Avian-to-Human Receptor-Binding Adaptation of Avian H7N9 Influenza Virus Hemagglutinin

Article

Avian-to-Human Receptor-Binding Adaptation of Avian H7N9 Influenza Virus Hemagglutinin Graphical Abstract

Authors Ying Xu, Ruchao Peng, Wei Zhang, ..., Jinghua Yan, Yi Shi, George F. Gao

Correspondence [email protected]

In Brief Residue substitution in hemagglutinin (HA) could be a determinant of interspecies transmission of influenza virus. Xu et al. report a unique evolutionary route behind the avian-tohuman receptor-binding adaptation in H7N9-subtype HA.

Highlights d

G186V mutation is key for H7N9 HA to acquire human receptor-binding capacity

d

Q226L mutation favors receptor binding only within a specific microenvironment

d

G186V mutation might be selected earlier than Q226L in evolution

d

Structural basis for G186V and Q226L mutation affecting the receptor tropism

Xu et al., 2019, Cell Reports 29, 2217–2228 November 19, 2019 ª 2019 The Authors. https://doi.org/10.1016/j.celrep.2019.10.047

Cell Reports

Article Avian-to-Human Receptor-Binding Adaptation of Avian H7N9 Influenza Virus Hemagglutinin Ying Xu,1,2 Ruchao Peng,2 Wei Zhang,2 Jianxun Qi,2 Hao Song,3 Sheng Liu,1,2 Haiyuan Wang,2,4 Min Wang,2 Haixia Xiao,5 Lifeng Fu,2,6 Zheng Fan,2 Yuhai Bi,2,6 Jinghua Yan,6,7,8 Yi Shi,2,6,7,8 and George F. Gao1,2,3,4,5,6,7,8,9,* 1School

of Life Sciences, University of Science and Technology of China, Hefei 230026, China Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences (CAS), Beijing 100101, China 3Research Network of Immunity and Health (RNIH), Beijing Institutes of Life Science, Chinese Academy of Sciences, Beijing 100101, China 4Laboratory of Animal Infectious Diseases, College of Animal Sciences and Veterinary Medicine, Guangxi University, Nanning 530004, China 5Laboratory of Protein Engineering and Vaccines, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China 6Center for Influenza Research and Early-Warning, Chinese Academy of Sciences (CASCIRE), Beijing 100101, China 7Savaid Medical School, University of Chinese Academy of Sciences, Beijing 101408, China 8Shenzhen Key Laboratory of Pathogen and Immunity, Shenzhen Third People’s Hospital, Shenzhen 518112, China 9Lead Contact *Correspondence: [email protected] https://doi.org/10.1016/j.celrep.2019.10.047 2CAS

SUMMARY

Since 2013, H7N9 avian influenza viruses (AIVs) have caused more than 1,600 human infections, posing a threat to public health. An emerging concern is whether H7N9 AIVs will cause pandemics among humans. Molecular analysis of hemagglutinin (HA), which is a critical determinant of interspecies transmission, shows that the current H7N9 AIVs are still dual-receptor tropic, indicating limited human-to-human transmission potency. Mutagenesis and structural studies reveal that a G186V substitution is sufficient for H7N9 AIVs to acquire human receptorbinding capacity, and a Q226L substitution would favor binding to both avian and human receptors only when paired with A138/V186/P221 hydrophobic residues. These data suggest a different evolutionary route of H7N9 viruses compared to other AIV-subtype HAs. INTRODUCTION Influenza A viruses (IAVs) are segmented, negative-sense RNA viruses that pose a great threat to public health and the economy and have caused multiple epidemics or pandemics among both humans and animals that have resulted in hundreds of thousands of human deaths (Bouvier and Palese, 2008; Gao, 2018; Taubenberger and Kash, 2010). A great concern in the prevention and control of IAVs is interspecies transmission, which significantly increases the ability of IAVs to cause pandemics in human populations (Shi et al., 2014; Wu et al., 2014). Many factors, from both viruses and their hosts, contribute to the interspecies transmission of IAVs (de Graaf and Fouchier, 2014; Medina and Garcı´a-Sastre, 2011; Taubenberger and Kash, 2010). One of the key factors is the receptor-binding preference of the IAV that

is determined by the hemagglutinin (HA) protein in the viral envelope (Bouvier and Palese, 2008; Shi et al., 2014; Sun et al., 2013; Wiley and Skehel, 1987). In general, pandemic IAV strains preferentially recognize the a2-6-(human receptor)-linked sialic acid receptor, while avian influenza virus (AIV) strains preferentially recognize the a2-3-(avian receptor)-linked sialic acid receptor or have dual-receptor-binding properties (Shi et al., 2014; Zhang et al., 2013a). In recent years, an increasing number of human infections with AIVs, including H5-, H6-, H7-, H9-, and H10-subtype AIVs, have been reported (Claas et al., 1998; Huo et al., 2018; Peiris et al., 1999; Qi et al., 2014; Sun et al., 2016; Yu et al., 2014; Yuan et al., 2013; Zhang et al., 2017). Among them, multiple outbreaks and human infections with H7-subtype AIVs, which mainly occurred in Europe, North America, and Asia, have been reported. Many of these virus isolates displayed very weak binding affinity to the human a2-6-linked sialic acid receptor (Gambaryan et al., 2012) and caused a limited number of human infections, including H7N1, H7N2, H7N3, H7N4, and H7N7 subtypes (Fouchier et al., 2004; Huo et al., 2018; Sutton et al., 2014; Tweed et al., 2004; Yang et al., 2010). Since the outbreak of human infections with H7N9 AIVs in China in 2013, with a high fatality rate of
30%, these viruses have been constantly infecting humans and have caused hundreds of hospitalizations and deaths each year (Bao et al., 2013; Liu et al., 2014). As of March 5, 2019, a total of 1,567 cases of human infections with H7N9 AIVs have been confirmed, including 615 deaths (https://www.who.int/ influenza/human_animal_interface/). In addition, several highly pathogenic strains of H7N9 AIVs (HPAIVs) were identified in 2017, highlighting the threat of H7N9 AIVs to public health (Qi et al., 2018; Quan et al., 2018; Zhang et al., 2017). Residue substitutions in the receptor-binding site (RBS) of HA molecules play crucial roles in the change of receptor-binding properties of influenza viruses (de Graaf and Fouchier, 2014; Shi et al., 2014; Webster et al., 1982). During the early stage of the avian-origin human-infecting H7N9 influenza virus outbreak in 2013, two strains of H7N9 AIV were isolated, the non-prevalent

Cell Reports 29, 2217–2228, November 19, 2019 ª 2019 The Authors. 2217 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

strain A/Shanghai/1/2013 (SH1-H7N9) and the prevalent strain A/Anhui/1/2013 (AH1-H7N9). Our previous studies and those of others (Shi et al., 2013; Xiong et al., 2013b; Xu et al., 2013) have identified that the SH1-H7N9 isolate has typical avian receptor-binding preference, while the AH1-H7N9 isolate has dual-receptor-binding capacity, and the four amino acid substitutions within the RBS (S138A/G186V/T221P/Q226L, H3 numbering throughout the article) in H7N9 HA are responsible for the acquisition of human receptor-binding capacity. We also showed that H7N9 AIVs should have a residue determinant for the receptor-binding change that is different from that of H5N1 AIV, which is determined by the Q226L substitution and the deglycosylation at position 158 (Shi et al., 2013). However, the precise key residue determinant of H7N9 AIVs obtaining human receptor-binding capacity has not been well characterized. Here, we present a systematic molecular analysis of H7N9 HA with different key amino acid combinations in the RBS to evaluate their receptor-binding properties and further infer the molecular evolutionary process of naturally occurring H7N9 AIV isolates. We found that the current avian-to-human transmissible H7N9 AIVs bind to both avian and human receptors. Mutagenesis, bioinformatics, and structural studies revealed that G186V substitution is key to enabling avian-specific H7N9 HA to acquire human receptor-binding capacity, which might be selected earlier than the Q226L substitution in evolution. These findings could help us to better understand the evolution of different subtypes of influenza viruses and provide important basis for prevention and control of potential epidemics or pandemics. RESULTS Receptor-Binding Properties of AH1- and SH1-H7N9 HA Proteins and Key Residue Determinants During the early stage of the avian-origin human-infecting H7N9 influenza virus outbreak in 2013, two strains of H7N9 AIV were isolated, the A/Shanghai/1/2013 (SH1-H7N9) and A/Anhui/1/ 2013 (AH1-H7N9) isolates. Sequence analysis revealed that the HAs of the two strains harbor four residue substitutions in the RBS (138S/A, 186G/V, 221T/P, and 226Q/L), which are located in the three key structural elements of the RBS (130-loop, 190-helix, and 220-loop, respectively) (Figure 1A). Our previous study (Shi et al., 2013) identified the SH1-H7N9 isolate as an avian-specific IAV and the AH1-H7N9 isolate with dual-receptor-binding capacity (Figures 1B and 1C). Based on previous knowledge, residue 226Q/L is well defined as the signature for avian- and human-specific AIVs in the H2, H3, and H5 subtypes (Imai and Kawaoka, 2012; Lu et al., 2013; Matrosovich et al., 2000; Stevens et al., 2006; Zhang et al., 2013a). However, the AH1-H7 mutant with L226Q substitution still retained high binding affinities to both human and avian receptors (Shi et al., 2013), which indicates that the key determinants of H7N9 AIV gaining human receptor-binding capacity could be attributed to the other three sites. To test whether the different receptor-binding properties of SH1- and AH1-H7N9 IAVs could be solely attributed to the four residue substitutions in the RBS (there are four other substitutions outside the RBS between the two strains;

