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

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 Relative fitness index for single, double, and triple mutants in WSN HA RBS (11 residues) by deep mutational scanning (Data S2) ; 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) ; Validation of WSN deep scan data by virus rescue and hemagglutination assays (log10 TCID50, log2 HA Titer) (Data S1_WSN) ; Validation of HK68 deep scan data by virus rescue and hemagglutination assays (log10 TCID50, log2 HA Titer) (Data S1_HK68) ; Kds for binding of bnAbs C05 IgG and S139/1 IgG to WSN HA variants (Table S1) ; Kds for binding of glycans 3'-SLNLN and 6'-SLNLN to WSN HA variants (Table S3_WSN) ; Kds for binding of glycans 3'-SLNLN and 6'-SLNLN to HK68 HA variants (Table S3_HK68) ; Kds for binding polymerized glycoconjugates (3'-SLNLN-PAA and 6'-SLNLN-PAA) to HK68 HA variants (Table S5_HK68) ; Off-rates of glycan 3'-SLNLN with WSN recombinant HA variants (Table S4_WSN) ; Kds of HK68 HA WT and escape mutant for binding to bnAb S139/1 IgG (Figure S4, B-C)
Sequence Assay Result Units