Diversity of Functionally Permissive Sequences in the Receptor-Binding Site of Influenza Hemagglutinin.


Abstract

Influenza A virus hemagglutinin (HA) initiates viral entry by engaging host receptor sialylated glycans via its receptor-binding site (RBS). The amino acid sequence of the RBS naturally varies across avian and human influenza virus subtypes and is also evolvable. However, functional sequence diversity in the RBS has not been fully explored. Here, we performed a large-scale mutational analysis of the RBS of A/WSN/33 (H1N1) and A/Hong Kong/1/1968 (H3N2) HAs. Many replication-competent mutants not yet observed in nature were identified, including some that could escape from an RBS-targeted broadly neutralizing antibody. This functional sequence diversity is made possible by pervasive epistasis in the RBS 220-loop and can be buffered by avidity in viral receptor binding. Overall, our study reveals that the HA RBS can accommodate a much greater range of sequence diversity than previously thought, which has significant implications for the complex evolutionary interrelationships between receptor specificity and immune escape.

Submission Details

ID: aSy5yPFi

Submitter: Marie Ary

Submission Date: June 22, 2018, 9:49 a.m.

Version: 1

Publication Details
Wu NC;Xie J;Zheng T;Nycholat CM;Grande G;Paulson JC;Lerner RA;Wilson IA,Cell Host Microbe (2017) Diversity of Functionally Permissive Sequences in the Receptor-Binding Site of Influenza Hemagglutinin. PMID:28618270
Additional Information

Study Summary

Number of data points 20882
Proteins Hemagglutinin A/HK68 ; Hemagglutinin A/WSN/33
Unique complexes 18725
Assays/Quantities/Protocols Experimental Assay: log10 TCID50, HK68 mutants ; Experimental Assay: HA RBS function by RF index, WSN ; Experimental Assay: Kd S139/1 IgG ; Experimental Assay: Kd1 3'-SLNLN ; Experimental Assay: HA RBS function by RF index, HK68 ; Experimental Assay: log2 HA titer, WSN mutants ; Experimental Assay: log10 TCID50, WSN mutants ; Experimental Assay: Kd1 6'-SLNLN ; Experimental Assay: Kd2 6'-SLNLN ; Experimental Assay: log2 HA titer, HK68 mutants ; Experimental Assay: Kd C05 IgG ; Experimental Assay: Kd2 3'-SLNLN ; Experimental Assay: koff1 3'-SLNLN ; Experimental Assay: koff2 3'-SLNLN ; Experimental Assay: Kd 3'-SLNLN-PAA ; Experimental Assay: Kd 6'-SLNLN-PAA ; Computational Protocol: R^2 (goodness of fit), 3'-SLNLN binding ; Computational Protocol: R^2 (goodness of fit), 3'-SLNLN-PAA binding ; Computational Protocol: R^2 (goodness of fit), 6'-SLNLN binding ; Computational Protocol: R^2 (goodness of fit), 6'-SLNLN-PAA binding ; Computational Protocol: R^2 (goodness of fit), C05 IgG binding ; Computational Protocol: R^2 (goodness of fit), S139/1 IgG binding
Libraries Kds of HK68 HA WT and escape mutant for binding to bnAb S139/1 IgG (Figure S4, B-C) ; Off-rates of glycan 3'-SLNLN with WSN recombinant HA variants (Table S4_WSN) ; Kds for binding polymerized glycoconjugates (3'-SLNLN-PAA and 6'-SLNLN-PAA) to HK68 HA variants (Table S5_HK68) ; Kds for binding of glycans 3'-SLNLN and 6'-SLNLN to HK68 HA variants (Table S3_HK68) ; Kds for binding of glycans 3'-SLNLN and 6'-SLNLN to WSN HA variants (Table S3_WSN) ; Kds for binding of bnAbs C05 IgG and S139/1 IgG to WSN HA variants (Table S1) ; Validation of HK68 deep scan data by virus rescue and hemagglutination assays (log10 TCID50, log2 HA Titer) (Data S1_HK68) ; Validation of WSN deep scan data by virus rescue and hemagglutination assays (log10 TCID50, log2 HA Titer) (Data S1_WSN) ; Relative fitness index for single, double, and triple mutants in HK68 HA 220 loop (residues 225, 226, 228) by deep mutational scanning (Data S2_HK68) ; Relative fitness index for single, double, and triple mutants in WSN HA RBS (11 residues) by deep mutational scanning (Data S2)