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Figure 1A), we first generated AH1-H7N9 HA mutant protein with the four residues replaced by those in the SH1-H7N9 isolate, and vice versa, to evaluate the receptor-binding properties of HA mutants by surface plasmon resonance (SPR) experiments. As expected, replacement of the four residues successfully reverted the receptor-binding properties mutually; i.e., introducing the four hydrophobic residues (A138/ V186/P221/L226) into SH1-H7N9 HA enabled it to bind to both human and avian receptors with high affinity, while the four hydrophilic substitutions (S138/G186/T221/Q226) in AH1-H7N9 HA impaired its binding to human receptors but retained its high affinity for avian receptor binding (Figures 1D and 1E). To further confirm the observation, we determined the crystal structures of both HA mutants with the four residues replaced by those in the other. Consistent with the biochemical assays, replacement of the four residues in AH1- or SH1-H7N9 HA completely restored all structural features within the RBS of the other, in which all these residues adopted almost identical conformations in both the wild-type (WT) and mutant protein contexts (Figure S1). These data together demonstrate that the different receptor-binding properties of the two strains are attributable to residues in these four sites. Identification of Determinant Residues for the Acquisition of Human Receptor-Binding Abilities of H7N9 HAs To better define the roles of these four residues in determining the receptor-binding property of H7-subtype HA, we generated a series of H7N9 HA mutants with different residue combinations in these four sites and evaluated their binding affinities to both human and avian receptors (Figure 2). For clarity, all H7N9 HA mutants are designated by the residues in these four sites (e.g., H7-SVTQ represents HA with S138/V186/T221/Q226). The single, double, and triple mutants were all generated based on the WT AH1-H7N9 HA. As shown by SPR experiments, a single G186V substitution could enable the avian-specific SH1-H7N9 HA to bind to human receptors and did not affect its affinity to avian receptors (Figure 2D). In contrast, the Q226L single substitution significantly abolished the binding of SH1-H7N9 HA to both avian and human receptors (Figure 2F). The single substitutions at the other two sites (S138A or T221P) did not cause obvious changes in the receptor-binding properties and retained avian receptor-binding specificity (Figures 2C and 2E). All of the mutants without a G186V substitution displayed negligible binding to the human receptor, even if the three other sites were replaced by hydrophobic residues (S138A, T221P, and Q226L) (Figure 2). These data demonstrated that V186 is the key determinant of avian-specific H7N9 HA obtaining human receptor-binding capacity. In addition, the Q226L substitution would severely decrease binding to both avian and human receptors when paired with hydrophilic residues in any of the other three sites (Figures 2F, 2I, 2K, 2L, and 2N-2P). Only if the other three sites are all hydrophobic residues (A138/V186/P221) would the Q226L substitution be favorable for binding to human receptors and still retain high affinity to avian receptors (Figure 2), consistent with the property of AH1-H7N9 isolates.

Figure 1. Amino Acid Substitutions and Receptor-Binding Properties of AH1-H7N9 and SH1-H7N9 HA and Variant Proteins (A) Ribbon representation of SH1-H7N9 (magenta, PDB: 4LCX) and AH1-H7N9 (green, PDB: 4KOL) HA structures with superposition of one protomer within the HA trimer. The other two HA protomers of SH1-H7N9 are colored in gray, while those of AH1-H7N9 are omitted for clarity. The eight residue substitutions between SH1-H7N9 and AH1-H7N9 HAs are shown as sphere models and indicated by black arrows; four of these substitutes are distributed within the RBS (138S/A, 186G/V, 221T/P, and 226Q/L), and the other four are in the stem region (174N/S, 276D/N, 283Y/H, and 400T/N). The position of the RBS is highlighted by a red dashed ellipse. (B–E) Receptor-binding profiles of SH1-H7N9 and AH1-H7N9 HAs and their quaternary mutants determined by SPR experiments. SH1-H7N9 wild-type HA (B) and the AH1-H7N9_HA-S138/G186/T221/Q226 mutant (D) preferentially bind to a2-3 glycans, while AH1-H7N9 wild-type HA (C) and the SH1-H7N9_HA-A138/ V186/P221/L226 mutant (E) bind to both glycans but show a preference for a2-3 glycans. The binding kinetic curves at different concentrations are plotted, and the KD values were calculated to quantify the binding affinities. KD values shown are the mean ± SEM of three independent experiments. The saturation curves for binding to a2-3 and a2-6 glycans are colored in blue and red, respectively. See also Figures S1 and S2 and Table S1.

Frequencies of Amino Acid Residues within the RBS of Naturally Isolated H7 HAs To investigate the natural evolving route of receptor binding for H7-subtype AIVs, we looked into all the HA sequences from natural H7-subtype virus isolates available in the GISAID (Global Initiative on Sharing All Influenza Data) database to analyze the amino acid substitutions in the RBS (including the 130-loop, 190-helix, and 220-loop and the aromatic residues at the bottom of the RBS) over the years. Among these sites, most of the residues, including 138 and 221, were highly conserved within the period (Table S1). However, the residues at sites 186, 189, and 226 exhibited obvious

distinctions before and after the H7N9 outbreak in 2013, which were dominated by G to V, T to A, and Q to L, respectively. Furthermore, the residue at site 135 transited from A to V since 2015. The substitutions in these four sites were particularly evident in the avian-to-human transmissible H7N9 AIVs (Figures S2A–S2D). However, A135V and A189T substitutions have no significant effect on the receptor-binding properties of AH1-H7N9 HAs, which still maintain dual-receptor tropism (Figures S2E and S2F). Thus, the residue substitutions at these two sites may render their effects by altering the antigenicity of the virus, as their side chains are fully exposed on the surface to form potential epitopes of HA.

Cell Reports 29, 2217–2228, November 19, 2019 2219

Figure 2. Receptor-Binding Profiles of H7N9 HA Mutants with a Single/Double/Triple Hydrophobic Residue Substitution among the Four Key Residues within the RBS The receptor-binding properties were assessed by SPR assays with glycans immobilized on the sensor chip. Binding to a2-3 and a2-6 glycans were both tested, with proteins flowing through the chip in different concentrations. The binding affinities were calculated based on the binding curves to facilitate quantitative comparison. KD values shown are the mean ± SEM of three independent experiments. The hydrophobic residue substitutions among the four key residues are highlighted in red. See also Figure 1 and Table 1.

Based on phylogenetic analysis, both AH1 and SH1 H7N9 strains originated from a common ancestor with H7-AGPQ residue combination in the RBS, which shows an avian-specific receptor-binding property (Figure S3). The SH1 strain is an occasional emergent variant and falls into a separate clade without further divergence, in contrast to the quite abundant AH1-like viruses with the H7-AVPL residue combination in the RBS. Thus, the evolution of H7H9 AIVs from avian-specific to dual-receptor tropism could be explained by the substitutions at sites 186 and 226, and the G186V substitution might occur earlier than the Q226L substitution to enable binding to human receptors. The Q226L substitution may play its role in the later stage

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to enhance the human receptor binding affinity, as it impairs binding to both human and avian receptors when paired with G186. We then investigated the occurrence of different residue combinations of HAs in all natural H7N9 IAV isolates. Among
1,900 sequences analyzed, more than 85% are similar to the AH1H7N9 isolate with A138/V186/P221/L226 residue combinations in these four sites (Table 1). The second most abundant population was H7-AVPQ, which has been shown to be dual-receptor tropic with slightly lower affinity to the human receptor than AH1-H7N9 HA (Figures 1C and 2M). The third largest group was H7-AGPQ, a group of highly avian-specific IAVs that do

Table 1. The Occurrence of Different Amino Acid Combinations in HAs from Naturally Occurring H7N9 IAV Isolates Key Residues in the RBS of H7N9 HA

Number of Natural Virus Isolates

138

186

221

226

Human

Avian

Other

Total

A

V

P

L

979

524

145

1648

A

G

P

Q

0

81

5

86

A

V

P

Q

42

36

40

118

A

A

P

L

9

11

2

22

A

V

P

I

15

2

8

25

A

V

P

P

1

0

5

6

A

G

P

L

1

0

0

1

T

V

P

L

1

0

0

1

T

G

P

Q

0

2

0

2

A

A

P

Q

0

2

0

2

S

G

T

Q

1

0

1

2

A

G

T

Q

1

0

0

1

A

I

P

H

1

0

0

1

A

V

P

H

1

0

0

1

A

I

P

Q

2

0

0

2

A

I

P

L

1

0

0

1

A

A

P

S

1

0

1

2

A

V

P

S

1

0

0

1

Other isolates include those from the environment and lab derived. See also Figures S2–S4.

