Investigation of the binding and cleavage characteristics of N1 neuraminidases from avian, seasonal, and pandemic influenza viruses using saturation transfer difference nuclear magnetic resonance

Abstract

ObjectivesThe main function of influenza neuraminidase (NA) involves enzymatic cleavage of sialic acid from the surface of host cells resulting in the release of the newly produced virions from infected cells, as well as aiding the movement of virions through sialylated mucus present in the respiratory tract. However, there has previously been little information on the binding affinity of different forms of sialylated glycan with NA. Our objectives were then to investigate both sialic acid binding and cleavage of neuraminidase at an atomic resolution level.DesignNuclear magnetic resonance (NMR) spectroscopy was used to investigate pH and temperature effects on binding and cleavage as well as to interrogate the selectivity of human-like or avian-like receptors for influenza neuraminidase N1 derived from a range of different influenza virus strains including human seasonal H1N1, H1N1pdm09 and avian H5N1.ResultsWe demonstrated that an acidic pH and physiological temperature are required for efficient NA enzymatic activity; however a change in the pH had a minimum effect on the NA-sialic acid binding affinity. Our data comparing α-2,3- and α-2,6-sialyllactose indicated that the variation in neuraminidase activity on different ligands correlated with a change in binding affinity. Epitope mapping of the sialylglycans interacting with NAs from different viral origin showed different binding profiles suggesting that different binding conformations were adopted.ConclusionsThe data presented in this study demonstrated that physicochemical conditions (pH in particular) could affect the NA enzymatic activity with minor effect on ligand binding. NA cleavage specificity seemed to be associated with a difference in binding affinity to different ligands, suggesting a relationship between the two events. These findings have implications regarding the replication cycle of influenza infection in the host where different sialidase activities would influence penetration through the respiratory mucin barrier and the release of the newly generated virus from the infected cells.

Ferrets exclusively synthesize Neu5Ac and express naturally humanized influenza A virus receptors

“…3a,b ). We and others have previously reported about using STD NMR to investigate ligand binding to intact virions 49 50 51 and virus-like particles 52 53 54 . The results presented in this study are the first using whole intact influenza virus particles.…”

Transmission of influenza A viruses

“…HA-mediated binding to, and NA-mediated release from sialic acids therefore must be balanced for efficient virus replication. The NA proteins of all influenza viruses exhibit significantly higher enzymatic activity against Siaα2,3Gal than against Siaα2,6Gal (164-166); nonetheless, the N1 NA of a 2009 pandemic virus had detectable activity against Siaα2,6Gal (166), similar to NA proteins derived from human N2 viruses (164, 165). Interestingly, several of the recently identified respiratory droplet-transmissible H5 viruses possess an NA gene derived from a human N2 virus Chen et al .…”

Replication of H9 influenza viruses in the human ex vivo respiratory tract, and the influence of neuraminidase on virus release

“…Nuclear magnetic resonance (NMR) spectroscopy. Enzymatic activity of viral NA against 3′-Sialyl-N-acetyllactosamine (3′SLN), 6′-Sialyl-N-acetyllactosamine (6′SLN), N-Acetylneuraminyl-galactose (Neu5Ac-Gal) and N-Glycolylneuraminicyl-galactose (Neu5Gc-Gal) was detected with NMR spectroscopy as detailed previously 43 . All NMR experiments were performed on a Varian 750 MHz spectrometer.…”