Influenza Virus Neuraminidases with Reduced Enzymatic Activity That Avidly Bind Sialic Acid Receptors

Abstract

Influenza virus neuraminidase (NA) cleaves off sialic acid from cellular receptors of hemagglutinin (HA) to enable progeny escape from infected cells. However, NA variants (D151G) of recent human H3N2 viruses have also been reported to bind receptors on red blood cells, but the nature of these receptors and the effect of the mutation on NA activity were not established. Here, we compare the functional and structural properties of a human H3N2 NA from A/Tanzania/205/2010 and its D151G mutant, which supports HA-independent receptor binding. While the wild-type NA efficiently cleaves sialic acid from both ␣2-6-and ␣2-3-linked glycans, the mutant exhibits much reduced enzymatic activity toward both types of sialosides. Conversely, while wild-type NA shows no detectable binding to sialosides, the D151G NA exhibits avid binding with broad specificity toward ␣2-3 sialosides. D151G NA binds the 3= sialyllactosamine (3=-SLN) and 6=-SLN sialosides with equilibrium dissociation constant (K D ) values of 30.0 M and 645 M, respectively, which correspond to much higher affinities than the corresponding affinities (low mM) of HA to these glycans. Crystal structures of wild-type and mutant NAs reveal the structural basis for glycan binding in the active site by exclusively impairing the glycosidic bond hydrolysis step. The general significance of D151 among influenza virus NAs was further explored by introducing the D151G mutation into three N1 NAs and one N2 NA, which all exhibited reduced enzymatic activity and preferential binding to ␣2-3 sialosides. Since the enzymatic and binding activities of NAs are not routinely assessed, the potential for NA receptor binding to contribute to influenza virus biology may be underappreciated.

Neuraminidase-mediated haemagglutination of recent human influenza A(H3N2) viruses is determined by arginine 150 flanking the neuraminidase catalytic site

“…Changes in the haemagglutination activity of A(H3N2) virus isolates from 2000 to 2016 Driven by our own observations and previous reports [18][19][20], the haemagglutination activity of 83 influenza A (H3N2) viruses isolated in MDCK cells from 2000 until 2016 was investigated (Table S1, available in the online Supplementary Material), from which a representative subset is shown in Table 1 …”

“…At the moment it is not clear what mechanism is involved in the 150HR-mediated binding of erythrocytes and what functional changes are caused in NA by this amino acid substitution. Amino acid substitution 151DG in NA has been shown to decrease NA activity, while at the same time increasing NA affinity to a-2,3SA [18,20]. Interestingly, another amino acid substitution located in the 150-loop of NA, 147GR, has been described to mediate subtype N1 NA binding to erythrocytes [32].…”

“…Since the early 2000s, several groups have observed changes in the haemagglutination behaviour of influenza A(H3N2) viruses, i.e. reduced haemagglutination of commonly used erythrocytes (turkey, chicken and guinea pig erythrocytes) and poor inhibition of haemagglutination by reference post-infection ferret antisera [11,[18][19][20][21][22]. In addition to the changes in the haemagglutination behaviour of HA, the NA activity of influenza A(H3N2) viruses isolated between 2005 and 2009 was also affected [18].…”

Substrate specificity of avian influenza H5N1 neuraminidase

“…Onsirisakul N et al . Substrate specificity of H5N1 NA [10,[21][22][23] . These methods required modification and purification on neuraminidase which is not the original forms of neuraminidase from influenza virus [21,22] .…”

“…Substrate specificity of H5N1 NA [10,[21][22][23] . These methods required modification and purification on neuraminidase which is not the original forms of neuraminidase from influenza virus [21,22] . To avoid the modification on influenza neuraminidase, the commercial Amplex Red ® assay was modified by changing the substrate.…”