not bind to the human receptor (Figure 2H). There were other two groups of AH1-H7N9-like viruses (H7-AAPL and H7-AVPI) with discernible abundance (22 and 25 isolates, respectively) (Table 1). Since A/V and L/I are both non-polar residue pairs with similar biochemical properties, it appeared that these two virus groups could be highly similar to the AH1-H7N9 virus isolate with dual-receptor binding capacity, and this observation was confirmed by SPR experiments (Figures S4A and S4B). Two small groups of isolates with infrequent substitution in 226 residues (H7-AVPP and H7-AVPS), which are both dual-receptor tropic as shown by SPR assays (Figures S4C and S4D), were observed. This observation indicates that the residue in 226 could be tolerated, to a certain extent, and it might be more crucial for affecting the affinity rather than the receptor-binding specificity of H7-subtype HA molecules. In all virus isolates with a L226 in the RBS, the other three sites were all hydrophobic residues (A138/V186/P221 or A138/A186/ P221), with only two exceptions (H7-TVPL and H7-AGPL) (Table 1). As threonine is biochemically comparable to serine, the H7-TVPL isolate was expected to possess properties similar to the H7-SVPL mutant. Both the H7-AGPL and H7-SVPL mutants were unable to bind to human receptors and showed very weak affinity to avian receptors, as shown by SPR experiments (Figures 2O and 2P). The existence of the two viral strains in nature indicated there must be some other evolutionary benefit gained by compromising their receptor-binding affinities. To further verify our observations made on different H7N9 HA proteins, we rescued a panel of recombinant H7N9 viruses with different residue combinations, which exist in natural isolates,

with reverse genetics technology (Sun et al., 2011) to evaluate their receptor-binding properties at the virus level (Figure 3). As shown by solid-phase binding assays, viruses with H7-AGPQ HA specifically recognized avian receptors and did not bind to human receptors, comparable to properties of the SH1-H7N9 strain (Figures 3A and 3C). In comparison, viruses with H7AVPQ HA (the sequence is identical to HA of A/Nanjing/5/2013) could bind to both human and avian receptors with a preference for the avian receptor (Figure 3D), whereas the viruses rescued by H7-AGPL (the sequence is identical to HA of A/Minhang/1/ 2013) and H7-SVPL HA displayed very weak binding to both human and avian receptors (Figures 3E and 3F), consistent with the properties of corresponding HAs determined by SPR experiments (Figures 2M, 2O, and 2P). For comparative analyses, the highly human-specific H1N1 and avian-specific H5N1 strains were selected as controls, which showed selective binding to human and avian receptors, respectively (Figures 3G and 3H). In addition, we also conducted an immunohistological fluorescence staining experiment to test the binding of these HA mutants to natural receptors (Figure S5). Again, the H7-AGPQ mutant selectively recognized avian receptors, and the H7-AVPQ mutant showed dual-receptor tropism (Figures S5E and S5J). The H7-SVTQ mutant binds both human and avian receptors (Figure S5F). In contrast, the H7-SVPL mutant showed moderate affinity to avian receptors and no detectable binding to human receptors (Figure S5H). The H7-SGTL and H7-AGPL mutants displayed negligible binding to both avian and human receptors (Figures S5G and S5I). These data further supported our hypothesis that G186V substitution is key for obtaining human receptor-binding capacity for H7N9 AIVs, and L226 would only be favorable for binding both human and avian receptors in the context of hydrophobic residues in the other three sites. Growth Kinetics of H7N9 Influenza Viruses with Different Residue Combinations in the HA RBS Based on the above analyses, the Q226L substitution could possibly be selected after all of the other three sites have been replaced by hydrophobic residues. The existence of the two ‘‘unexpected’’ natural isolates, H7-AGPL and H7-SVPL, suggested that viruses could benefit in some way by encompassing unfavorable receptor-binding properties. We then tested the growth kinetics of the two virus strains compared with the AH1-H7N9 strain. The second and third most abundant virus populations (H7-AVPQ and H7-AGPQ) were also tested for comparison. By propagating the viruses in MDCK cells, which contain both human and avian receptors, we found that the H7-SVPL mutant virus showed a slower growth rate than all other strains tested, implying that this strain might have emerged by chance with very low selection benefits (Figure S6). In contrast, the H7AGPL mutant virus displayed similar growth kinetics to the AH1-H7N9 strain (Figure S6). The H7-AVPQ mutant virus (the second most abundant population in nature), which is dual-receptor tropic, showed the highest growth rate compared to all other strains, and the H7-AGPQ virus (the third most abundant population in natural isolates) showed similar growth kinetics to the AH1 strain (Figure S6). These observations suggest that the receptor-binding property is a crucial, but not the only, determinant of viral fitness. The residue combination of H7-AVPQ

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Figure 3. Receptor-Binding Properties of Rescued AH1-H7N9 and SH1-H7N9 Viruses and Mutants Determined by Solid-Phase Binding Assays Sialylglycopolymers containing either a2-3 glycans (blue) or a2-6 glycans (red) sialic acids were immobilized on 96-well plates, and virus preparations were added to test binding following an ELISA-based protocol. Error bars represent the SD of the mean, which was calculated from three independent repeats. (A–F) Rescued (A) SH1-H7N9 (rSH1-H7N9), (B) AH1-H7N9 (rAH1-H7N9), (C) H7-AGPQ (rH7N9-AGPQ) mutant, (D) H7-AVPQ (rH7N9-AVPQ) mutant, (E) H7-AGPL (rH7N9-AGPL) mutant, and (F) H7-SVPL (rH7N9-SVPL) mutant viruses were analyzed against both human and avian receptor analogs. (G and H) Avian-specific (G) AH05-H5N1 and human-specific (H) CA04-H1N1 virus isolates were selected as controls. See also Figures 1, 2, and S5 and Table 1.

might represent an optimal solution to balance the selection benefits of different determinant factors. The failure of occurrence for other viruses with different residue combinations might imply the low fitness of the cooperative outcomes. Structural Basis for the Evolving Receptor-Binding Properties To uncover the underlying mechanisms of the evolving receptorbinding properties of H7N9 HAs in different residue contexts, we determined the crystal structures of a series of HA mutants with different residue combinations and compared them with the structures of AH1- and SH1-H7N9 HAs in complex with receptor analogs (Figures 4, 5, and 6). Our previous study defined the basic principles for receptor recognition of H7N9 HAs (Shi et al., 2013). Basically, conserved hydrogen bonds, contributed by Y98, A135, T136, S137, and E190, stabilize the terminal sialic acid (Sia-1) in the glycan receptors. On the other hand, several other residues in the 220-loop (Q222 and G225) further stabilize Gal-2 and GlcNAc-3 by additional hydrogen bonds with the polar hydroxyl groups in the glycan rings. In addition, the four hydrophobic residues (A138/V186/P221/L226) in AH1-H7N9 HA form a hydrophobic platform to accommodate the non-polar portion of glycan rings in which the side chain of L226 forms substantive van der Waals contacts with the carbon ring of Gal-2 in the avian receptor but fewer interactions in the human receptor. The other three residues, however, were not involved in direct interactions with the receptors (Shi et al., 2013).

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To figure out the mechanism of G186V substitution enabling avian H7N9 HA to bind to the human receptor, we determined the structures of H7-SVTQ mutant bound to human and avian receptors to analyze the structural basis for its receptor-binding properties (Figure 4). In general, the binding modes of H7SVTQ mutant to both receptors are quite similar to AH1-H7N9 HA, where both human and avian receptors adopt cis conformations. All interactions with the anchoring residues are well preserved in the mutant compared with AH1-H7N9 HA, except for the additional hydrogen bonds between Sia-1 and Q226, which further stabilize the interactions with both human and avian receptors (Figures 4A and 4D). The conformation of the avian receptor analog bound by the H7-SVTQ mutant is almost identical to that seen in the complex with AH1-H7N9 HA, suggesting that the microenvironment around residue 186 and the 190-helix greatly impacts the contacting mode of H7 HAs with the receptors. This could be attributed to the flexible conformations of the E190 side chain in different contexts. It is commonly seen that the side chain of E190 adopts an upward conformation in the free HAs but moves downward when bound to glycan receptors to form hydrogen bonds with either Sia-1 or GlcNAc-3 (Figure 5). In the hydrophobic context of V186 (e.g., AH1-H7N9 HA), the polar oxygens point vertically toward the hydroxyl groups of Sia-1 to avoid encountering the non-polar side chain of V186 (Figures 4A and 4B). In contrast, when paired with hydrophilic G186, as seen in SH1-H7N9 HA, the polar side chain of E190 could be well accommodated in this area so that the two terminal oxygens

Figure 4. Structural Basis for the H7-SVTQ Mutant to Bind Avian/Human Receptors and Comparison with AH1-H7N9 and SH1-H7N9 HAs (A and D) The molecular interactions for the H7-SVTQ mutant to bind avian (A) and human (D) receptor analogs are shown. The position of the 130-loop, 150-loop, 190-helix, and 220-loop are labeled accordingly. The key residues involved in interactions with the receptors are shown as sticks and labeled by the side. Hydrogen bonds between HA and glycans are represented by yellow dashed lines. Water molecules are represented by red spheres. (B, C, E, and F) The structures of the H7-SVTQ mutant in complex with receptors were superimposed with the complex structures of AH1-H7N9 and SH1-H7N9 HAs bound to avian (B and C) or human (E and F) receptor analogs for comparison (PDB: 4LKG, 4LKI, 4KOM, and 4KON, respectively). Wild-type AH1-H7N9 and SH1-H7N9 HAs and the H7-SVTQ mutant are colored in green, magenta, and cyan, respectively, and the four key residues in the RBS are shown as sticks and labeled with the same color code used for the molecules. See also Figure 1 and Table S4.

