Molecular Mechanisms of Serum Resistance of Human Influenza H3N2 Virus and Their Involvement in Virus Adaptation in a New Host

Abstract

H3N2 human influenza viruses that are resistant to horse, pig, or rabbit serum possess unique amino acid mutations in their hemagglutinin (HA) protein. To determine the molecular mechanisms of this resistance, we characterized the receptor-binding properties of these mutants by measuring their affinity for total serum protein inhibitors and for soluble receptor analogs. Pig serum-resistant variants displayed a markedly decreased affinity for total pig serum sialylglycoproteins (which contain predominantly 2-6 linkage between sialic acid and galactose residues) and for the sialyloligosaccharide 6′-sialyl(N-acetyllactosamine). These properties correlated with the substitution 186S→I in HA1. The major inhibitory activity in rabbit serum was found to be a β inhibitor with characteristics of mannose-binding lectins. Rabbit serum-resistant variants exhibited decreased sensitivity to this inhibitor due to the loss of a glycosylation sequon at positions 246 to 248 of the HA. In addition to a somewhat reduced affinity for 6′-sialyl(N-acetyllactosamine)-containing receptors, horse serum-resistant variants lost the ability to bind the viral neuraminidase-resistant 4-O-acetylated sialic acid moieties of equine α2-macroglobulin because of the mutation 145N→K/D in their HA1. These results indicate that influenza viruses become resistant to serum inhibitors because their affinity for these inhibitors is reduced. To determine whether natural inhibitors play a role in viral evolution during interspecies transmission, we compared the receptor-binding properties of H3N8 avian and equine viruses, including two strains isolated during the 1989 to 1990 equine influenza outbreak, which was caused by an avian virus in China. Avian strains bound 4-O-acetylated sialic acid residues of equine α2-macroglobulin, whereas equine strains did not. The earliest avian-like isolate from a horse influenza outbreak bound to this sialic acid with an affinity similar to that of avian viruses; a later isolate, however, displayed binding properties more similar to those of classical equine strains. These data suggest that the neuraminidase-resistant sialylglycoconjugates present in horses exert selective pressure on the receptor-binding properties of avian virus HA after its introduction into this host.

Cross-Species Virus Transmission and the Emergence of New Epidemic Diseases

“…For example, glycans or lectins (often called serum or tissue inhibitors) may bind and eliminate incoming viruses. This was seen for human influenza viruses, which may bind to sialylated ␣-2macroglobulin in porcine plasma and to alternative sialylated glycoproteins in other animals (78,97,98). Viruses which lack efficient neuraminidase or esterase activity for the glycans of the new hosts may be bound and inactivated, requiring that viruses infecting those hosts rapidly adapt.…”

Effects of Sialic Acid Modifications on Virus Binding and Infection

“…The natural in vivo activity of human or avian influenza NA towards modified Sia appears to be consistently reduced, while 'drift' in linkage specificity has been observed over large influenza evolutionary timescales [57]. It appears that, for influenza viruses, HA compensatory mutations are commonly used to avoid inhibitory or 'decoy' Sias, rather than changes in the sialidase specificity of NA, and also that sialidases cannot readily gain the ability to avoid inhibition by at least the 4-O-Ac Sia [58]. Similarly, NA resistance alleles to sialidase inhibitors (e.g., Oseltamivirresistant influenza) can arise, but may require favorable compensatory epistatic alleles in HA to allow fixation [59,60].…”

“…For example, mutations that allow human influenza viruses to replicate in the presence of high levels of 4-O-Ac Sia occurred in the Sia binding site of the HA and prevented HA binding to the 4-O-Ac Sia. Those mutations were seen in the H3N8 and H7N7 equine influenza viruses, as well as in human influenza viruses experimentally adapted to grow in the presence of horse serum proteins [58]. It is not known whether selection of mutations in HA to avoid these inhibitory modified Sias also alters HA receptor binding specificities for other Sias, or alters antigenic epitopes, and thereby shapes the adaptive potential of the viruses in different hosts.…”

Bitter-sweet symphony: glycan–lectin interactions in virus biology

“…SP-A (cfr. supra) or a2-macroglobulincan interfere with the viral hemagglutinating activity that is crucial for receptor binding (Rogers et al, 1983;Pritchett & Paulson, 1989;Ryan-Poirier & Kawaoka, 1991;Matrosovich et al, 1992;Ryan-Poirier & Kawaoka, 1993;Hartshorn et al, 1994;Malhotra et al, 1994;Benne et al, 1995;Gimsa et al, 1996;Benne et al, 1997;Hartshorn et al, 1997;Matrosovich et al, 1998;van Eijk et al, 2003;Mikerov et al, 2008;Chen et al, 2010;Cwach et al, 2012).…”