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
2DCI 2006-01-24 NMR structure of influenza HA fusion peptide mutant W14A in DPC in pH5
1IBN 2001-08-08 NMR STRUCTURE OF HEMAGGLUTININ FUSION PEPTIDE IN DPC MICELLES AT PH 5
1IBO 2001-08-08 NMR STRUCTURE OF HEMAGGLUTININ FUSION PEPTIDE IN DPC MICELLES AT PH 7.4
5VLI 2017-09-27 1.8 Computationally designed inhibitor peptide HB1.6928.2.3 in complex with influenza hemagglutinin (A/PuertoRico/8/1934)
1QU1 2000-01-05 1.9 CRYSTAL STRUCTURE OF EHA2 (23-185)
4FNK 2012-09-19 1.9 Crystal structure of the A/Hong Kong/1/1968 (H3N2) influenza virus hemagglutinin
5UMN 2017-05-24 1.97 Crystal structure of C05 VPGSGW mutant bound to H3 influenza hemagglutinin, HA1 subunit
2G9H 2006-07-11 2.0 Crystal Structure of Staphylococcal Enterotoxin I (SEI) in Complex with a Human MHC class II Molecule
6E56 2019-05-22 2.0 Human antibody H2214 in complex with influenza hemagglutinin A/Aichi/2/1968 (X-31) (H3N2)
6OCB 2019-05-29 2.1 Crystal structure of FluA-20 Fab in complex with the head domain of H3 (A/Hong Kong/1/1968)
4WE5 2015-02-11 2.1 The crystal structure of hemagglutinin from A/Port Chalmers/1/1973 influenza virus
3S5L 2011-09-21 2.1 Crystal structure of CD4 mutant bound to HLA-DR1
1PYW 2003-12-09 2.1 Human class II MHC protein HLA-DR1 bound to a designed peptide related to influenza virus hemagglutinin, FVKQNA(MAA)AL, in complex with staphylococcal enterotoxin C3 variant 3B2 (SEC3-3B2)
5VTZ 2017-06-28 2.15 Crystal structure of the A/Hong Kong/1/1968 (H3N2) influenza virus hemagglutinin G225Q/L226A mutant apo form
6BKM 2018-02-28 2.2 Crystal structure of the A/Hong Kong/1/1968 (H3N2) influenza virus hemagglutinin E190D mutant apo form
1RVX 2004-03-30 2.2 1934 H1 Hemagglutinin in complex with LSTA
6BKQ 2018-02-28 2.25 Crystal structure of the A/Hong Kong/1/1968 (H3N2) influenza virus hemagglutinin E190D mutant in complex with 6'-SLN
5VTV 2017-06-28 2.25 Crystal structure of the A/Hong Kong/1/1968 (H3N2) influenza virus hemagglutinin G225M/L226T/S228A mutant in complex with 3'-SLN
5VU4 2017-06-28 2.25 Crystal structure of the A/Hong Kong/1/1968 (H3N2) influenza virus hemagglutinin G225Q/L226A mutant in complex with 6'-SLN
1RVZ 2004-03-30 2.25 1934 H1 Hemagglutinin in complex with LSTC
5W5S 2017-10-04 2.28 Crystal structure of the A/Puerto Rico/8/1934 (H1N1) influenza virus hemagglutinin in complex with cyclic peptide CP141019 (P5)
1RU7 2004-03-30 2.3 1934 Human H1 Hemagglutinin
2XN9 2010-11-24 2.3 Crystal structure of the ternary complex between human T cell receptor, staphylococcal enterotoxin H and human major histocompatibility complex class II
4WE4 2015-02-11 2.35 The crystal structure of hemagglutinin from 1968 H3N2 influenza virus
5VTW 2017-06-28 2.35 Crystal structure of the A/Hong Kong/1/1968 (H3N2) influenza virus hemagglutinin G225M/L226T/S228A mutant in complex with 6'-SLN