are horizontally positioned to reach Sia-1 and GlcNAc-3, respectively. In this context, GlcNAc-3 is anchored by a hydrogen bond with E190, resulting in a flatter conformation of glycans compared to that in the complex with AH1-H7N9 HA (Figures 4A–4C). In the structure of H7-SVTQ bound to the human receptor analog, three glycan residues were well resolved with an orientation similar to that observed in the complex with AH1-H7N9 HA, except that Gal-2 slightly rotated toward the 190-helix (Figures 4D and 4E). This is probably due to hydrophilic platform created by Q226 and S138 underneath the Gal-2 glycan ring; thus, the hydroxyl groups in the peripheral of Gal-2 were oriented to face the polar termini of Q226, and the non-polar carbon ring moves toward the hydrophobic V186. Besides, the GlcNAc-3 is further stabilized by one direct and two water-bridged hydrogen bonds to K193 in the 190-helix (Figures 4D and 5B). Interestingly, the side chain of S138 forms a hydrogen bond with the main chain of N224 (direct hydrogen bond or water bridges) to lock the 130-loop in vicinity with 220-loop, which highly resembles the feature of SH1-H7N9 HA (Figures 4A and 4D). This observation indicates that the hydrophobic environment created by V186 plays a more important role in determining

the receptor binding specificity of H7 HAs than residues in the 130- and 220-loops (Figures 5B–5D). To explain the highly compromised capacity of L226-containing H7N9 HAs to bind to both human and avian receptors in the context of hydrophilic residues in the other three sites, we compared the structure of the H7-SGTL mutant with both AH1- and SH1-H7N9 HAs bound to both receptor analogs (Figure 6). Due to the presence of hydrophilic S138, the isolated non-polar L226 moved back to leave the 130-loop so that the 220-loop in the H7-SGTL mutant was farther from the 130-loop as compared with either AH1- or SH1-H7N9 HAs. In this context, the side chain of L226 in H7-SGTL retracted by
1 A˚ and became closer to the Gal-2 glycan ring in both human and avian receptors (Figure 6). As S138 and G186 are both hydrophilic, the Gal-2 in human receptor cannot adopt a conformation similar to that in the complex with AH1-H7N9 HA, where the non-polar carbon ring faces the hydrophobic V186 (Figure 6C). Instead, Gal-2 could possibly rotate to make the peripheral hydroxyl groups point to the hydrophilic G186, which, as a result, might impair its hydrogen bonding with the main chain of G225. Thus, the Q226L substitution alone could not enable avian-specific H7 to bind to the human receptor. On

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Figure 5. Conformation of E190 in Different Contexts (A and B) Molecular interaction details of the H7-SVTQ mutant bound to avian (A) and human (B) receptors. The hydrogen bonds between receptors and residues within the RBS are represented as yellow dashed lines. Water molecules are represented by red spheres. The position of E190 is highlighted by a red ellipse. (C–E) The flexible side chain conformations of E190 in the native and receptor-bound states. In AH1-H7N9 HA (C) and the H7-SVTQ mutant (E), the terminal oxygen of the E190 side chain adopts different conformations in the native (upward) and receptor-bound (downward and vertical) states. In SH1-H7N9 HA (D), the side chain of E190 displays a similar horizontal conformation in both the native and receptor-bound states. See also Figure 4 and Table S4.

the other hand, when compared with the SH1-H7N9 HA, the side chain of L226 in H7-SGTL reached out to a similar position of the polar termini of Q226 in SH1-H7N9 HA to abolish the hydrophilic platform, which would again cause Gal-2 in the avian receptor to rotate or move upward to leave the hydrophobic side chain of L226 (Figure 6B). In addition, the replacement of Q226L would cause the loss of two hydrogen bonds with Sia-1 to further destabilize the binding of both human and avian receptors (Figures 4 and 6). Interestingly, when paired with a hydrophobic V186 or P221, the side chain of L226 would be lifted by
2 A˚ due to incompatibility with polar S138, which would thus render steric hindrance to the glycan ring of Gal-2 (Figures S7A and S7B) and significantly decrease binding to both receptors. When paired with a single A138 or two hydrophobic residues in the other three sites, the side chain of L226 would be placed at a position similar to that of the polar termini of Q226 to subvert the hydrophilic environment to be hydrophobic (Figure S7), which would still be incompatible for the binding of both human and avian receptors.

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In addition, the 220-loop of H7N9-subtype HA in the context of all hydrophobic (e.g., AH1 strain) or all hydrophilic (e.g., SH1 strain) residues in these four sites could be well stabilized, as shown by the normalized B factor analyses on this region, very similar to that seen in other subtypes of HA structures such as H2, H3, and H5 (Table S2). In contrast, the introduction of L226 in combination with hydrophilic residues in any of the other three sites would greatly increase the B factor of the entire 220-loop, which indicates higher flexibility of this region and may therefore impair binding to both human and avian receptors (Table S2). DISCUSSION Interspecies transmission is a key determinant of the emergence of influenza epidemics/pandemics and has long been a hot topic in influenza virus research. To cross the host barrier, viruses should be able to bind to receptors on the surface of new host cells, which is the first step for virus entry (de Graaf and Fouchier,

Figure 6. Structural Comparison of the H7SGTL Mutant with AH1-H7N9 and SH1-H7N9 HAs Bound to Avian and Human Receptor Analogs The H7-SGTL mutant structures were superimposed with the structures of SH1-H7N9 HA in complex with avian (A and B) or human (C and D) receptor analogs for comparison. Wild-type AH1H7N9 and SH1-H7N9 HAs and the H7-SGTL mutant are colored in green, magenta, and orange, respectively, and the four key residues in the RBS are shown as sticks and labeled with the same color code used for the molecules. The movement of the 220-loop in the H7-SGTL mutant relative to AH1H7N9 and SH1-H7N9 HAs is indicated by red dashed ellipses in the superposition. See also Figures 4 and S7 and Tables S2 and S4.

2014; Shi et al., 2014; Sun et al., 2013; Wiley and Skehel, 1987; Wu et al., 2014). The molecular mechanisms by which avian-specific HAs gain human receptor-binding avidity vary among different subtypes, and there could be more than one path for a specific HA subtype to evolve from avian-receptor specific to dual-receptor tropic and even subverting to human specific viruses (Shi et al., 2014; Xiong et al., 2014). For the H1 subtype, residues in 190 and 225 play crucial roles in the switch of receptor-binding specificity. The avian isolates with E190 and G225 features preferentially bind to the avian receptor, whereas the D190 and G225 combination switches the binding preference from the avian receptor to the human receptor yet maintains avian receptor-binding property. H1 HAs with D190 and 225D/E can only bind to the human receptor (Gamblin et al., 2004; Matrosovich et al., 2000; Xu et al., 2012; Zhang et al., 2013b). In the evolving ways from avian to human tropism for H2- and H3-subtype IAVs, Q226L and G228S substitutions are key determinants, and the Q226L substitution appears to be more critical for human receptor recognition (Matrosovich et al., 2000; Rogers et al., 1983; Xu et al., 2010). Recent research revealed that Q226L substitution also plays a pivotal role in altering receptor-binding specificity for H4 HAs, and G228S substitution would decrease the affinity for avian receptors while enhancing binding to human receptors (Song et al., 2017). Although H5-subtype AIVs have not evolved a human receptor-binding preference in nature, several tropismswitching substitutions have been identified in laboratories

(Herfst et al., 2012; Imai et al., 2012). The key determinants of receptor tropism of H5-subtype HA fall into the Q226L substitution and the loss of a glycosylation site on position 158, which pose their influences by altering the hydrophobicity and glycosylation of corresponding areas in the RBS (Lu et al., 2013; Xiong et al., 2013a; Zhang et al., 2013a). However, the molecular determinants of the host jump of H7N9-subtype IAVs are not well defined. In this work, we identified that the G186V single mutation is sufficient for avian-specific H7N9 influenza viruses to gain human receptor-binding capacity. Surprisingly, the well-known avian/human signature residue in 226 (Q/L) is not crucial for the acquisition of human receptor-binding capacity, but it seems to be harmful for receptor binding without the assistance of other hydrophobic residues in the RBS that may affect the stability of the 220-loop. Our study has further complemented the knowledge of H7N9 receptorbinding properties presented by our previous study and those of others (Shi et al., 2013; Xiong et al., 2013b; Xu et al., 2013). In the context accompanied by residues in 138, 186, and 221 and possibly other sites as well, the outcome of Q226L substitution could be substantially different, demonstrating the cooperative effects of residues in multiple sites to alter the receptorbinding properties and stabilities of HAs, which cooperatively contribute to the fitness of influenza viruses. Indeed, residues A138 and P221 were selected in avian-specific H7-subtype IAVs before the H7N9 outbreak in 2013; thus, the evolution of receptor-binding properties of H7-subtype HAs mainly involves substitutions of G186V and Q226L. Compared with other HA subtypes (e.g., H1, H2, H3, and H5), H7-subtype IAVs could likely adopt a different evolutionary route to gain human receptor specificity in naturally occurring viral isolates. Based on our analyses of viruses isolated at different times, the current H7N9 AIVs are highly similar to the AH1-H7N9 isolates with dual-receptor tropism, and no evidence has indicated the ability for efficient human-to-human transmissions.

