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

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. [Display omitted] •Large-scale mutational analysis of influenza hemagglutinin receptor-binding site (RBS)•Many new replication-competent RBS mutants were identified•Some previously unobserved RBS mutants escape from a broadly neutralizing antibody•Epistasis is common in the 220-loop of the hemagglutinin receptor-binding site Wu et al. performed a large-scale mutational analysis of the influenza hemagglutinin receptor-binding site (RBS). A large number of replication-competent RBS mutants were observed. Such functional sequence diversity is made possible by pervasive epistasis in the RBS 220-loop and can be buffered by avidity in viral receptor binding.