5VTY 2017-06-28 2.36 Crystal structure of the A/Hong Kong/1/1968 (H3N2) influenza virus hemagglutinin G225Q/L226A mutant in complex with 3'-SLN
3S4S 2011-09-21 2.4 Crystal structure of CD4 mutant bound to HLA-DR1
1J8H 2002-03-13 2.4 Crystal Structure of a Complex of a Human alpha/beta-T cell Receptor, Influenza HA Antigen Peptide, and MHC Class II Molecule, HLA-DR4
5VTU 2017-06-28 2.45 Crystal structure of the A/Hong Kong/1/1968 (H3N2) influenza virus hemagglutinin G225L/L226S mutant apo form
5W5U 2017-10-04 2.46 Crystal structure of the A/Puerto Rico/8/1934 (H1N1) influenza virus hemagglutinin in complex with cyclic peptide CP141037 (P4)
5VTR 2017-06-28 2.5 Crystal structure of the A/Hong Kong/1/1968 (H3N2) influenza virus hemagglutinin G225L/L226S mutant in complex with 6'-SLN
1HTM 1995-02-14 2.5 STRUCTURE OF INFLUENZA HAEMAGGLUTININ AT THE PH OF MEMBRANE FUSION
5K9Q 2016-11-30 2.5 Crystal structure of multidonor HV1-18-class broadly neutralizing Influenza A antibody 16.a.26 in complex with A/Hong Kong/1-4-MA21-1/1968 (H3N2) Hemagglutinin
2VIU 1998-04-29 2.5 INFLUENZA VIRUS HEMAGGLUTININ
5T6N 2017-01-04 2.54 Crystal structure of the A/Hong Kong/1/1968 (H3N2) influenza virus hemagglutinin in complex with the antiviral drug arbidol
6CEX 2018-04-04 2.57 Crystal structure of the A/Hong Kong/1/1968 (H3N2) influenza virus hemagglutinin in complex with small molecule N-Cyclohexyltaurine
5W6T 2017-10-04 2.59 Crystal structure of the A/Puerto Rico/8/1934 (H1N1) influenza virus hemagglutinin in complex with cyclic peptide CP151070 (P7)
5THF 2017-04-05 2.59 Crystal structure of H3 hemagglutinin with insertion of two amino acids in the 150-loop from the A/Hong Kong/1/1968 (H3N2) influenza virus
1FYT 2000-11-08 2.6 CRYSTAL STRUCTURE OF A COMPLEX OF A HUMAN ALPHA/BETA-T CELL RECEPTOR, INFLUENZA HA ANTIGEN PEPTIDE, AND MHC CLASS II MOLECULE, HLA-DR1
1HGE 1994-01-31 2.6 BINDING OF INFLUENZA VIRUS HEMAGGLUTININ TO ANALOGS OF ITS CELL-SURFACE RECEPTOR, SIALIC ACID: ANALYSIS BY PROTON NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY AND X-RAY CRYSTALLOGRAPHY
1R5I 2004-03-16 2.6 Crystal structure of the MAM-MHC complex
1MQM 2003-08-26 2.6 BHA/LSTa
1HXY 2001-06-27 2.6 CRYSTAL STRUCTURE OF STAPHYLOCOCCAL ENTEROTOXIN H IN COMPLEX WITH HUMAN MHC CLASS II
5KUY 2017-06-14 2.6 Influenza hemagglutinin H3 A/Hong Kong/1/1968 in complex with designed inhibitor protein HSB.2A
1KG0 2002-03-27 2.65 Structure of the Epstein-Barr Virus gp42 Protein Bound to the MHC class II Receptor HLA-DR1
5VTX 2017-06-28 2.65 Crystal structure of the A/Hong Kong/1/1968 (H3N2) influenza virus hemagglutinin G225M/L226T/S228A mutant apo form
1HGH 1994-01-31 2.7 BINDING OF INFLUENZA VIRUS HEMAGGLUTININ TO ANALOGS OF ITS CELL-SURFACE RECEPTOR, SIALIC ACID: ANALYSIS BY PROTON NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY AND X-RAY CRYSTALLOGRAPHY
