Role of Neuraminidase in Influenza A(H7N9) Virus Receptor Binding

Abstract

Influenza A(H7N9) viruses have caused a large number of zoonotic infections since their emergence in 2013. They remain a public health concern due to the repeated high levels of infection with these viruses and their perceived pandemic potential. A major factor that determines influenza A virus fitness and therefore transmissibility is the interaction of the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) with the cell surface receptor sialic acid. Typically, the HA is responsible for binding to the sialic acid to allow virus internalization and the NA is a sialidase responsible for cleaving sialic acid to aid virus spread and release. N9 NA has previously been shown to have receptor binding properties mediated by a sialic acid binding site, termed the hemadsorption (Hb) site, which is discrete from the enzymatically active sialidase site. This study investigated the N9 NA from a zoonotic H7N9 virus strain in order to determine its possible role in virus receptor binding. We demonstrate that this N9 NA has an active Hb site which binds to sialic acid, which enhances overall virus binding to sialic acid receptor analogues. We also show that the N9 NA can also contribute to receptor binding due to unusual kinetic characteristics of the sialidase site which specifically enhance binding to human-like α2,6-linked sialic acid receptors.IMPORTANCE The interaction of influenza A virus glycoproteins with cell surface receptors is a major determinant of infectivity and therefore transmissibility. Understanding these interactions is important for understanding which factors are necessary to determine pandemic potential. Influenza A viruses generally mediate binding to cell surface sialic acid receptors via the hemagglutinin (HA) glycoprotein, with the neuraminidase (NA) glycoprotein being responsible for cleaving the receptor to allow virus release. Previous studies showed that the NA proteins of the N9 subtype can bind sialic acid via a separate binding site distinct from the sialidase active site. This study demonstrates for purified protein and virus that the NA of the zoonotic H7N9 viruses has a binding capacity via both the secondary binding site and unusual kinetic properties of the sialidase site which promote receptor binding via this site and which enhance binding to human-like receptors. This could have implications for understanding human-to-human transmission of these viruses.

Keywords: biophysics; enzyme kinetics; hemagglutinin; influenza A virus; neuraminidase; receptor analogues; receptor binding.

FIG 1

Red blood cell capture assay by WT (Hb⁺) and S367N (Hb⁻) N9 NAs. Insect cell-expressed N9 NA was attached to His tag binding magnetic beads. The beads were incubated with turkey red blood cells (TRBCs) and rapidly pelleted with a magnet. The supernatant was removed and left to settle in a microtiter plate (A), or TRBCs were lysed with SDS and the released hemoglobin was quantified by measuring the A₅₄₀ (B). The results of control experiments without protein attached to beads (no protein) and without incubation of beads with TRBCs (no beads) are also shown. Absorbance measurements were normalized to those for the no-protein control and are shown as the means from three independent measurements, with error bars showing standard deviations from the mean. When present, oseltamivir carboxylate was added at a concentration of 100 μM. **, P < 0.01.

FIG 2

Biolayer interferometry curves of influenza A virus (100 pM) binding to the receptor analogues α2,6-sialyl-N-acetyllactosamine (6SLN) and α2,3-sialyl-N-acetyllactosamine (3SLN). The binding of H7N9 (A) and H1N9 (B) viruses to both wild-type (Hb⁺) and S367N mutant (Hb⁻) N9 NAs was measured. The plotted data are the fractional saturation of virus binding as a function of sugar loading.

FIG 3

(A and B) Binding of H3N9 viruses to Hb⁺ (A) and Hb⁻ (B) NAs determined by biolayer interferometry. Binding to a range of different sugars was measured: α2,6- and α2,3-sialyl-N-acetyllactosamine (6SLN and 3SLN, respectively), α2,6- and α2,3-sialyllactose (6SL and 3SL, respectively), 3SLN 6′ sulfated on GlcNAc (6-Su-3SLN), sialyl-Lewis X (Sia Lex), and sialyl Lewis X 6′ sulfated on GlcNAc (6-Su-Sia Lex). The data shown are experimental response traces. (C) Plaque assay of H3N9 viruses with Hb⁺ and Hb⁻ NAs. The results of assays carried out using both MDCK and MDCK-SIAT cells are shown in duplicate. The plaques shown for MDCK cells used 10 times the concentration of each input virus compared to that used for MDCK-SIAT cells.

FIG 4

Biolayer interferometry curves of binding of H7N9 viruses with wild-type (Hb⁺) and S367N (Hb⁻) NA to the receptor analogues α2,6-sialyl-N-acetyllactosamine (6SLN) and α2,3-sialyl-N-acetyllactosamine (3SLN). Measurements were made in the presence (solid lines) and absence (dashed lines) of NA inhibitors (Inhib).

FIG 5

Biolayer interferometry curves of H1N9 virus (100 pM) binding to the receptor analogues α2,6-sialyl-N-acetyllactosamine (6SLN) and α2,3-sialyl-N-acetyllactosamine (3SLN). Measurements were made in the presence (solid lines) and absence (dashed lines) of NA inhibitors. The binding of viruses to wild-type (Hb⁺) and S367N (Hb⁻) N9 NAs was measured.