Influenza hemagglutinin subtypes have different sequence constraints despite sharing extremely similar structures

Area of Science:

• Virology
• Structural Biology
• Evolutionary Biology

Background:

• Influenza A virus hemagglutinins (HAs) exhibit significant sequence divergence (~40% identity) across subtypes.
• Despite sequence variability, HA protein structure and cell entry function remain highly conserved.

Purpose of the Study:

• To investigate the differences in sequence constraints on hemagglutinin (HA) across three influenza A virus subtypes.
• To compare site-specific evolutionary preferences for amino acid mutations in H7, H3, and H5 HAs.

Main Methods:

• Utilized pseudovirus deep mutational scanning to assess the impact of all amino-acid mutations on H7 HA cell entry.
• Compared H7 HA mutational data with existing data for H3 and H5 HAs.

Main Results:

• Approximately 50% of HA sites demonstrated significantly different amino acid preferences across subtypes.
• Divergent sites were often buried within the protein structure and featured distinct wildtype amino acids.
• Rewiring of residue interactions explained shifts in tolerated amino acids at specific sites.

Conclusions:

• Influenza HA subtypes experience distinct site-specific evolutionary constraints despite conserved structure and function.
• Understanding these divergent constraints is crucial for influenza virus evolution studies.