Abstract
The hemagglutinin (HA) receptor-binding site (RBS) in human influenza A viruses is critical for attachment to host cells, which imposes a functional constraint on its natural evolution. On the other hand, being part of the major antigenic sites, the HA RBS of human H3N2 viruses needs to constantly mutate to evade the immune system. From large-scale mutagenesis experiments, we here show that several of the natural RBS substitutions become integrated into an extensive epistatic network that prevents substitution reversion. X-ray structural analysis reveals the mechanistic consequences as well as changes in the mode of receptor binding. Further studies are necessary to elucidate whether such entrenchment limits future options for immune escape or adversely affect long-term viral fitness.
Highlights
The hemagglutinin (HA) receptor-binding site (RBS) in human influenza A viruses is critical for attachment to host cells, which imposes a functional constraint on its natural evolution
While E190D and G225D substitutions were thought to be irrelevant in human adaptation of the H3N2 subtype, recent human H3N2 strains have acquired these human H1N1 signature RBS mutations in addition to other substitutions within the HA RBS, likely as a result of antigenic drift to escape from the immune system[2,3,9,10]
Intragenic epistasis has been empirically shown to constrain the natural evolution of proteins from a variety of viruses and organisms[12,29,30,31,32,33,34,35,36]
Summary
The hemagglutinin (HA) receptor-binding site (RBS) in human influenza A viruses is critical for attachment to host cells, which imposes a functional constraint on its natural evolution. For avian viruses to transmit in humans, the receptor specificity has to switch to α2–6 linked sialosides Such a change in tropism of the H3N2 subtype occurred in 1968 through a double substitution Q226L/G228S within the HA RBS, whereas a different double substitution, E190D/G225D, was employed by the H1N1 subtype in 1918 A series of deep mutational scanning and virus rescue experiments based on reverting H3N2 A/Brisbane/10/2007 (Bris07) to sequences present in the RBS of earlier strains revealed that the subsequent entrenchment of Asp at residue 190 was attributable to a number of other substitutions that arose within and surrounding the HA RBS. Our work elucidates the importance of intragenic epistasis and its complexity in the HA RBS in human H3N2 viruses, and of the potential consequences for escape from immune pressure
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