1HGJ 1994-01-31 2.7 BINDING OF INFLUENZA VIRUS HEMAGGLUTININ TO ANALOGS OF ITS CELL-SURFACE RECEPTOR, SIALIC ACID: ANALYSIS BY PROTON NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY AND X-RAY CRYSTALLOGRAPHY
1HGI 1994-01-31 2.7 BINDING OF INFLUENZA VIRUS HEMAGGLUTININ TO ANALOGS OF ITS CELL-SURFACE RECEPTOR, SIALIC ACID: ANALYSIS BY PROTON NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY AND X-RAY CRYSTALLOGRAPHY
1HGD 1994-01-31 2.7 BINDING OF INFLUENZA VIRUS HEMAGGLUTININ TO ANALOGS OF ITS CELL-SURFACE RECEPTOR, SIALIC ACID: ANALYSIS BY PROTON NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY AND X-RAY CRYSTALLOGRAPHY
5W6R 2017-10-04 2.73 Crystal structure of the A/Puerto Rico/8/1934 (H1N1) influenza virus hemagglutinin in complex with cyclic peptide CP141099 (P6)
4O58 2014-04-16 2.75 Crystal structure of broadly neutralizing antibody F045-092 in complex with A/Victoria/3/1975 (H3N2) influenza hemagglutinin
5KAN 2016-11-30 2.79 Crystal structure of multidonor HV1-18-class broadly neutralizing Influenza A antibody 16.g.07 in complex with A/Hong Kong/1-4-MA21-1/1968 (H3N2) Hemagglutinin
1QFU 1999-04-16 2.8 INFLUENZA VIRUS HEMAGGLUTININ COMPLEXED WITH A NEUTRALIZING ANTIBODY
1HA0 1998-10-12 2.8 HEMAGGLUTININ PRECURSOR HA0
1EO8 2000-04-12 2.8 INFLUENZA VIRUS HEMAGGLUTININ COMPLEXED WITH A NEUTRALIZING ANTIBODY
1DLH 1994-06-22 2.8 CRYSTAL STRUCTURE OF THE HUMAN CLASS II MHC PROTEIN HLA-DR1 COMPLEXED WITH AN INFLUENZA VIRUS PEPTIDE
3EYM 2009-01-13 2.8 Structure of Influenza Haemagglutinin in complex with an inhibitor of membrane fusion
3SDY 2011-07-20 2.85 Crystal Structure of Broadly Neutralizing Antibody CR8020 Bound to the Influenza A H3 Hemagglutinin
2YPG 2012-11-07 2.85 Haemagglutinin of 1968 Human H3N2 Virus in Complex with Human Receptor Analogue LSTc
5W6U 2017-10-04 2.88 Crystal structure of the A/Puerto Rico/8/1934 (H1N1) influenza virus hemagglutinin in complex with cyclic peptide CP121068 (P2)
3HMG 1991-01-15 2.9 REFINEMENT OF THE INFLUENZA VIRUS HEMAGGLUTININ BY SIMULATED ANNEALING
4C56 2014-07-23 2.9 X-ray structure of the complex between staphylococcal enterotoxin B, T cell receptor and major histocompatibility complex class II
5Y2L 2019-01-30 2.9 Crystal structure of a group 2 HA binding antibody AF4H1K1 Fab in complex with the 1968 H3N2 pandemic (H3-AC/68) hemagglutinin
1HGG 1994-01-31 2.9 BINDING OF INFLUENZA VIRUS HEMAGGLUTININ TO ANALOGS OF ITS CELL-SURFACE RECEPTOR, SIALIC ACID: ANALYSIS BY PROTON NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY AND X-RAY CRYSTALLOGRAPHY
1MQL 2003-08-26 2.9 BHA of Ukr/63
4FP8 2012-09-19 2.95 Crystal structure of broadly neutralizing antibody C05 bound to H3 influenza hemagglutinin, HA1 subunit
