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Amino Acid Substitutions That Affect Receptor Binding and Stability of the Hemagglutinin of Influenza A/H7N9 Virus Eefje J. A. Schrauwen,a Mathilde Richard,a David F. Burke,b Guus F. Rimmelzwaan,a Sander Herfst,a Ron A. M. Fouchiera Department of Viroscience, Postgraduate School Molecular Medicine, Erasmus MC, Rotterdam, The Netherlandsa; Department of Zoology, University of Cambridge, Cambridge, United Kingdomb
Receptor-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.
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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 (AN1WT) virus binds to both avian and human influenza virus receptors (Fig. 1A and F), as shown previously (6, 15). AN1L217Q virus predominantly bound to avian receptors but showed residual binding to human receptors (Fig. 1B and F). This is in agreement with the results of a study by Shi et al., which showed that the L217Q substitution in the AN1 HA did not completely abro-
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gate human receptor binding, implying that other substitutions in the RBS might contribute to the ␣2,6-linked SA binding of AN1 (3, 16). Moreover, the G219S substitution significantly increased overall binding affinities to ␣2,3- and ␣2,6-linked SA (Fig. 1C and F), as previously described (12, 13). Recombinant viruses containing the HA of the avian H5N1 virus A/Vietnam/1194/04 (A/VN/1194/04) or the human H3N2 virus A/Netherlands/213/03 (A/NL/213/03) were used as positive controls for binding to ␣2,3- or ␣2,6-linked SA, respectively (Fig. 1D, E, and F). HA-mediated cell-to-cell fusion was assessed using a syncytium formation assay in Vero cells, upon transfection of pCAGGS expression plasmids expressing various HAs and subsequent exposure to different pH levels (11). The pH threshold at which cell-to-cell fusion was triggered by AN1WT HA was 5.6, which is relatively high compared to results seen with human influenza viruses (17) (Fig. 2A). For AN1L217Q HA, the fusion threshold decreased to pH 5.4, while AN1G219S HA showed a pH threshold of fusion of ⬎6.0. The conformational change of HA from a nonfusogenic to a fusogenic state can also be triggered at neutral pH when the HA is exposed to increasing temperature. Therefore, the heat stability was assessed using a temperature sensitivity assay to further assess HA stability (11). In agreement with the results of the fusion assay, AN1L217Q HA showed increased temperature stability, whereas AN1G219S HA was less stable than AN1WT HA (Fig. 2B). Next, substitutions that could potentially increase the HA stability of AN1WT to levels comparable to those seen with human viruses or airborne avian viruses were investigated. In addition, since the G219S substitution increased overall binding to avian and human receptors at the expense of HA stability, putative stability substitutions that would compensate for the decrease in HA stability
Received 3 December 2015 Accepted 8 January 2016 Accepted manuscript posted online 20 January 2016 Citation Schrauwen EJA, Richard M, Burke DF, Rimmelzwaan GF, Herfst S, Fouchier RAM. 2016. Amino acid substitutions that affect receptor binding and stability of the hemagglutinin of influenza A/H7N9 virus. J Virol 90:3794 –3799. doi:10.1128/JVI.03052-15. Editor: A. García-Sastre Address correspondence to Mathilde Richard, [email protected]. E.J.A.S. and M.R. contributed equally to this article. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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FIG 1 Binding specificity and affinity of the AN1WT virus and AN1 viruses carrying substitutions to sialylglycopolymers in the receptor binding site. (A to E)
Ligand binding curves representing binding of AN1WT (A), AN1L217Q (B), AN1G219S (C), A/VN/1194/04 (D), and A/NL/213/03 (E) to ␣2,3-sialyllactosamine (3=SLN) (in red) and ␣2,6-sialyllactosamine (6=SLN) (in blue). Binding curves were fitted using a one-site nonlinear regression in GraphPad [Y ⫽ Bmax ⫻ X/ (Kd ⫹ X)]. The values correspond to the means ⫾ standard deviations calculated from the results of two independent assays performed in duplicate. OD, optical density. (F) The values corresponding to the dissociation constant at equilibrium (Kd) were calculated from the nonlinear regression curves as the concentration of sialylglycopolymers needed to achieve half-maximal binding at equilibrium. P values were calculated using an unpaired two-tailed t test comparing the Kd values for the AN1WT virus to the values determined for the mutant viruses. Statistical significance (P values under 0.05) is indicated by an asterisk.
