Adaptation of novel H7N9 influenza A virus to human receptors

bReceptor-binding preference and stability of hemagglutinin have been implicated as crucial determinants of airborne transmission of influenza viruses. Here, amino acid substitutions previously identified to affect these traits were tested in the context of an A/H7N9 virus. Some combinations of substitutions, most notably G219S and K58I, resulted in relatively high affinity for ␣2,6-linked sialic acid receptor and acid and temperature stability. Thus, the hemagglutinin of the A/H7N9 virus may adopt traits associated with airborne transmission. R ecent human infections with influenza A/H7N9 viruses in China have raised concern of a pandemic threat emerging from avian reservoirs. As of November 2015, 681 laboratory-confirmed human cases of infection with A/H7N9 have been reported, including 275 fatalities (1). To date, no sustained humanto-human transmission of A/H7N9 viruses has been detected, although some of these viruses possess known mammalian adaption markers in the hemagglutinin (HA) and in the polymerase genes (2). Most A/H7N9 virus isolates can bind both avian and human influenza virus receptors (3), as the result of a leucine at amino acid position 217 (H7 numbering, corresponding to position 226 in H3 numbering [4]). Studies in ferrets have demonstrated that the airborne transmissibility of A/H7N9 viruses was more efficient than that of other avian influenza viruses but less efficient than that of pandemic and seasonal human influenza viruses (2, 5-9). Phenotypic traits of HA such as human receptor binding preference and increased acid or temperature stability have been shown to be critical for airborne transmission of avian A/H5N1 viruses between ferrets (10, 11). Here, we describe several amino acid substitutions that affect both of these properties of the HA of A/H7N9 A/Anhui/1/13 virus (AN1).The impact of two substitutions in the receptor-binding site (RBS), L217Q and G219S, on binding affinity and HA stability was investigated first. The L217Q substitution was previously detected in a minor virus population that emerged in ferrets upon inoculation with the AN1 virus isolate (6). The G219S substitution has been previously shown to increase the binding of A/H7N9 viruses to human trachea, alveolar epithelium, and synthetic glycans (12, 13). Recombinant viruses containing 7 gene segments of the mouse-adapted virus A/Puerto-Rico/8/34 and HA of AN1 with or without substitutions of interest were produced in 293T cells and propagated in Madin-Darby canine kidney (MDCK) cells by reverse genetics according to standard procedures (14). Properties of binding of recombinant viruses to ␣2,3-and ␣2,6-linked sialic acids (SA), the avian and human receptors, respectively, were assessed using a solid-phase binding assay (adjusted from the method described in reference 11). The latter analysis confirmed that the wild-type AN1 (AN1 WT ) virus binds to both avian and human influenza virus receptors (Fig. 1A and F), as shown previously (6, 15). AN1 L217Q virus predominantly bound to avian receptors but showed residual b...

Section: R Ecent Human Infections With Influenza A/h7n9 Viruses Insupporting

confidence: 92%

Amino Acid Substitutions That Affect Receptor Binding and Stability of the Hemagglutinin of Influenza A/H7N9 Virus

Schrauwen

Richard

Burke

et al. 2016

“…Previously, we and others generated recombinant soluble NA proteins for structure determination, enzymatic activity, and immunogenicity analyses (22)(23)(24)(25)(26)(27)(28)(29)(30). The use of recombinant soluble glycoproteins provides several advantages.…”

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confidence: 99%

“…In view of the importance of NA oligomerization for enzymatic activity, N-terminal tetramerization domains are generally fused to the recombinant soluble NA proteins. These tetramerization domains may be artificial, like GCN4-pLI (22,23), or derived from bacterial or mammalian proteins, such as the Staphylothermus marinus Tetrabrachion protein (27,28) or human vasodilatorstimulated phosphoprotein (VASP) (24,29,30). Although the recombinant soluble NA proteins have been used for different analyses, it is not known how the different N-terminal oligomerization domains affect NA protein assembly and enzymatic properties.…”