5VTQ 2017-06-28 2.95 Crystal structure of the A/Hong Kong/1/1968 (H3N2) influenza virus hemagglutinin G225L/L226S mutant in complex with 3'-SLN
6NZ7 2019-05-08 2.95 Crystal structure of broadly neutralizing Influenza A antibody 429 B01 in complex with Hemagglutinin Hong Kong 1968
4GMS 2012-10-03 2.95 Crystal structure of heterosubtypic Fab S139/1 in complex with influenza A H3 hemagglutinin
5K9K 2016-12-21 2.97 Crystal structure of multidonor HV6-1-class broadly neutralizing Influenza A antibody 56.a.09 in complex with Hemagglutinin Hong Kong 1968.
2HMG 1991-01-15 3.0 REFINEMENT OF THE INFLUENZA VIRUS HEMAGGLUTININ BY SIMULATED ANNEALING
1HGF 1994-01-31 3.0 BINDING OF INFLUENZA VIRUS HEMAGGLUTININ TO ANALOGS OF ITS CELL-SURFACE RECEPTOR, SIALIC ACID: ANALYSIS BY PROTON NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY AND X-RAY CRYSTALLOGRAPHY
3VUN 2012-08-01 3.0 Crystal structure of a influenza A virus (A/Aichi/2/1968 H3N2) hemagglutinin in C2 space group.
4ZCJ 2015-05-13 3.0 Crystal structure of the A/Hong Kong/1/1968 (H3N2) influenza virus hemagglutinin HA1 Cys30, HA2 Cys47 mutant
6N5D 2019-06-05 3.0 Broadly protective antibodies directed to a subdominant influenza hemagglutinin epitope
4HMG 1991-01-15 3.0 REFINEMENT OF THE INFLUENZA VIRUS HEMAGGLUTININ BY SIMULATED ANNEALING
6N5E 2019-06-05 3.0 Broadly protective antibodies directed to a subdominant influenza hemagglutinin epitope
5W6I 2017-10-04 3.1 Crystal structure of the A/Puerto Rico/8/1934 (H1N1) influenza virus hemagglutinin in complex with cyclic peptide CP141046 (P3)
1MQN 2003-08-26 3.2 BHA/LSTc
5HMG 1991-01-15 3.2 REFINEMENT OF THE INFLUENZA VIRUS HEMAGGLUTININ BY SIMULATED ANNEALING
2VIR 1998-04-29 3.25 INFLUENZA VIRUS HEMAGGLUTININ COMPLEXED WITH A NEUTRALIZING ANTIBODY
2VIS 1998-04-29 3.25 INFLUENZA VIRUS HEMAGGLUTININ, (ESCAPE) MUTANT WITH THR 131 REPLACED BY ILE, COMPLEXED WITH A NEUTRALIZING ANTIBODY
6D0U 2019-01-16 3.25 Crystal structure of C05 V110P/A117E mutant bound to H3 influenza hemagglutinin, HA1 subunit
2VIT 1998-04-29 3.25 INFLUENZA VIRUS HEMAGGLUTININ, MUTANT WITH THR 155 REPLACED BY ILE, COMPLEXED WITH A NEUTRALIZING ANTIBODY
3ZTJ 2011-08-10 3.41 Structure of influenza A neutralizing antibody selected from cultures of single human plasma cells in complex with human H3 Influenza haemagglutinin.
4UBD 2015-06-24 3.5 Crystal structure of a neutralizing human monoclonal antibody with 1968 H3 HA
1KEN 2002-04-24 3.5 INFLUENZA VIRUS HEMAGGLUTININ COMPLEXED WITH AN ANTIBODY THAT PREVENTS THE HEMAGGLUTININ LOW PH FUSOGENIC TRANSITION
4NM8 2013-12-25 4.0 Crystal structure of broadly neutralizing antibody CR8043 bound to H3 influenza hemagglutinin
4FQR 2012-09-19 4.1 Crystal structure of broadly neutralizing antibody C05 bound to H3 influenza hemagglutinin
4FQY 2012-08-22 5.25 Crystal structure of broadly neutralizing antibody CR9114 bound to H3 influenza hemagglutinin