caused by the G219S substitution were tested. Two substitutions— H103Y and T315I (H5 numbering)—were previously shown to increase the stability of A/H5N1 viruses via different mechanisms (10, 11, 18–20). Using computational modeling (21), a N94K substitution located at the trimer interface of AN1 HA which could potentially have an impact similar to that seen with the H103Y substitution in HA of A/H5N1 was identified. Substitution N94K was predicted to interact with E74 of HA2 in the neighboring monomer (Fig. 3A and B). However, this would come at the expense of E74 losing an interaction with Q76 in HA2 of another monomer, resulting in little overall change in stability. An A210E substitution, also located at the trimer interface although more distal to the stalk, was previously detected as a minor variant in ferrets upon inoculation with the AN1 virus and was also tested here (6). The A210E substitution was predicted to yield interactions with the side chains of both T156 and S237
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on the neighboring monomer, thus increasing its stability (Fig. 3A and C). These interactions are presumably affected when this mutation is combined with G219S, due to their close proximity. A K58I substitution (HA2 numbering), which is known to increase the HA stability of A/H5N1 and A/H7N1 viruses (22, 23), was the third substitution investigated here. In AN1, amino acid K58 is predicted to interact with nearby N282, causing a kink in the peptide backbone and a less-than-optimal interaction between the adjacent monomers (Fig. 3A and D). The K58I mutation would, surprisingly, lead to a loss of this interaction but presumably allows the movement of the backbone, improving interactions of the neighboring charged R54 and E57 amino acids in HA2 with the adjacent monomer. Substitutions N94K, K58I, and A210E were assessed for their ability to alter the stability of both AN1WT and AN1G219S. Substitution
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FIG 2 pH threshold of fusion and thermostability of AN1WT HA and mutant HAs. (A) Syncytium formation in Vero cells expressing wild-type or mutant AN1 HA proteins after exposure to different levels of pH. Cells were fixed using 80% ice-cold acetone, washed, and stained using a 20% Giemsa solution. The black boxes represent the range of pH values at which fusion was detected microscopically. (B) HA protein stability as measured by the ability of viruses to agglutinate turkey red blood cells after incubation at 50°C for the indicated times (minutes). Colors indicate the HA titers upon treatment at various time points at 50°C as shown in the legend.
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FIG 3 Cartoon representation of a model of the trimer structure of HA of AN1 at different positions. (A) HA of AN1 with the substitutions studied annotated. (B) Mutant N94K (cyan) is predicted to interact with E74 in HA2 of the neighboring monomer, but its presence results in the loss of an interaction between E74 and Q76. (C) Mutant A210E (shown as orange sticks), close to G219 (magenta), is predicted to interact with the side chains of both T156 and S237 on the neighboring monomer. (D) At position 58, there is a gap between the monomers due to the interaction of K58, kinking the peptide backbone. Substitution K58I could allow the backbone to straighten and form additional intermonomer interactions. Mutations were introduced using the program Andante into the crystal structure of the HA of A/Shanghai/2/2013 H7N9 (PDB code 4LN6). The sequence is identical to that of AN1.
