Date Published: June 10, 2019
Publisher: Public Library of Science
Author(s): Wenjuan Du, Hongbo Guo, Vera S. Nijman, Jennifer Doedt, Erhard van der Vries, Joline van der Lee, Zeshi Li, Geert-Jan Boons, Frank J. M. van Kuppeveld, Erik de Vries, Mikhail Matrosovich, Cornelis A. M. de Haan, Florian Krammer.
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 2nd 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.
Influenza A virus (IAV) particles contain hemagglutinin (HA) and neuraminidase (NA) glycoproteins. HA functions as a sialic acid (SIA)-binding and fusion protein. NA has receptor-destroying activity by cleaving SIAs from sialoglycans. The HA and NA protein functionalities are critical for host tropism, and need to be well balanced in relation to the host receptor repertoire for optimal in vivo viral fitness [1–3]. However, there is no standard assay and unit for measuring a functional balance and the precise mode by which HA- and NA-receptor interactions contribute to the balance at the molecular level remains mostly unexplored. An optimal HA-NA balance is hypothesized to allow virions to penetrate the heavily sialylated mucus layer, to attach to host cells prior to virus entry, and to be released from cells after assembly [4–7].
Since the discovery of hemadsorption activity in NA 1984  and the structural evidence of the 2SBS in N9 1997 , only few studies have addressed 2SBS-mediated receptor binding and the functional consequences thereof for NA activity [26, 28, 33, 34, 59]. We now show that the 2SBS is an important factor in the complex interplay between HA, NA and receptors, referred to as the HA-NA-receptor balance. A functional 2SBS in N2 was shown to prefer binding to α2,3-linked sialosides similarly to N1  and N9 . In agreement herewith, it enhances catalytic activity against substrates carrying α2,3-linked SIAs. The contribution of the 2SBS to the HA-NA-receptor balance of virus particles was shown to be receptor- and HA protein-dependent as demonstrated by kinetic analysis of receptor-binding and -cleavage of virions using BLI. The 2SBS was shown to contribute to receptor binding also when NA was combined with a receptor-binding HA in IAV virions, as well as to cleavage of receptors by virions and to virion self-elution from a receptor-coated surface. The absence or presence of a functional 2SBS also affected virus replication in a cell type- and HA-dependent manner. Our results indicate that mutation of the 2SBS as observed in early human pandemic viruses negatively affects the catalytic activity of NA and may serve to restore the HA-NA-receptor balance of viruses carrying HA proteins with altered receptor-binding properties in relation to a novel host sialome. Conservation of the 2SBS in most avian strains, with the notable exception of H9N2 viruses, is lost in human [26, 29, 30, 34], swine and canine variants (S2 Fig). Strong conservation usually reflects a critical function. It would be very interesting to investigate in depth whether a critical function for the 2SBS in avian strains, for instance related to the HA-NA-receptor balance, is not required for efficient replication and transmission of human, canine and swine strains.