Enhanced Human-Type Receptor Binding by Ferret-Transmissible H5N1 with a K193T Mutation

Abstract

All human influenza pandemics have originated from avian influenza viruses. Although multiple changes are needed for an avian virus to be able to transmit between humans, binding to human-type receptors is essential. Several research groups have reported mutations in H5N1 viruses that exhibit specificity for human-type receptors and promote respiratory droplet transmission between ferrets. Upon detailed analysis, we have found that these mutants exhibit significant differences in fine receptor specificity compared to human H1N1 and H3N2 and retain avian-type receptor binding. We have recently shown that human influenza viruses preferentially bind to α2-6-sialylated branched N-linked glycans, where the sialic acids on each branch can bind to receptor sites on two protomers of the same hemagglutinin (HA) trimer. In this binding mode, the glycan projects over the 190 helix at the top of the receptor-binding pocket, which in H5N1 would create a stearic clash with lysine at position 193. Thus, we hypothesized that a K193T mutation would improve binding to branched N-linked receptors. Indeed, the addition of the K193T mutation to the H5 HA of a respiratory-droplet-transmissible virus dramatically improves both binding to human trachea epithelial cells and specificity for extended α2-6-sialylated N-linked glycans recognized by human influenza viruses. Infections by avian H5N1 viruses are associated with a high mortality rate in several species, including humans. Fortunately, H5N1 viruses do not transmit between humans because they do not bind to human-type receptors. In 2012, three seminal papers have shown how these viruses can be engineered to transmit between ferrets, the human model for influenza virus infection. Receptor binding, among others, was changed, and the viruses now bind to human-type receptors. Receptor specificity was still markedly different compared to that of human influenza viruses. Here we report an additional mutation in ferret-transmissible H5N1 that increases human-type receptor binding. K193T seems to be a common receptor specificity determinant, as it increases human-type receptor binding in multiple subtypes. The K193T mutation can now be used as a marker during surveillance of emerging viruses to assess potential pandemic risk.

N-Glycolylneuraminic Acid as a Receptor for Influenza A Viruses

“…While rescuing this virus again, we found that a large majority of the viruses contained a second mutation, T160A. This T160A mutation results in the loss of an N-glycosylation site in the head of HA that has been implicated to be important in receptor binding and specificity for H5 HA (de Vries et al, 2014; Paulson and de Vries, 2013; Peng et al, 2018). We therefore analyzed H5 wild-type versus H5 Y161A and H5 T160A Y161A for replication efficiency in Mardin Darby canine kidney (MDCK) cells.…”

Onward transmission of viruses: how do viruses emerge to cause epidemics after spillover?

“…The switch in specificity from the α2,3 to the α2,6 Sia linkage seems to be a requirement for efficient human-to-human transmission [ 30 ]. A third receptor-associated inter-host difference is the length and branching of the glycan, and the number of Sia that are displayed [ 32 , 33 ]. This may affect the binding of HA to the receptor as more branched and multi-Sia glycans can bind virus with higher affinity.…”

Diversity of A(H5N1) clade 2.3.2.1c avian influenza viruses with evidence of reassortment in Cambodia, 2014-2016

“…Though, numerous substitutions were also identified that are associated with a binding preference for α2,3 receptors (S2d Table). The well-known Q222L and G224S substitutions associated with increased binding to α2,6 receptors and decreased α2,3 binding were not observed [55,56].…”