N94K caused an increase in acid stability in both AN1WT and AN1G219S HA (Fig. 2A). However, the temperature stability of AN1N94K and AN1G219S N94K viruses was not increased compared to that of AN1WT and AN1G219S viruses (Fig. 2B). The K58I substitution resulted in a marked increase in the acid and temperature stability of AN1 HA with and without the G219S substitution (Fig. 2). AN1A210E HA presented a pH threshold for fusion similar to that seen with the AN1WT HA. However, the A210E substitution resulted in a decrease of the pH threshold for fusion of AN1G219S by pH 0.2 (Fig. 2A) and a higher HA thermostability with and without the G219S substitution (Fig. 2B). Next, we investigated whether the N94K, A210E, and K58I substitutions would affect the binding properties of AN1WT and AN1G219S. The N94K substitution significantly increased affinity to both ␣2,3- and ␣2,6-linked SA to levels comparable to those
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seen with the G219S substitution (Fig. 4A, D, and G). The A210E substitution did not affect the receptor binding affinity or the specificity (Fig. 4C, F, and G). Surprisingly, the K58I substitution, only in combination with G219S, significantly increased the receptor binding affinity to both ␣2,3- and ␣2,6-linked SA compared to AN1WT and AN1G219S (Fig. 4B, E, and G). Here, specific substitutions in the RBS of AN1 were shown to affect HA stability. Moreover, several compensatory amino acid substitutions resulted in increased acid and/or temperature stability of AN1G219S HA and of AN1WT HA. Remarkably, the K58I substitution was the only one to increase both acid stability and heat stability. The K58I substitution was first identified in a A/H7N1 virus, in which it was selected as a consequence of the inhibition of M2 protein by amantadine (23). In A/H5N1, the K58I substitution has been associated with an increase in virus
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FIG 4 Binding specificity and affinity of the AN1WT virus and AN1 viruses carrying substitutions that affect HA stability to sialylglycopolymers. (A to F) Ligand binding
curves representing binding of AN1N94K (A), AN1K58I (B), AN1A210E (C), AN1G219S N94K (D), AN1G219S K58I (E), and AN1G219S A210E (F) to ␣2,3-sialyllactosamine (3=SLN) (in red) and ␣2,6-sialyllactosamine (6=SLN) (in blue). Binding curves were fitted using a one-site nonlinear regression in GraphPad [Y ⫽ Bmax ⫻ X/(Kd ⫹ X)]. The values correspond to the means ⫾ standard deviations calculated from the results of two independent assays performed in duplicate. (G) The values corresponding to the dissociation constant at equilibrium (Kd) were calculated from the nonlinear regression curves as the concentration of sialylglycopolymers needed to achieve half-maximal binding at equilibrium. P values were calculated using an unpaired two-tailed t test comparing the Kd values of the AN1WT virus or the AN1G219S virus with the values determined for the mutant viruses. Statistical significance (P values under 0.05) is indicated by an asterisk.
replication in the upper respiratory tract of mice and ferrets (22, 24–26). Interestingly, the K58I substitution also increased the overall affinities of binding to ␣2,3- and ␣2,6-linked SA of the AN1G219S virus. Therefore, it would be interesting to study the impact of these substitutions on replication and transmission of A/H7N9 viruses in ferrets. Keeping in mind that (acid) stability may be only a surrogate marker for another phenotype (e.g., stability in aerosols or respiratory droplets), increased knowledge of amino acid substitutions that alter the HA stability across HA subtypes would help to better understand stability as a biological trait for replication and transmission of influenza viruses.
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This study identified amino acid substitutions that alter the HA stability and binding affinity of A/H7N9 viruses, properties that have been shown to be critical for airborne transmission of avian influenza viruses between mammals. These data may be useful for surveillance and assessment of the pandemic potential of zoonotic viruses, upon confirmation of the virus phenotypes in appropriate animal models. ACKNOWLEDGMENTS We thank Pascal Lexmond, Theo Bestebroer, and Miranda de Graaf for excellent technical assistance.
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FUNDING INFORMATION HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID) provided funding to Eefje J. A. Schrauwen, Mathilde Richard, David Burke, Sander Herfst, and Ron A. M. Fouchier under grant number HHSN272201400008C.
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EU FP7 provided funding to Eefje J. A. Schrauwen, Mathilde Richard, Guus F Rimmelzwaan, Sander Herfst, and Ron A. M. Fouchier under grant number 278976 (7th Framework Programme ANTIGONE).
