Hemagglutinins (HA) from different influenza A virus subtypes share as little as ∼40% amino acid identity, yet their protein structure and cell entry function are highly conserved. Here we examine the extent that sequence constraints on HA differ across three subtypes. To do this, we first use pseudovirus deep mutational scanning to measure how all amino-acid mutations to an H7 HA affect its cell entry function. We then compare these new measurements to previously described measurements of how all mutations to H3 and H5 HAs affect cell entry function. We find that ∼50% of HA sites display substantially diverged preferences for different amino acids across the HA subtypes. The sites with the most divergent amino-acid preferences tend to be buried and have biochemically distinct wildtype amino acids in the different HA subtypes. We provide an example of how rewiring the interactions among contacting residues has dramatically shifted which amino acids are tolerated at specific sites. Overall, our results show how proteins with the same structure and function can become subject to very different site-specific evolutionary constraints as their sequences diverge. ### Competing Interest Statement JDB consults for Apriori Bio, Invivyd, Pfizer, GSK, and the Vaccine Company. JDB and BD are inventors on Fred Hutch licensed patents related to the deep mutational scanning of viral proteins. National Institute of Allergy and Infectious Diseases, R01AI165821, 75N93021C00015 U.S. National Science Foundation, DGE-2140004 Howard Hughes Medical Institute, https://ror.org/006w34k90

The evolution of human influenza virus hemagglutinin (HA) involves simultaneous selection to acquire antigenic mutations that escape population immunity while preserving protein function and stability...

Human influenza viruses rapidly acquire mutations in their hemagglutinin (HA) protein that erode neutralization by antibodies from prior exposures. Here, we use a sequencing-based assay to measure neu...