Calcium binding in the N2 neuraminidase low-affinity calcium-binding pocket affects interspecies transmission of Influenza A viruses

Transmission of human influenza A viruses (FLUAV) to pigs is frequently reported but only a small set of viruses has become stablished in the swine population. Although mutations in the hemagglutinin protein (HA) have been pointed out as critical for successful interspecies transmission, such mutations can also be detrimental by affecting the HA/neuraminidase (NA) balance of the virus. However, the molecular mechanisms used by FLUAV to regulate the NA activity and HA/NA balance remain poorly understood. In this study, we used a reassortant virus containing human-origin H3N2 surface gene segments (HA and NA) and swine-origin internal genes to investigate how human-origin FLUAVs adapt to pigs. This reassortant virus contained an adaptative mutation in the HA (A138S, hVIC/11A138S). After two serial passages of hVIC/11A138S in pigs, a mutation in the NA protein (D113A) emerged. This mutation prevented calcium-binding in the low-affinity calcium-binding pocket and enhanced NA enzymatic activity and thermostability under calcium-depleted conditions, mimicking the behavior of an endemic swine NA. Interestingly, most swine-adapted H3N2 viruses contain D113, but available NA crystal structures revealed that they most likely do not bind calcium. Further analysis showed a key amino acid difference at position 93 in the NA protein: human N2 isolates have G93, while in swine N2s N93 is prevalent. This difference alters the NA's interaction with calcium and leads to enhanced activity and thermostability in swine NAs even when D113 is present. Ultimately, these changes altered different kinetic parameters of the NA enzyme and distinctly affected substrate affinity and the reaction velocity. This discovery provides valuable insights into influenza A virus evolution and provides evidence into the mechanisms utilized by FLUAV to modulate NA activity.