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Amino Acid Substitutions That Affect Receptor Binding and Stability of the Hemagglutinin of Influenza A/H7N9 Virus Eefje J. A. Schrauwen,a Mathilde Richard,a David F. Burke,b Guus F. Rimmelzwaan,a Sander Herfst,a Ron A. M. Fouchiera Department of Viroscience, Postgraduate School Molecular Medicine, Erasmus MC, Rotterdam, The Netherlandsa; Department of Zoology, University of Cambridge, Cambridge, United Kingdomb
Receptor-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.
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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 (AN1WT) virus binds to both avian and human influenza virus receptors (Fig. 1A and F), as shown previously (6, 15). AN1L217Q virus predominantly bound to avian receptors but showed residual binding to human receptors (Fig. 1B and F). This is in agreement with the results of a study by Shi et al., which showed that the L217Q substitution in the AN1 HA did not completely abro-
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gate human receptor binding, implying that other substitutions in the RBS might contribute to the ␣2,6-linked SA binding of AN1 (3, 16). Moreover, the G219S substitution significantly increased overall binding affinities to ␣2,3- and ␣2,6-linked SA (Fig. 1C and F), as previously described (12, 13). Recombinant viruses containing the HA of the avian H5N1 virus A/Vietnam/1194/04 (A/VN/1194/04) or the human H3N2 virus A/Netherlands/213/03 (A/NL/213/03) were used as positive controls for binding to ␣2,3- or ␣2,6-linked SA, respectively (Fig. 1D, E, and F). HA-mediated cell-to-cell fusion was assessed using a syncytium formation assay in Vero cells, upon transfection of pCAGGS expression plasmids expressing various HAs and subsequent exposure to different pH levels (11). The pH threshold at which cell-to-cell fusion was triggered by AN1WT HA was 5.6, which is relatively high compared to results seen with human influenza viruses (17) (Fig. 2A). For AN1L217Q HA, the fusion threshold decreased to pH 5.4, while AN1G219S HA showed a pH threshold of fusion of ⬎6.0. The conformational change of HA from a nonfusogenic to a fusogenic state can also be triggered at neutral pH when the HA is exposed to increasing temperature. Therefore, the heat stability was assessed using a temperature sensitivity assay to further assess HA stability (11). In agreement with the results of the fusion assay, AN1L217Q HA showed increased temperature stability, whereas AN1G219S HA was less stable than AN1WT HA (Fig. 2B). Next, substitutions that could potentially increase the HA stability of AN1WT to levels comparable to those seen with human viruses or airborne avian viruses were investigated. In addition, since the G219S substitution increased overall binding to avian and human receptors at the expense of HA stability, putative stability substitutions that would compensate for the decrease in HA stability
Received 3 December 2015 Accepted 8 January 2016 Accepted manuscript posted online 20 January 2016 Citation Schrauwen EJA, Richard M, Burke DF, Rimmelzwaan GF, Herfst S, Fouchier RAM. 2016. Amino acid substitutions that affect receptor binding and stability of the hemagglutinin of influenza A/H7N9 virus. J Virol 90:3794 –3799. doi:10.1128/JVI.03052-15. Editor: A. García-Sastre Address correspondence to Mathilde Richard, [email protected]. E.J.A.S. and M.R. contributed equally to this article. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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FIG 1 Binding specificity and affinity of the AN1WT virus and AN1 viruses carrying substitutions to sialylglycopolymers in the receptor binding site. (A to E)
Ligand binding curves representing binding of AN1WT (A), AN1L217Q (B), AN1G219S (C), A/VN/1194/04 (D), and A/NL/213/03 (E) to ␣2,3-sialyllactosamine (3=SLN) (in red) and ␣2,6-sialyllactosamine (6=SLN) (in blue). Binding curves were fitted using a one-site nonlinear regression in GraphPad [Y ⫽ Bmax ⫻ X/ (Kd ⫹ X)]. The values correspond to the means ⫾ standard deviations calculated from the results of two independent assays performed in duplicate. OD, optical density. (F) The values corresponding to the dissociation constant at equilibrium (Kd) were calculated from the nonlinear regression curves as the concentration of sialylglycopolymers needed to achieve half-maximal binding at equilibrium. P values were calculated using an unpaired two-tailed t test comparing the Kd values for the AN1WT virus to the values determined for the mutant viruses. Statistical significance (P values under 0.05) is indicated by an asterisk.