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Thus, H7N9-subtype AIVs have not yet been well adapted for human specificity but indeed are constantly evolving. Recently, three artificial site mutations (V186K/K193T/G228S or V186N/N224K/G228S) in H7N9 HA were identified to subvert avian-specific H7N9 AIVs to human receptor specificity (de Vries et al., 2017). It might indicate the next stage of H7N9 virus evolution and raises the concern that H7N9 viruses could become human adapted to enable efficient human-to-human transmission, resulting in pandemics among human populations. Therefore, more attention should be paid to the surveillance and prevention of H7N9-subtype AIVs. STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d d d d

d d

KEY RESOURCES TABLE LEAD CONTACT AND MATERIALS AVAILABILITY EXPERIMENTAL MODEL AND SUBJECT DETAILS METHOD DETAILS B Protein Expression and Purification B Crystallization, Data Collection and Structure Determination B SPR Experiments B Phylogenetic Analysis B Virus Preparation B Solid-phase Binding Assay B Immunofluorescence Staining Assays B Growth Kinetics Analysis QUANTIFICATION AND STATISTICAL ANALYSIS DATA AND CODE AVAILABILITY

AUTHOR CONTRIBUTIONS G.F.G. and Y.S. conveyed the study. Y.X., W.Z., H.S., S.L., H.W., M.W., H.X., L.F., and Z.F. conducted the experiments. R.P. and J.Q. collected X-ray diffraction data and solved the structures. Y.X., R.P., and G.F.G. wrote the manuscript. H.S., H.X., Y.S., and G.F.G. participated in manuscript editing and discussion. G.F.G. supervised the study. DECLARATION OF INTERESTS The authors declare no competing interests. Received: September 7, 2018 Revised: August 23, 2019 Accepted: October 10, 2019 Published: November 19, 2019 REFERENCES Adams, P.D., Afonine, P.V., Bunko´czi, G., Chen, V.B., Davis, I.W., Echols, N., Headd, J.J., Hung, L.W., Kapral, G.J., Grosse-Kunstleve, R.W., et al. (2010). PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221. Bao, C.J., Cui, L.B., Zhou, M.H., Hong, L., Gao, G.F., and Wang, H. (2013). Live-animal markets and influenza A (H7N9) virus infection. N. Engl. J. Med. 368, 2337–2339. Bouvier, N.M., and Palese, P. (2008). The biology of influenza viruses. Vaccine 26 (Suppl 4), D49–D53. Chen, V.B., Arendall, W.B., 3rd, Headd, J.J., Keedy, D.A., Immormino, R.M., Kapral, G.J., Murray, L.W., Richardson, J.S., and Richardson, D.C. (2010). MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21. Claas, E.C., Osterhaus, A.D., van Beek, R., De Jong, J.C., Rimmelzwaan, G.F., Senne, D.A., Krauss, S., Shortridge, K.F., and Webster, R.G. (1998). Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus. Lancet 351, 472–477. Collaborative Computational Project (1994). The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763.

SUPPLEMENTAL INFORMATION

de Graaf, M., and Fouchier, R.A. (2014). Role of receptor binding specificity in influenza A virus transmission and pathogenesis. EMBO J. 33, 823–841.

Supplemental Information can be found online at https://doi.org/10.1016/j. celrep.2019.10.047.

de Vries, R.P., Peng, W., Grant, O.C., Thompson, A.J., Zhu, X., Bouwman, K.M., de la Pena, A.T.T., van Breemen, M.J., Ambepitiya Wickramasinghe, I.N., de Haan, C.A.M., et al. (2017). Three mutations switch H7N9 influenza to human-type receptor specificity. PLoS Pathog. 13, e1006390.

ACKNOWLEDGMENTS We thank the staff at SSRF beamline 17U/19U and the Consortium for Functional Glycomics (Scripps Research Institute, Department of Molecular Biology, La Jolla, CA) for their kind help with data collection and for kindly providing biotinylated 30 SLNLN and 6’SLNLN, respectively. This work was supported by the National Key Research and Development Program of China (2016YFC1200305); the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB29010292); the National Science and Technology Major Project (2018ZX10101004, 2018ZX10733403, 2015ZX09102024, and 2020ZX10001016-001); the Tianjin Key Technologies Research and Development Program (17YFZCSY01090); the National Natural Science Foundation of China (NSFC, 31771009); and the Emergency Technology Research Issue on Prevention and Control for Human Infection with A (H7N9) Avian Influenza Virus (10600100000015001206). G.F.G. is supported as a leading principal investigator of the NSFC Innovative Research Group (81621091). R.P. is supported by the Young Elite Scientist Sponsorship (YESS) Program of the China Association for Science and Technology (CAST; 2018QNRC001). Y.S. is supported by the Excellent Young Scientist Program of the NSFC (81622031), the Excellent Young Scientist Program of CAS, and the Youth Innovation Promotion Association of CAS (2015078).

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Edgar, R.C. (2004). MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797. Emsley, P., and Cowtan, K. (2004). Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132. Fouchier, R.A., Schneeberger, P.M., Rozendaal, F.W., Broekman, J.M., Kemink, S.A., Munster, V., Kuiken, T., Rimmelzwaan, G.F., Schutten, M., Van Doornum, G.J., et al. (2004). Avian influenza A virus (H7N7) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome. Proc. Natl. Acad. Sci. USA 101, 1356–1361. Gambaryan, A.S., Matrosovich, T.Y., Philipp, J., Munster, V.J., Fouchier, R.A., Cattoli, G., Capua, I., Krauss, S.L., Webster, R.G., Banks, J., et al. (2012). Receptor-binding profiles of H7 subtype influenza viruses in different host species. J. Virol. 86, 4370–4379. Gamblin, S.J., Haire, L.F., Russell, R.J., Stevens, D.J., Xiao, B., Ha, Y., Vasisht, N., Steinhauer, D.A., Daniels, R.S., Elliot, A., et al. (2004). The structure and receptor binding properties of the 1918 influenza hemagglutinin. Science 303, 1838–1842. Gao, G.F. (2018). From ‘‘A’’IV to ‘‘Z’’IKV: attacks from emerging and reemerging pathogens. Cell 172, 1157–1159.

Herfst, S., Schrauwen, E.J., Linster, M., Chutinimitkul, S., de Wit, E., Munster, V.J., Sorrell, E.M., Bestebroer, T.M., Burke, D.F., Smith, D.J., et al. (2012). Airborne transmission of influenza A/H5N1 virus between ferrets. Science 336, 1534–1541. Hoffmann, E., Stech, J., Guan, Y., Webster, R.G., and Perez, D.R. (2001). Universal primer set for the full-length amplification of all influenza A viruses. Arch. Virol. 146, 2275–2289. Huo, X., Cui, L., Chen, C., Wang, D., Qi, X., Zhou, M.-h., Guo, X., Wang, F., Liu, W.J., Kong, W., et al. (2018). Severe human infection with a novel avian-origin influenza A (H7N4) virus. Sci. Bull. (Beijing) 63, 1043–1050. Imai, M., and Kawaoka, Y. (2012). The role of receptor binding specificity in interspecies transmission of influenza viruses. Curr. Opin. Virol. 2, 160–167. Imai, M., Watanabe, T., Hatta, M., Das, S.C., Ozawa, M., Shinya, K., Zhong, G., Hanson, A., Katsura, H., Watanabe, S., et al. (2012). Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets. Nature 486, 420–428. Li, Q., Sun, X., Li, Z., Liu, Y., Vavricka, C.J., Qi, J., and Gao, G.F. (2012). Structural and functional characterization of neuraminidase-like molecule N10 derived from bat influenza A virus. Proc. Natl. Acad. Sci. USA 109, 18897– 18902. Liu, J., Xiao, H., Wu, Y., Liu, D., Qi, X., Shi, Y., and Gao, G.F. (2014). H7N9: a low pathogenic avian influenza A virus infecting humans. Curr. Opin. Virol. 5, 91–97. Lu, X., Shi, Y., Zhang, W., Zhang, Y., Qi, J., and Gao, G.F. (2013). Structure and receptor-binding properties of an airborne transmissible avian influenza A virus hemagglutinin H5 (VN1203mut). Protein Cell 4, 502–511. Lv, X., Cao, H., Lin, B., Wang, W., Zhang, W., Duan, Q., Tao, Y., Liu, X.W., and Li, X. (2017). Synthesis of sialic acids, their derivatives, and analogs by using a whole-cell catalyst. Chemistry 23, 15143–15149. Matrosovich, M., Tuzikov, A., Bovin, N., Gambaryan, A., Klimov, A., Castrucci, M.R., Donatelli, I., and Kawaoka, Y. (2000). Early alterations of the receptorbinding properties of H1, H2, and H3 avian influenza virus hemagglutinins after their introduction into mammals. J. Virol. 74, 8502–8512. Medina, R.A., and Garcı´a-Sastre, A. (2011). Influenza A viruses: new research developments. Nat. Rev. Microbiol. 9, 590–603. Murshudov, G.N., Vagin, A.A., and Dodson, E.J. (1997). Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255. Otwinowski, Z., and Minor, W. (1997). Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326. Parthasarathy, S., and Murthy, M.R.N. (2000). Protein thermal stability: insights from atomic displacement parameters (B values). Protein Eng. 13, 9–13. Peiris, M., Yuen, K.Y., Leung, C.W., Chan, K.H., Ip, P.L., Lai, R.W., Orr, W.K., and Shortridge, K.F. (1999). Human infection with influenza H9N2. Lancet 354, 916–917. Pettersen, E.F., Goddard, T.D., Huang, C.C., Couch, G.S., Greenblatt, D.M., Meng, E.C., and Ferrin, T.E. (2004). UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612. Qi, W., Zhou, X., Shi, W., Huang, L., Xia, W., Liu, D., Li, H., Chen, S., Lei, F., Cao, L., et al. (2014). Genesis of the novel human-infecting influenza A(H10N8) virus and potential genetic diversity of the virus in poultry, China. Euro Surveill. 19, 50–62. Qi, W., Jia, W., Liu, D., Li, J., Bi, Y., Xie, S., Li, B., Hu, T., Du, Y., Xing, L., et al. (2018). Emergence and adaptation of a novel highly pathogenic H7N9 influenza virus in birds and humans from a 2013 human-infecting low-pathogenic ancestor. J. Virol. 92, 1–17. Quan, C., Shi, W., Yang, Y., Yang, Y., Liu, X., Xu, W., Li, H., Li, J., Wang, Q., Tong, Z., et al. (2018). New threads of H7N9 influenza virus: the spread and evolution of highly and low pathogenic variants with high genomic diversity in Wave Five. J. Virol. 92, 1–18. https://doi.org/10.1128/JVI.00301-18. Read, R.J. (2001). Pushing the boundaries of molecular replacement with maximum likelihood. Acta Crystallogr. D Biol. Crystallogr. 57, 1373–1382.

