Structural Characterization of the 1918 Influenza Virus H1N1 Neuraminidase

Abstract

Influenza virus neuraminidase (NA) plays a crucial role in facilitating the spread of newly synthesized virus in the host and is an important target for controlling disease progression. The NA crystal structure from the 1918 "Spanish flu" (A/Brevig Mission/1/18 H1N1) and that of its complex with zanamivir (Relenza) at 1.65-Å and 1.45-Å resolutions, respectively, corroborated the successful expression of correctly folded NA tetramers in a baculovirus expression system. An additional cavity adjacent to the substrate-binding site is observed in N1, compared to N2 and N9 NAs, including H5N1. This cavity arises from an open conformation of the 150 loop (Gly147 to Asp151) and appears to be conserved among group 1 NAs (N1, N4, N5, and N8). It closes upon zanamivir binding. Three calcium sites were identified, including a novel site that may be conserved in N1 and N4. Thus, these high-resolution structures, combined with our recombinant expression system, provide new opportunities to augment the limited arsenal of therapeutics against influenza.

Influenza neuraminidase operates via a nucleophilic mechanism and can be targeted by covalent inhibitors

“…AX4 cells, MDCK cells with increased levels of sialyl-a2,6-galactose moieties, were grown and maintained following previous protocols 36,37 Expression and purification of influenza NAs. Recombinant NAs were purified to homogeneity after being expressed in a baculovirus expression system, based on the original method by Xu et al [38][39][40][41] with modifications. For rN2-Tyr406Asp and 2009 pandemic rN1-His274Tyr, point mutations were introduced by site-directed mutagenesis based upon the corresponding wild-type NAs (Generay, China).…”

Influenza neuraminidase

“…A sequence alignment of the nine NA subtypes indicating the location of key structural elements is shown in Supplementary Figure 1. Crystal structures of NA encompass the catalytically active heads (Figure 1A), either proteolytically cleaved from the virus 7,8 or engineered as a soluble secreted protein 9 . The intact NA has not been crystallized, but a cryo-electron microscopy study of the X-31 (A/Aichi/68, H3N2) reassortant virus has revealed considerable detail at near atomic resolution 10 .…”

“…Recent determinations of N1 NA structures from H5N1, 1918 H1N1 and 2009 swine-origin H1N1 viruses 7,9,31 , and also N4 7 , N6 32 , and N8 7 have greatly expanded our understanding of the dynamics of the head domain. NA sequences fall into two distinct groups 33 ; Group 1 contains N1, N4, N5 and N8 and Group 2 contains N2, N3, N6, N7, and N9 (Supplementary Figure 1).…”

In-silico Virtual Screening and ADMET Study to Find Novel Neuraminidase N1 Inhibitors Extended to the 150-Cavity

“…Given its role in cleaving SA residues on the cellular receptor, which bind to the newly formed virions, Influenza virus neuraminidase inhibitors (NAIs) have emerged as promising therapeutic agents for the treatment of influenza. Current treatment exists for influenza infections with the newer class of neuraminidase inhibitors, such as zanamivir (Relenza) and oseltamivir (Tamiflu) (Von Itzstein, 2007;Xu et al, 2008).…”

“…Considering the fact that changes from open to close form at the active site of group-1 NAs when the inhibitor binding to the enzyme, this encouraging to study the conformational transformation that open insights to find specific inhibitor would be effective against the group-1 NAs that can be used to inhibiting the NAs by the conservative active site as well as 150-cavity, simultaneously (Amro et al, 2011;Li et al, 2010) Different studies reported that all group-I neuraminidases having an open conformation and all group-II neuraminidases have a closed conformation (Xu et al, 2008;Russell et al, 2006).…”