caused by the G219S substitution were tested. Two substitutions— H103Y and T315I (H5 numbering)—were previously shown to increase the stability of A/H5N1 viruses via different mechanisms (10, 11, 18–20). Using computational modeling (21), a N94K substitution located at the trimer interface of AN1 HA which could potentially have an impact similar to that seen with the H103Y substitution in HA of A/H5N1 was identified. Substitution N94K was predicted to interact with E74 of HA2 in the neighboring monomer (Fig. 3A and B). However, this would come at the expense of E74 losing an interaction with Q76 in HA2 of another monomer, resulting in little overall change in stability. An A210E substitution, also located at the trimer interface although more distal to the stalk, was previously detected as a minor variant in ferrets upon inoculation with the AN1 virus and was also tested here (6). The A210E substitution was predicted to yield interactions with the side chains of both T156 and S237
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on the neighboring monomer, thus increasing its stability (Fig. 3A and C). These interactions are presumably affected when this mutation is combined with G219S, due to their close proximity. A K58I substitution (HA2 numbering), which is known to increase the HA stability of A/H5N1 and A/H7N1 viruses (22, 23), was the third substitution investigated here. In AN1, amino acid K58 is predicted to interact with nearby N282, causing a kink in the peptide backbone and a less-than-optimal interaction between the adjacent monomers (Fig. 3A and D). The K58I mutation would, surprisingly, lead to a loss of this interaction but presumably allows the movement of the backbone, improving interactions of the neighboring charged R54 and E57 amino acids in HA2 with the adjacent monomer. Substitutions N94K, K58I, and A210E were assessed for their ability to alter the stability of both AN1WT and AN1G219S. Substitution
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FIG 2 pH threshold of fusion and thermostability of AN1WT HA and mutant HAs. (A) Syncytium formation in Vero cells expressing wild-type or mutant AN1 HA proteins after exposure to different levels of pH. Cells were fixed using 80% ice-cold acetone, washed, and stained using a 20% Giemsa solution. The black boxes represent the range of pH values at which fusion was detected microscopically. (B) HA protein stability as measured by the ability of viruses to agglutinate turkey red blood cells after incubation at 50°C for the indicated times (minutes). Colors indicate the HA titers upon treatment at various time points at 50°C as shown in the legend.
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FIG 3 Cartoon representation of a model of the trimer structure of HA of AN1 at different positions. (A) HA of AN1 with the substitutions studied annotated. (B) Mutant N94K (cyan) is predicted to interact with E74 in HA2 of the neighboring monomer, but its presence results in the loss of an interaction between E74 and Q76. (C) Mutant A210E (shown as orange sticks), close to G219 (magenta), is predicted to interact with the side chains of both T156 and S237 on the neighboring monomer. (D) At position 58, there is a gap between the monomers due to the interaction of K58, kinking the peptide backbone. Substitution K58I could allow the backbone to straighten and form additional intermonomer interactions. Mutations were introduced using the program Andante into the crystal structure of the HA of A/Shanghai/2/2013 H7N9 (PDB code 4LN6). The sequence is identical to that of AN1.