Reed, L.J., and Muench, H. (1938). A simple method of estimating fifty per cent endpoints. Am. J. Epidemiol. 27, 493–497. Rogers, G.N., Paulson, J.C., Daniels, R.S., Skehel, J.J., Wilson, I.A., and Wiley, D.C. (1983). Single amino acid substitutions in influenza haemagglutinin change receptor binding specificity. Nature 304, 76–78. Shi, Y., Zhang, W., Wang, F., Qi, J., Wu, Y., Song, H., Gao, F., Bi, Y., Zhang, Y., Fan, Z., et al. (2013). Structures and receptor binding of hemagglutinins from human-infecting H7N9 influenza viruses. Science 342, 243–247. Shi, Y., Wu, Y., Zhang, W., Qi, J., and Gao, G.F. (2014). Enabling the ‘host jump’: structural determinants of receptor-binding specificity in influenza A viruses. Nat. Rev. Microbiol. 12, 822–831. Song, H., Qi, J., Xiao, H., Bi, Y., Zhang, W., Xu, Y., Wang, F., Shi, Y., and Gao, G.F. (2017). Avian-to-human receptor-binding adaptation by influenza a virus hemagglutinin H4. Cell Rep. 20, 1201–1214. Stevens, J., Blixt, O., Tumpey, T.M., Taubenberger, J.K., Paulson, J.C., and Wilson, I.A. (2006). Structure and receptor specificity of the hemagglutinin from an H5N1 influenza virus. Science 312, 404–410. Sun, Y., Qin, K., Wang, J., Pu, J., Tang, Q., Hu, Y., Bi, Y., Zhao, X., Yang, H., Shu, Y., and Liu, J. (2011). High genetic compatibility and increased pathogenicity of reassortants derived from avian H9N2 and pandemic H1N1/2009 influenza viruses. Proc. Natl. Acad. Sci. USA 108, 4164–4169. Sun, X., Shi, Y., Lu, X., He, J., Gao, F., Yan, J., Qi, J., and Gao, G.F. (2013). Batderived influenza hemagglutinin H17 does not bind canonical avian or human receptors and most likely uses a unique entry mechanism. Cell Rep. 3, 769–778. Sun, H., Pu, J., Wei, Y., Sun, Y., Hu, J., Liu, L., Xu, G., Gao, W., Li, C., Zhang, X., et al. (2016). Highly pathogenic avian influenza H5N6 viruses exhibit enhanced affinity for human type sialic acid receptor and in-contact transmission in model ferrets. J. Virol. 90, 6235–6243. Sutton, T.C., Finch, C., Shao, H., Angel, M., Chen, H., Capua, I., Cattoli, G., Monne, I., and Perez, D.R. (2014). Airborne transmission of highly pathogenic H7N1 influenza virus in ferrets. J. Virol. 88, 6623–6635. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., and Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739. Taubenberger, J.K., and Kash, J.C. (2010). Influenza virus evolution, host adaptation, and pandemic formation. Cell Host Microbe 7, 440–451. Tweed, S.A., Skowronski, D.M., David, S.T., Larder, A., Petric, M., Lees, W., Li, Y., Katz, J., Krajden, M., Tellier, R., et al. (2004). Human illness from avian influenza H7N3, British Columbia. Emerg. Infect. Dis. 10, 2196–2199. Wang, M., Zhang, W., Qi, J., Wang, F., Zhou, J., Bi, Y., Wu, Y., Sun, H., Liu, J., Huang, C., et al. (2015). Structural basis for preferential avian receptor binding by the human-infecting H10N8 avian influenza virus. Nat. Commun. 6, 5600. Watanabe, Y., Ibrahim, M.S., Ellakany, H.F., Kawashita, N., Mizuike, R., Hiramatsu, H., Sriwilaijaroen, N., Takagi, T., Suzuki, Y., and Ikuta, K. (2011). Acquisition of human-type receptor binding specificity by new H5N1 influenza virus sublineages during their emergence in birds in Egypt. PLoS Pathog. 7, e1002068. Webster, R.G., Laver, W.G., Air, G.M., and Schild, G.C. (1982). Molecular mechanisms of variation in influenza viruses. Nature 296, 115–121. Wiley, D.C., and Skehel, J.J. (1987). The structure and function of the hemagglutinin membrane glycoprotein of influenza virus. Annu. Rev. Biochem. 56, 365–394. Wu, Y., Bi, Y., Vavricka, C.J., Sun, X., Zhang, Y., Gao, F., Zhao, M., Xiao, H., Qin, C., He, J., et al. (2013). Characterization of two distinct neuraminidases from avian-origin human-infecting H7N9 influenza viruses. Cell Res. 23, 1347–1355. Wu, Y., Wu, Y., Tefsen, B., Shi, Y., and Gao, G.F. (2014). Bat-derived influenzalike viruses H17N10 and H18N11. Trends Microbiol. 22, 183–191. Xiong, X., Coombs, P.J., Martin, S.R., Liu, J., Xiao, H., McCauley, J.W., Locher, K., Walker, P.A., Collins, P.J., Kawaoka, Y., et al. (2013a). Receptor binding by a ferret-transmissible H5 avian influenza virus. Nature 497, 392–396.

Cell Reports 29, 2217–2228, November 19, 2019 2227

Xiong, X., Martin, S.R., Haire, L.F., Wharton, S.A., Daniels, R.S., Bennett, M.S., McCauley, J.W., Collins, P.J., Walker, P.A., Skehel, J.J., and Gamblin, S.J. (2013b). Receptor binding by an H7N9 influenza virus from humans. Nature 499, 496–499. Xiong, X., McCauley, J.W., and Steinhauer, D.A. (2014). Receptor binding properties of the influenza virus hemagglutinin as a determinant of host range. Curr. Top. Microbiol. Immunol. 385, 63–91. Xu, R., McBride, R., Paulson, J.C., Basler, C.F., and Wilson, I.A. (2010). Structure, receptor binding, and antigenicity of influenza virus hemagglutinins from the 1957 H2N2 pandemic. J. Virol. 84, 1715–1721. Xu, R., McBride, R., Nycholat, C.M., Paulson, J.C., and Wilson, I.A. (2012). Structural characterization of the hemagglutinin receptor specificity from the 2009 H1N1 influenza pandemic. J. Virol. 86, 982–990.

Yu, X., Jin, T., Cui, Y., Pu, X., Li, J., Xu, J., Liu, G., Jia, H., Liu, D., Song, S., et al. (2014). Influenza H7N9 and H9N2 viruses: coexistence in poultry linked to human H7N9 infection and genome characteristics. J. Virol. 88, 3423–3431. Yuan, J., Zhang, L., Kan, X., Jiang, L., Yang, J., Guo, Z., and Ren, Q. (2013). Origin and molecular characteristics of a novel 2013 avian influenza A(H6N1) virus causing human infection in Taiwan. Clin. Infect. Dis. 57, 1367–1368. Zhang, W., Qi, J., Shi, Y., Li, Q., Gao, F., Sun, Y., Lu, X., Lu, Q., Vavricka, C.J., Liu, D., et al. (2010). Crystal structure of the swine-origin A (H1N1)-2009 influenza A virus hemagglutinin (HA) reveals similar antigenicity to that of the 1918 pandemic virus. Protein Cell 1, 459–467. Zhang, W., Shi, Y., Lu, X., Shu, Y., Qi, J., and Gao, G.F. (2013a). An airborne transmissible avian influenza H5 hemagglutinin seen at the atomic level. Science 340, 1463–1467.

Xu, R., de Vries, R.P., Zhu, X., Nycholat, C.M., McBride, R., Yu, W., Paulson, J.C., and Wilson, I.A. (2013). Preferential recognition of avian-like receptors in human influenza A H7N9 viruses. Science 342, 1230–1235.