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
91.5 Hemagglutinin A/HK68 P03440 HEMA_I77A8
90.9 Hemagglutinin A/HK68 P26141 HEMA_I84A5
91.8 Hemagglutinin A/HK68 Q2VNF2 HEMA_I78A7
91.8 Hemagglutinin A/HK68 Q2VND2 HEMA_I78A8
91.8 Hemagglutinin A/HK68 Q03909 HEMA_I89A7
92.2 Hemagglutinin A/HK68 P26139 HEMA_I77A4
92.5 Hemagglutinin A/HK68 Q30NQ1 HEMA_I75A0
92.5 Hemagglutinin A/HK68 Q2RFA5 HEMA_I76A6
93.7 Hemagglutinin A/HK68 P03435 HEMA_I75A3
95.0 Hemagglutinin A/HK68 P43259 HEMA_I77A7
95.3 Hemagglutinin A/HK68 P12586 HEMA_I82A6
95.6 Hemagglutinin A/HK68 P03442 HEMA_I63A3
95.0 Hemagglutinin A/HK68 P26138 HEMA_I78A4
95.9 Hemagglutinin A/HK68 P43260 HEMA_I76AC
95.6 Hemagglutinin A/HK68 P26135 HEMA_I74A2
95.6 Hemagglutinin A/HK68 P11133 HEMA_I78A9
95.9 Hemagglutinin A/HK68 P26134 HEMA_I76A3
95.6 Hemagglutinin A/HK68 P43257 HEMA_I75A2
96.2 Hemagglutinin A/HK68 Q1PUD9 HEMA_I73A5
96.2 Hemagglutinin A/HK68 P43258 HEMA_I76AD
95.9 Hemagglutinin A/HK68 P11134 HEMA_I82A4
96.6 Hemagglutinin A/HK68 P12582 HEMA_I77A6
96.6 Hemagglutinin A/HK68 P12583 HEMA_I80A6
97.2 Hemagglutinin A/HK68 Q2ICR0 HEMA_I72A4
97.2 Hemagglutinin A/HK68 P03439 HEMA_I72A3
97.2 Hemagglutinin A/HK68 P19106 HEMA_I72A2
97.2 Hemagglutinin A/HK68 P12587 HEMA_I85A1
97.2 Hemagglutinin A/HK68 P12584 HEMA_I80A7
97.5 Hemagglutinin A/HK68 P12588 HEMA_I85A2
97.2 Hemagglutinin A/HK68 P12585 HEMA_I82A5
98.7 Hemagglutinin A/HK68 P04663 HEMA_I70A0
98.4 Hemagglutinin A/HK68 P03438 HEMA_I000X
98.7 Hemagglutinin A/HK68 P04664 HEMA_I69A0
99.1 Hemagglutinin A/HK68 P03449 HEMA_I71A1
99.4 Hemagglutinin A/HK68 P03436 HEMA_I68A6
99.7 Hemagglutinin A/HK68 P03437 HEMA_I68A0
100.0 Hemagglutinin A/HK68 Q91MA7 HEMA_I68A4
90.8 Hemagglutinin A/WSN/33 P03452 HEMA_I34A1
91.9 Hemagglutinin A/WSN/33 A4GCI6 HEMA_I36A0
92.8 Hemagglutinin A/WSN/33 A4GCL9 HEMA_I35A3
99.5 Hemagglutinin A/WSN/33 P03454 HEMA_I33A0