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confidence: 99%

Identification of Residues That Affect Oligomerization and/or Enzymatic Activity of Influenza Virus H5N1 Neuraminidase Proteins

Dai

Guo

Dortmans

et al. 2016

Influenza A virus (IAV) attachment to and release from sialoside receptors is determined by the balance between hemagglutinin (HA) and neuraminidase (NA). The molecular determinants that mediate the specificity and activity of NA are still poorly understood. In this study, we aimed to design the optimal recombinant soluble NA protein to identify residues that affect NA enzymatic activity. To this end, recombinant soluble versions of four different NA proteins from H5N1 viruses were compared with their full-length counterparts. The soluble NA ectodomains were fused to three commonly used tetramerization domains. Our results indicate that the particular oligomerization domain used does not affect the K m value but may affect the specific enzymatic activity. This particularly holds true when the stalk domain is included and for NA ectodomains that display a low intrinsic ability to oligomerize. NA ectodomains extended with a Tetrabrachion domain, which forms a nearly parallel four-helix bundle, better mimicked the enzymatic properties of full-length proteins than when other coiled-coil tetramerization domains were used, which probably distort the stalk domain. Comparison of different NA proteins and mutagenic analysis of recombinant soluble versions thereof resulted in the identification of several residues that affected oligomerization of the NA head domain (position 95) and therefore the specific activity or sialic acid binding affinity (K m value; positions 252 and 347). This study demonstrates the potential of using recombinant soluble NA proteins to reveal determinants of NA assembly and enzymatic activity. IMPORTANCEThe IAV HA and NA glycoproteins are important determinants of host tropism and pathogenicity. However, NA is relatively understudied compared to HA. Analysis of soluble versions of these glycoproteins is an attractive way to study their activities, as they are easily purified from cell culture media and applied in downstream assays. In the present study, we analyzed the enzymatic activity of different NA ectodomains with three commonly used tetramerization domains and compared them with fulllength NA proteins. By performing a mutagenic analysis, we identified several residues that affected NA assembly, activity, and/or substrate binding. In addition, our results indicate that the design of the recombinant soluble NA protein, including the particular tetramerization domain, is an important determinant for maintaining the enzymatic properties within the head domain. NA ectodomains extended with a Tetrabrachion domain better mimicked the full-length proteins than when the other tetramerization domains were used. V irus particles contain dedicated proteins that recognize cell surface molecules. While some viruses evolved to bind specific protein receptors, others bind carbohydrate moieties, such as sialic acids (SIAs), that are omnipresent on the cell surface as well as in the mucus. Several of the latter viruses, including influenza A virus (IAV), carry receptor-destroying activities in addit...

“…Structural and receptor binding analyses have demonstrated that the H7N9 viruses bind to both avian-like ␣2,3-linked sialic acid (SA) receptors and mammalian-like ␣2,6-linked SA receptors. The Q226L change, which has been associated with reduced binding to ␣2,3-SA and increased binding to ␣2,6-SA (16,17), and other residues in the H7N9 HA contribute to this receptor binding specificity (13,(18)(19)(20). Although sustained human-tohuman transmission has not been reported, the H7N9 virus can be transmitted via aerosol in ferrets, raising concerns about its pandemic potential (21)(22)(23).…”

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confidence: 99%

Development of a High-Yield Live Attenuated H7N9 Influenza Virus Vaccine That Provides Protection against Homologous and Heterologous H7 Wild-Type Viruses in Ferrets

Chen

Baz

et al. 2014

IMPORTANCEIn response to the recent avian H7N9 influenza virus infection in humans, we developed a live attenuated H7N9 influenza vaccine (LAIV) with two amino acid substitutions in the viral HA protein that improved vaccine yield by 10-fold in chicken embryonated eggs, the substrate for vaccine manufacture. The two amino acids also improved the antigen yield for inactivated H7N9 vaccines, demonstrating that this finding could great facilitate the efficiency of H7N9 vaccine manufacture. The candidate H7N9 LAIV was immunogenic and protected ferrets against homologous and heterologous wild-type H7 virus challenge, making it suitable for use in protecting humans from H7 infection.