Zhang, W., Shi, Y., Qi, J., Gao, F., Li, Q., Fan, Z., Yan, J., and Gao, G.F. (2013b). Molecular basis of the receptor binding specificity switch of the hemagglutinins from both the 1918 and 2009 pandemic influenza A viruses by a D225G substitution. J. Virol. 87, 5949–5958.

Yang, H., Chen, L.M., Carney, P.J., Donis, R.O., and Stevens, J. (2010). Structures of receptor complexes of a North American H7N2 influenza hemagglutinin with a loop deletion in the receptor binding site. PLoS Pathog. 6, e1001081.

Zhang, F., Bi, Y., Wang, J., Wong, G., Shi, W., Hu, F., Yang, Y., Yang, L., Deng, X., Jiang, S., et al. (2017). Human infections with recently-emerging highly pathogenic H7N9 avian influenza virus in China. J. Infect. 75, 71–75.

2228 Cell Reports 29, 2217–2228, November 19, 2019

STAR+METHODS KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Primary mouse monoclonal antibody against His tag

ZSGB-BIO

Cat# TA-02; RRID:AB_2801388

Goat anti-rabbit IgG/HRP

ZSGB-BIO

Cat# ZB-2301; RRID:AB_2747412

Goat anti-mouse IgG/HRP

ZSGB-BIO

Cat# ZB-2305; RRID:AB_2747415

Influenza H1N1 (A/California/04/2009) Hemagglutinin / HA Antibody, Rabbit mAb

Sino biological

Cat# 11055-RM10-100 mg

Fluorescein-Conjugated Goat anti-Mouse IgG (H+L)

ZSGB-BIO

Cat# ZF-0312; RRID:AB_2716306

Antibodies

Bacterial and Virus Strains Escherichia coli (E. coli) DH5a strain

TIANGEN

Cat# CB101-02

MAX Efficiency DH10Bac Competent Cells

Invitrogen

Cat# 10361-012

Virus: A/California/04/2009 (H1N1)

Shi et al., 2013

N/A

Virus: A/Anhui/1/2005 (H5N1)

Shi et al., 2013

N/A

Virus: A/Anhui/1/2013 (H7N9)

Shi et al., 2013

N/A

Virus: A/Shanghai/1/2013 (H7N9)

Shi et al., 2013

N/A

Biological Samples Human: Human tracheal sections

Auragene Bioscience Corporation

Cat# N0100

Duck: Duck duodenum tissue sections

Auragene Bioscience Corporation

Cat# N0016

Chemicals, Peptides, and Recombinant Proteins DMEM basic

Thermo Fisher Scientific

Cat# C11995500BT

FBS

Invitrogen

Cat# 10270106

Insect-XPRESS

LONZA

Cat# 12-730Q

Lipofectamine 2000

Invitrogen

Cat# 11668019

TPCK-trypsin

Worthington

Cat# LS003740

DAPI

Beyotime

Cat# C1002

Thrombin

Sigma

Cat# T4648

30 SLN (Neu5Aca2-3-Galb1-4-GlcNAcb1propyn)

Lv et al., 2017

N/A

6’SLN (Neu5Aca2-6-Galb1-4-GlcNAcb1propyn)

Lv et al., 2017

N/A

Biotinylated 30 SLNLN (NeuAca2-3Galb14GlcNAcb1-3Galb1-4GlcNAcb1-SpNHLC-LC-Biotin)

Consortium for Functional Glycomics

N/A

Biotinylated 6’SLNLN (NeuAca2-6Galb14GlcNAcb1-3Galb1-4Glc-NAcb1-SpNHLC-LC-Biotin)

Consortium for Functional Glycomics

N/A

pFastBac1-AH1-H7N9 HA, GISAID isolate ID: EPI_ISL_138739

Shi et al., 2013

N/A

pFastBac1-SH1-H7N9 HA, GISAID isolate ID: EPI_ISL_138737

Shi et al., 2013

N/A

Critical Commercial Assays Mut ExpressIIFast Mutagenesis Kit V2

Vazyme Biotech

Cat# C214-02

HisTrap HP 5 ml column

GE Healthcare

Cat# 17524802 (Continued on next page)

Cell Reports 29, 2217–2228.e1–e5, November 19, 2019 e1

Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER

RESOURCE Q, 6 ML

GE Healthcare

Cat# 17117901

Hiload 16/600 Superdex 200 PG column

GE Healthcare

Cat# 28989335

Streptavidin Coated Plates (HBC), 96-well

Pierce

Cat# 15500

Crystallization kits

Hampton Research and Molecular Dimensions

https://www.hamptonresearch.com/ https://www.moleculardimensions.com/

Sensor Chip SA

GE Healthcare

Cat# BR100032

Crystal structure of H7 hemagglutinin mutant AH-SGTQ

This paper

PDB: 6ICW

Crystal structure of H7 hemagglutinin mutant H7-AGPL

This paper

PDB: 6ICX

Crystal structure of H7 hemagglutinin mutant H7-AGTL

This paper

PDB: 6ICY

Crystal structure of H7 hemagglutinin mutant H7-AVTL

This paper

PDB: 6ID2

Crystal structure of H7 hemagglutinin mutant H7-SGPL

This paper

PDB: 6ID3

Crystal structure of H7 hemagglutinin mutant H7-SGTL

This paper

PDB: 6ID9

Crystal structure of H7 hemagglutinin mutant H7-SVPL

This paper

PDB: 6ID5

Crystal structure of H7 hemagglutinin mutant H7-SVTL

This paper

PDB: 6ID8

Crystal structure of H7 hemagglutinin mutant H7-SVTQ

This paper

PDB: 6IDA

Crystal structure of H7 hemagglutinin mutant H7-SVTQ with 30 SLN

This paper

PDB: 6IDZ

Crystal structure of H7 hemagglutinin mutant H7-SVTQ with 6’SLN

This paper

PDB: 6IDB

Crystal structure of H7 hemagglutinin mutant SH1-AVPL

This paper

PDB: 6IDD

Sf9 Cells, SFM Adapted

Invitrogen

Cat# 11496015

High Five cells

Invitrogen

Cat# B85502

HEK293T cells

ATCC

Cat# ATCC CRL-3216

MDCK cells

ATCC

Cat# ATCC CCL-34

Primers for Reverse Transcription: U12: AGCAAAAGCAGG

Hoffmann et al., 2001

N/A

Forward primers for HA amplication: Bm-HA-1: TATTCGTCTCAG GGAGCAAAAGCAGGGG

Hoffmann et al., 2001

N/A

Reverse primers for HA amplication: Bm-NS-890R: ATATCGTCTCGTATTAG TAGAAACAAGGGTGTTTT

Hoffmann et al., 2001

N/A

pFastBac1

Invitrogen

Cat# 10359016

pFastBac1-H7N9_HA, various mutants

This paper

N/A

MEGA program (version 5.1)

Tamura et al., 2011

N/A

FigTree (v1.4.3)

Graphical viewer of phylogenetic trees

http://tree.bio.ed.ac.uk/

Deposited Data

Experimental Models: Cell Lines

Oligonucleotides

Recombinant DNA

Software and Algorithms

(Continued on next page)

