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

Abstract

The 1918 influenza A virus (IAV) caused the most severe flu pandemic in recorded human history. Nonstructural protein 1 (NS1) is an important virulence factor of the 1918 IAV. NS1 antagonizes host defense mechanisms through interactions with multiple host factors. One pathway by which NS1 increases virulence is through the activation of phosphoinositide 3-kinase (PI3K) by binding to its p85β subunit. Here we present the mechanism underlying the molecular recognition of the p85β subunit by 1918 NS1. Using X-ray crystallography, we determine the structure of 1918 NS1 complexed with p85β of human PI3K. We find that the 1918 NS1 effector domain (1918 NS1ED) undergoes a conformational change to bind p85β. Using NMR relaxation dispersion and molecular dynamics simulation, we identify that free 1918 NS1EDexists in a dynamic equilibrium between p85β-binding–competent and –incompetent conformations in the submillisecond timescale. Moreover, we discover that NS1EDproteins of 1918 (H1N1) and Udorn (H3N2) strains exhibit drastically different conformational dynamics and binding kinetics to p85β. These results provide evidence of strain-dependent conformational dynamics of NS1. Using kinetic modeling based on the experimental data, we demonstrate that 1918 NS1EDcan result in the faster hijacking of p85β compared to Ud NS1ED, although the former has a lower affinity to p85β than the latter. Our results suggest that the difference in binding kinetics may impact the competition with cellular antiviral responses for the activation of PI3K. We anticipate that our findings will increase the understanding of the strain-dependent behaviors of influenza NS1 proteins.

Molecular Basis of the Ternary Interaction between NS1 of the 1918 Influenza A Virus, PI3K, and CRK

“…Here, we characterize the binary and ternary interactions mediated by 1918 NS1 using a combination of biophysical approaches, including biolayer interferometry (BLI) and nuclear magnetic resonance (NMR). Our laboratory recently determined the crystal structure of 1918 NS1 complexed with the p85 subunit [27]. In the present study, we find that 1918 NS1 complexed with p85 hijacks CRK through a fuzzy electrostatic interaction mediated by its CTT.…”

“…For example, Jureka et al showed that the RBD of 1918 NS1 directly interacts with the RIG-I CARD domain while the interaction was not observed for the protein from the Udorn strain [25]. Moreover, our laboratory revealed that the ED of 1918 NS1 (1918 NS1 ED ) binds to p85 iSH2 with drastically different binding characteristics to those from the ED of the Udorn strain [27].…”

“…To study the interaction of 1918 NS1 with PI3K and CRK, we employed full-length CRK proteins (CRK-II and CRK-L), the iSH2 domain of p85β subunit of PI3K (p85β iSH2 ), and 1918 NS1 containing ED and CTT (1918 NS1 ED-CTT ) ( Figure 1). Previous studies indicated that the interaction with PI3K is mediated by monomeric form of NS1 ED [11,13,27]. The isolated NS1 ED forms a W187-mediated homodimer [36][37][38].…”

“…It should be noted that W187 is located on the opposite side of the p85β-binding site (Figure 2A). Moreover, it was shown that W187R substitution in 1918 NS1 did not affect binding to p85β iSH2 [27].…”

“…These results are direct evidence that 1918 NS1 can form a ternary complex with CRK and PI3K through the so-called NS1-bridged ternary complex. To further understand the structural basis of the ternary interaction, we examined the crystal structure of the 1918 NS1 ED-CTT :p85β iSH2 complex, which was recently determined by our research group (PDB ID: 6U28) [27]. Although the 1918 NS1 in the crystal structure contained the full-length CTT (residues 204-230), the electron densities of residues 213-230 in the CTT were missing in the crystal structure ( Figure 5A); the missing region also included the PRM (residues 211-221).…”