Substitutions T200A and E227A in the Hemagglutinin of Pandemic 2009 Influenza A Virus Increase Lethality but Decrease Transmission

Abstract

Modifications in the hemagglutinin (HA) glycoprotein of the influenza A virus are believed to be a catalyst for previous world pandemics (1, 2). This includes reassortment between cocirculating animal and human viruses and mutations in the HA which grant better transmissibility between hosts (3). The pandemic influenza A virus from 2009, A(H1N1)pdm09, originated from reassortment among three cocirculating swine and avian-like viruses; hence, its HA comes from a swine virus origin (4). Although little is known about the specific requirements for the swine influenza virus to adopt human-to-human transmissibility, minimal adaptation in the HA sequence might suffice (5).Different residues within and near the receptor-binding site of the HA in A(H1N1)pdm09 viruses have been found to specifically alter receptor affinity. Residues 200 and 227 were recently reported to be responsible for the difference in the binding affinity for the sialic acid receptors between the HAs of swine and A(H1N1)pdm09 influenza virus isolates (6). Furthermore, recent publications have shed light on the role of HA residue 227 in cell tropism and transmission, confirming its role in sialic acid recognition (7). In swine influenza virus isolates, amino acids 200 and 227 of the HA are alanines (A), while in some A(H1N1)pdm09 viruses, residue 200 is a threonine (T) and 227 is invariably a glutamic acid (E). In fetuin binding and glycan array assays, it was observed that swine virus-derived HA showed stronger binding to ␣2,6-linked sialic acids. When substitutions T200A and E227A were introduced into A(H1N1)pdm09 HA, its receptor binding increased to the same level as its swine HA counterpart (6).We sought to characterize the effect of these two substitutions in viral replication, pathogenicity, and transmission. Using plasmid-based reverse genetics (8), we generated recombinant influenza viruses with HA mutations T200A and E227A by using the A/California/04/2009 strain as the backbone. A wildtype (WT) recombinant (rCal/09 WT) was generated, as well as three HA substitution mutants, one bearing T200A (rCal/09 HA T200A), another with E227A (rCal/09 HA E227A), and a third containing both substitutions (rCal/09 HA T200A/ E227A). All recombinant viruses were plaque purified at least two times, and full-genome sequencing of the viruses was conducted to confirm the presence of both substitutions, as well as the absence of any other, undesired, mutation. We determined the hemagglutination titer of our recombinant virus stocks (10 6 PFU for each virus) in 1% turkey red blood cells (Fig. 1). Viruses with T200A and/or E227A showed a clear increase in hemagglutination activity compared to that of rCal/09 WT. We 6 PFU, and then 2-fold dilutions were mixed with a 1% turkey red blood cell solution in phosphatebuffered saline. Hemagglutination activity was measured after 45 min of incubation at 4°C.

Evolution of the Hemagglutinin Protein of the New Pandemic H1N1 Influenza Virus: Maintaining Optimal Receptor Binding by Compensatory Substitutions

“…Modeling of the mutations in a structure model of pH1N1 HA showed that changes in receptor binding are likely explained by the effect of these mutations on hydrogen bond formation either with the 190 helix or with the sialylated glycan directly. Importantly, mutations in HA that affected receptor binding, as detected with the recombinant protein approach, were also shown to affect receptor binding in the context of complete virus particles (13,16; this study). Several of the substitutions that affected receptor binding in vitro were shown by others to affect the , and blue (GlcNAc).…”

“…Remarkably, formation of this new bond decreases binding avidity, whereas the disruption of the water-supported network by S186P increases binding. pathogenicity or transmission of pH1N1 viruses in animal models (16,31), demonstrating that changes in receptor binding identified with our recombinant protein approach are of biological significance in vivo, but the specific impact on viral phenotypes of the HA substitutions studied here, with the exception of A200T, remains to be determined.…”

“…Optimal adaptation of HA binding to human receptors in the swine host is therefore plausible. In a previous study, we showed that two mutations in HA of pH1N1 (strain Cal04), including T200A, that result in enhanced binding (13) also decrease transmission in a ferret model (16). Decreased transmission may explain the selection of compensatory mutations that restore the binding properties of HA to their original values.…”

“…A wild-type recombinant virus of strain A/California/04/ 2009 (Cal04) and an HA substitution mutant (Cal04 T200A) (16) were grown in MDCKII cells in Opti-MEM/GlutaMAX (Gibco) medium containing 2% bovine serum albumin and 1 g/ml trypsin-TPCK (tosylsulfonyl phenylalanyl chloromethyl ketone) (Sigma-Aldrich). Viruses were inoculated at a multiplicity of infection of 0.01 and harvested after 48 h in four independent batches.…”

Hemagglutinin Stalk Immunity Reduces Influenza Virus Replication and Transmission in Ferrets

“…All animal experiments were conducted by using protocols approved by the Icahn School of Medicine at Mount Sinai Institutional Animal Care and Use Committee (IACUC). Ferrets were provided access to food and water ad libitum (9,10,22,23).…”