e2 Cell Reports 29, 2217–2228.e1–e5, November 19, 2019

Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER N/A

MUSCLE

Edgar, 2004

BIAcore 3000 Evaluation software

GE Healthcare

N/A

Graphpad Prism 6

GraphPad Software

https://www.graphpad.com/ scientificsoftware/prism/ N/A

HKL2000

Otwinowski and Minor, 1997

PHASER

Read, 2001

http://www.ccp4.ac.uk/html/phaser.html

CCP4 program suit

Collaborative Computational Project

http://www.ccp4.ac.uk/

REFMAC5

Murshudov et al., 1997

http://www.ccp4.ac.uk/dist/html/ refmac5.html

PyMOL software

DeLano WL PyMOL molecular graphics system

https://pymol.org/2/

Chimera

Pettersen et al., 2004

N/A

Coot

Emsley and Cowtan, 2004

https://www2.mrc-lmb.cam.ac.uk/ personal/pemsley/coot/

Phenix

Adams et al., 2010

http://www.phenix-online.org/

MolProbity

Chen et al., 2010

https://www.phenix-online.org/ documentation/tutorials/molprobity.html

Leica TCS SP8 laser scanning confocal microscopy

Leica

https://www.leica-microsystems.com

Influenza virus resource

Influenza Research Database

https://www.fludb.org/brc/home.spg? decorator=influenza

Influenza virus resource

Global Initiative on Sharing All Influenza Data

http://platform.gisaid.org/epi2/

Other

LEAD CONTACT AND MATERIALS AVAILABILITY This study did not generate new unique reagents. Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, George F. Gao ([email protected]). EXPERIMENTAL MODEL AND SUBJECT DETAILS Escherichia coli DH5a strain (CB101-02) and MAX Efficiency DH10Bac Competent cells (10361-012) were incubated in LB medium with corresponding antibiotics at 37 C in a shaking incubator (180 rpm). Adherent cultured HEK293T (ATCC CRL-3216) and MDCK (ATCC CCL-34) cells were cultured at 37 C in Dulbecco’s Modified Eagle medium (DMEM) supplemented containing 10% fetal bovine serum (FBS) with 5% CO2. Sf9 (11496015) and High Five (B85502) cells were cultured at 27 C in Insect-XPRESS (12-730Q) with continuous shaking (120
130 rpm). METHOD DETAILS Protein Expression and Purification The codon-optimized coding sequences for the ectodomain of the AH1-H7N9 HA (A/Anhui/1/2013/H7N9, Global Initiative on Sharing All Influenza Data (GISAID: EPI_ISL_138739) and SH1-H7N9 HA (A/Shanghai/1/2013/H7N9, GISAID: EPI_ISL_138737) were synthesized by GENWIZ company and incorporated into the pFastBacTM 1(Invitrogen) plasmid for protein production using the Bac-to-Bac baculovirus expression system as previous described (Li et al., 2012; Zhang et al., 2010). The proteins were fused with an N-terminal gp67 secretive signal peptide, a thrombin-cleavable C-terminal trimerization motif and a 6 3 His affinity tag to facilitate protein expression and purification. The HA mutant expression plasmids were generated by Mut Express II Fast Mutagenesis Kit V2 based on the wild-type plasmids (Vazyme Biotech Co., Ltd). Both the wild-type HA and mutant proteins were expressed using Hi5 cells (Invitrogen) as soluble proteins and were captured from the cell culture supernatant by immobilized metal affinity chromatography (IMAC) with a HisTrap HP 5 mL column (GE Healthcare). The eluted fractions were pooled and further purified by ion-exchange chromatography using a RESOURCETM Q 6 mL column (GE Healthcare). After overnight thrombin-cleavage at 4 C, the proteins were subjected to size-exclusion chromatography (SEC) with a Hiload Superdex 200 16/600 pg column (GE Healthcare) equilibrated

Cell Reports 29, 2217–2228.e1–e5, November 19, 2019 e3

with a buffer containing 20mM Tris-HCl (pH8.0), 50mM NaCl for final purification. The resulting products reached a purity of more than 95% and were concentrated to 8 mg/mL for crystal screen. Crystallization, Data Collection and Structure Determination Initial crystallization trials were performed at 18 C using the sitting drop vapor-diffusion method by mixing 1 mL protein with 1 mL reservoir solution. Crystals were observed in many conditions containing PEG with various shapes (Table S3). For the complexes structure with receptor analog, high resolution crystals were soaked in a reservoir solution containing 7 mM 30 LSN or 50 LSN (Lv et al., 2017) for 5 h. The crystals were cryo-protected and flash-cooled in liquid nitrogen for data collection at Shanghai Synchrotron Radiation Facilities (SSRF: 17U/19U). All diffraction data were processed with HKL2000 software (Otwinowski and Minor, 1997) and the structures were determined by molecular replacement method with PHASER (Read, 2001) in the CCP4 program suit (Collaborative Computational Project, 1994) using the structure of wild-type AH1-H7N9 HA (PDB: 4KOL) as the search model. Initial rigid body rigid body refinement was performed using REFMAC5 (Murshudov et al., 1997). Iterative rounds of manual model building and refinement were further carried out using COOT (Emsley and Cowtan, 2004) and PHENIX (Adams et al., 2010) with energy minimization, isotropic ADP refinement and bulk solvent modeling. The stereochemical quality of final models was assessed with MOLPROBITY (Chen et al., 2010). All statistics for data collection and refinement are summarized in Table S4. All structural figures were generated with PyMOL (http://pymol. org/2) or Chimera (Pettersen et al., 2004). The normalized B factors of 220-loop and residue 226 for each structure were calculated as previous mentioned (Parthasarathy and Murthy, 2000). SPR Experiments The affinities and kinetics of HAs binding to avian and human receptor analogs were analyzed using the BIAcore 3000 system at 25 C using a streptavidin chip (SA chip, GE Healthcare). The flowing buffer system was PBS supplemented with 0.005% Tween-20 (PBST). Two biotinylated sialic acid receptor analogs, 6’SLNLN and 30 SLNLN, were immobilized on the chip, and a blank channel was set as the negative control. Association of the HA analytes (0 mM, 0.125 mM, 0.25 mM, 0.5 mM, 1 mM, 2 mM, 4 mM, and 8 mM) was measured at a flow rate of 30 mL/min. The resulting SPR sensorgrams were corrected with the blank (0 mM analyte) curves, and fitted globally with a 1:1 Langmuir binding model using the BIAevaluation (version 4.1) software. The affinity values were calculated with a simultaneous kinetic Ka (association rate) / Kd (dissociation rate) model. Phylogenetic Analysis The H7 HA sequences used for phylogenetic tree construct were obtained from GISAID (https://www.gisaid.org), then aligned by MUSCLE (Edgar, 2004) algorithm in influenza research database (IRD, http://www.fludb.org/brc/home.spg?decorator=influenza ). Phylogenetic trees were constructed by neighbor-joining method using the MEGA program (version 5.1) (Tamura et al., 2011) and decorated by FigTree (v1.4.3). The sequence edit was also performed by MEGA program to remove short or ambigious sequence. Virus Preparation The experiments were performed in the approved bio-safety level-3 (BSL-3) laboratory. The AH1-/SH1-H7N9 and mutant viruses were generated by plasmid-based reverse genetics technology (Sun et al., 2011; Wu et al., 2013) using the six internal genes of PR8/H1N1 strain as the backbone. The rescued viruses were named by rAH1-H7N9, rH7-AVPQ, rH7-AGPL, rH7-AGPQ, and rSH1-H7N9. All rescued viruses were verified by sequencing (Hoffmann et al., 2001). The CA04-H1N1 (A/California/04/2009 (H1N1)) and AH05-H5N1 (A/Anhui/1/2005 (H5N1)) are original virus isolates stocked in our laboratory. All virus stocks were propagated in MDCK cells and the concentrations were determined by hemagglutination assays with 1% (vol/vol) chicken red blood cells. Solid-phase Binding Assay The experiments were performed in the approved BSL-3 laboratory following the protocol as previously described (Wang et al., 2015; Watanabe et al., 2011). Briefly, serial dilutions (0.15625, 0.3125, 0.625, 1.25, 2.5 and 5 mg/mL) of biotinylated glycans 30 SLNLN and 6’SLNLN were prepared in PBS, and 50 mL was added to the wells of the streptavidin-coated high binding capacity 96-well plates (Pierce). After incubation overnight at 4 C, the plates were washed with PBST (PBS containing 0.05% Tween-20), and blocked with PBS containing 2% bovine serum albumin (BSA) at room temperature for 2 h, then washed with PBST again. 32 HA units different influenza viruses (prepared in PBST containing 10 mM oseltamivir and 10 mM zanamivir) were added to the wells and incubated overnight at 4 C. After washing with PBST, the wells were incubated with rabbit antisera against AH1-H7N9 HA, CA04-H1N1 HA or AH05-H5N1 HA, separately, at 4 C for 5 h. The wells were then washed with PBST and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody at 4 C for 2 h. After extensive washes with PBST, the plates were incubated with 3,30 ,5,50 -tetramethylbenzidine (TMB) solution at room temperature for 6 min at room temperature, and the reaction was stopped with 2 M H2SO4. The absorbance was measured at 450 nm. Immunofluorescence Staining Assays Paraffin-embedded human tracheal and duck duodenum tissue sections were purchased from Auragene Bioscience Corporation (China) with an ethics statement provided. Paraffinized human and duck duodenum tissue sections were deparaffinized with xylene,

e4 Cell Reports 29, 2217–2228.e1–e5, November 19, 2019

rehydrated using graded ethanol and incubated with 1% BSA in PBS for 1 hour at room temperature (RT) to prevent nonspecific binding. Purified HA with trimerization foldon sequence and His6-tag was precomplexed with primary antibody (mouse anti-His-tag, ZSGB-BIO) and secondary antibody (Fluorescein-conjugated goat anti-mouse IgG, ZSGB-BIO) in a molar ratio of 4:2:1, respectively, for 30 min on ice. Tissue sections were then incubated with the HA-antibody complexes overnight at 4 C. Sections were then washed and counterstained with DAPI (Beyotime; 1:1,000 in PBS) for nuclei for 10 min at RT. After thorough washing, the tissue sections were mounted and then examined by using confocal laser scanning microscopy (Leica TCS SP8 laser scanning confocal microscopy). Growth Kinetics Analysis MDCK cells were inoculated at an MOI of 0.005. Supernatants were collected at 6 h, 12 h, 24 h, 36 h, 48 h, 60 h and 72 h and titrated by 50% tissue culture infectious dose (TCID50) in MDCK cells using the Reed-Muench method (Reed and Muench, 1938). QUANTIFICATION AND STATISTICAL ANALYSIS Data are presented as the means ± SD and were analyzed with GraphPad Prism 6. The independent-samples t test was used for analysis, and p values less than 0.05 were considered significant. Statistical differences are indicated as (*p < 0.05; **p < 0.01). Biological replicates are indicated in the figure legends. DATA AND CODE AVAILABILITY The accession numbers for the atomic coordinates and structure factors of H7N9 subtype HAs and mutants alone or in complex with receptors reported in this paper are Protein Data Bank (PDB): 6ICW, 6ICX, 6ICY, 6ID2, 6ID3, 6ID9, 6ID5, 6ID8, 6IDA, 6IDZ, 6IDB, 6IDD.

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