral NS1 to cellular CPSF30, thus indicating this interaction as a potential therapeutic target [400]. Peptidemediated inhibition of NS1-CPSF30 binding has been shown to limit virus replication in tissue culture [399,401,402]. However, this strategy is specific to influenza A and is yet to be tested in vivo [399].…”