N94K caused an increase in acid stability in both AN1WT and AN1G219S HA (Fig. 2A). However, the temperature stability of AN1N94K and AN1G219S N94K viruses was not increased compared to that of AN1WT and AN1G219S viruses (Fig. 2B). The K58I substitution resulted in a marked increase in the acid and temperature stability of AN1 HA with and without the G219S substitution (Fig. 2). AN1A210E HA presented a pH threshold for fusion similar to that seen with the AN1WT HA. However, the A210E substitution resulted in a decrease of the pH threshold for fusion of AN1G219S by pH 0.2 (Fig. 2A) and a higher HA thermostability with and without the G219S substitution (Fig. 2B). Next, we investigated whether the N94K, A210E, and K58I substitutions would affect the binding properties of AN1WT and AN1G219S. The N94K substitution significantly increased affinity to both ␣2,3- and ␣2,6-linked SA to levels comparable to those
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seen with the G219S substitution (Fig. 4A, D, and G). The A210E substitution did not affect the receptor binding affinity or the specificity (Fig. 4C, F, and G). Surprisingly, the K58I substitution, only in combination with G219S, significantly increased the receptor binding affinity to both ␣2,3- and ␣2,6-linked SA compared to AN1WT and AN1G219S (Fig. 4B, E, and G). Here, specific substitutions in the RBS of AN1 were shown to affect HA stability. Moreover, several compensatory amino acid substitutions resulted in increased acid and/or temperature stability of AN1G219S HA and of AN1WT HA. Remarkably, the K58I substitution was the only one to increase both acid stability and heat stability. The K58I substitution was first identified in a A/H7N1 virus, in which it was selected as a consequence of the inhibition of M2 protein by amantadine (23). In A/H5N1, the K58I substitution has been associated with an increase in virus
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FIG 4 Binding specificity and affinity of the AN1WT virus and AN1 viruses carrying substitutions that affect HA stability to sialylglycopolymers. (A to F) Ligand binding
curves representing binding of AN1N94K (A), AN1K58I (B), AN1A210E (C), AN1G219S N94K (D), AN1G219S K58I (E), and AN1G219S A210E (F) to ␣2,3-sialyllactosamine (3=SLN) (in red) and ␣2,6-sialyllactosamine (6=SLN) (in blue). Binding curves were fitted using a one-site nonlinear regression in GraphPad [Y ⫽ Bmax ⫻ X/(Kd ⫹ X)]. The values correspond to the means ⫾ standard deviations calculated from the results of two independent assays performed in duplicate. (G) The values corresponding to the dissociation constant at equilibrium (Kd) were calculated from the nonlinear regression curves as the concentration of sialylglycopolymers needed to achieve half-maximal binding at equilibrium. P values were calculated using an unpaired two-tailed t test comparing the Kd values of the AN1WT virus or the AN1G219S virus with the values determined for the mutant viruses. Statistical significance (P values under 0.05) is indicated by an asterisk.
replication in the upper respiratory tract of mice and ferrets (22, 24–26). Interestingly, the K58I substitution also increased the overall affinities of binding to ␣2,3- and ␣2,6-linked SA of the AN1G219S virus. Therefore, it would be interesting to study the impact of these substitutions on replication and transmission of A/H7N9 viruses in ferrets. Keeping in mind that (acid) stability may be only a surrogate marker for another phenotype (e.g., stability in aerosols or respiratory droplets), increased knowledge of amino acid substitutions that alter the HA stability across HA subtypes would help to better understand stability as a biological trait for replication and transmission of influenza viruses.
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This study identified amino acid substitutions that alter the HA stability and binding affinity of A/H7N9 viruses, properties that have been shown to be critical for airborne transmission of avian influenza viruses between mammals. These data may be useful for surveillance and assessment of the pandemic potential of zoonotic viruses, upon confirmation of the virus phenotypes in appropriate animal models. ACKNOWLEDGMENTS We thank Pascal Lexmond, Theo Bestebroer, and Miranda de Graaf for excellent technical assistance.
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FUNDING INFORMATION HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID) provided funding to Eefje J. A. Schrauwen, Mathilde Richard, David Burke, Sander Herfst, and Ron A. M. Fouchier under grant number HHSN272201400008C.
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EU FP7 provided funding to Eefje J. A. Schrauwen, Mathilde Richard, Guus F Rimmelzwaan, Sander Herfst, and Ron A. M. Fouchier under grant number 278976 (7th Framework Programme ANTIGONE).
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