The 2nd sialic acid-binding site of influenza A virus neuraminidase is an important determinant of the hemagglutinin-neuraminidase-receptor balance

Abstract

Influenza A virus (IAV) neuraminidase (NA) receptor-destroying activity and hemagglutinin (HA) receptor-binding affinity need to be balanced with the host receptor repertoire for optimal viral fitness. NAs of avian, but not human viruses, contain a functional 2 nd sialic acid (SIA)-binding site (2SBS) adjacent to the catalytic site, which contributes to sialidase activity against multivalent substrates. The receptor-binding specificity and potentially crucial contribution of the 2SBS to the HA-NA balance of virus particles is, however, poorly characterized. Here, we elucidated the receptor-binding specificity of the 2SBS of N2 NA and established an important role for this site in the virion HA-NA-receptor balance. NAs of H2N2/1957 pandemic virus with or without a functional 2SBS and viruses containing this NA were analysed. Avian-like N2, with a restored 2SBS due to an amino acid substitution at position 367, was more active than human N2 on multivalent substrates containing α2,3-linked SIAs, corresponding with the pronounced binding-specificity of avian-like N2 for these receptors. When introduced into human viruses, avian-like N2 gave rise to altered plaque morphology and decreased replication compared to human N2. An opposite replication phenotype was observed when N2 was combined with avian-like HA. Specific bio-layer interferometry assays revealed a clear effect of the 2SBS on the dynamic interaction of virus particles with receptors. The absence or presence of a functional 2SBS affected virion-receptor binding and receptor cleavage required for particle movement on a receptor-coated surface and subsequent NA-dependent self-elution. The contribution of the 2SBS to virus-receptor interactions depended on the receptor-binding properties of HA and the identity of the receptors used. We conclude that the 2SBS is an important and underappreciated determinant of the HA-NA-receptor balance. The rapid loss of a functional 2SBS in pandemic viruses may have served to balance the novel host receptor-repertoire and altered receptor-binding properties of the corresponding HA protein.

Host receptors: the key to establishing cells with broad viral tropism for vaccine production

“…In this process, haemagglutinin (HA) mediates viral entry through binding to the cellular receptors and facilitates fusion of the virion membrane with the endosomal membrane (Byrd-Leotis et al 2017). Conversely, neuraminidase (NA) can cleave off the SAs from the cellular membrane and viral glycoproteins, facilitating the egress of nascent virions (Byrd-Leotis et al 2017;Du et al 2019). HA and NA glycoproteins collectively determine the optimal level of HA affinity and NA enzymatic cleavage to allow for productive viral infection (Byrd-Leotis et al 2017).…”

Antiviral Activity of Chicken Cathelicidin B1 Against Influenza A Virus

“…HA functions as a receptor binding and fusion protein, while the NA protein is involed in release of (nascent) virus particles from decoy receptors or the cell surface (Gamblin and Skehel, 2010;Dou et al, 2018). Recently, it has been reported that NA activity of IAV is influenced by virus-receptor binding (Guo et al, 2018;Du et al, 2019;Lai et al, 2019). NA activity is altered based on enhanced or a reduced HA-receptor binding property.…”

“…In short, IAV was incubated with CATH-B1 (0-40 µM) for 1 h at 37 • C, followed by addition of MUNANA for another 1 h at 37 • C. Next, the reaction was stopped, and fluorescence intensity was measured using a FLUOstar Omega microplate reader. The activity of NA toward the sialylated glycoprotein fetuin was analyzed in a solid phase cleavage assay using a previously described enzyme linked lectin assay (ELLA) (Dai et al, 2017;Du et al, 2019). Fetuin (2.5 ug/mL) was coated on Maxisorp Nunc 96-well plates (Thermo Fisher Scientific).…”

Coronavirus hemagglutinin-esterase and spike proteins co-evolve for functional balance and optimal virion avidity

“…Different from HE, NA may do so via its catalytic pocket which doubles as a Sia-binding site 72 . However, NA also possesses a second Sia binding site 73,74 , which like the HE lectin domain, regulates NA activity and which, in further analogy, is conserved or lost in apparent correlation with host tropism 75–77 . Finally, among many other similarities to embecoviruses, influenza A variants with different set points in their HA-NA functional balance may cooperate to support their propagation in cultured cells 48 .…”