Modulation of human-to-swine influenza a virus adaptation by the neuraminidase low-affinity calcium-binding pocket

Authors: CARDENAS, MATIAS - University Of Georgia; SEIBERT, BRITTANY - University Of Georgia; COWAN, BRIANNA - University Of Georgia; CACERES, JOAQUIN - University Of Georgia; GAY, CLAIRE - University Of Georgia; FACCIN, FLAVIO CARGNIN - University Of Georgia; PEREZ, DANIEL - University Of Georgia; Baker, Amy; Anderson, Tavis; RAJAO, DANIELA - University Of Georgia

Submitted to: Communications Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/19/2024
Publication Date: 10/1/2024
Citation: Cardenas, M., Seibert, B., Cowan, B., Caceres, J.C., Gay, C.L., Faccin, F., Perez, D.R., Baker, A.L., Anderson, T.K., Rajao, D.S. 2024. Modulation of human-to-swine influenza a virus adaptation by the neuraminidase low-affinity calcium-binding pocket. Communications Biology. 7. Article 1230. https://doi.org/10.1038/s42003-024-06928-6.
DOI: https://doi.org/10.1038/s42003-024-06928-6

Interpretive Summary: Human-to-swine influenza A virus (IAV) spillover events are relatively common and have led to the establishment of multiple evolutionarily distinct virus lineages in the swine population worldwide. The frequent interspecies transmission events increase the risk of reassortment and emergence of novel IAV that may retain the ability to infect and transmit in humans, an event exemplified by the H1N1 pandemic in 2009. However, the molecular mechanisms driving IAV adaptation to pigs following the introduction of a human virus into the host are not understood. In this study we found that a single point mutation in a human-origin neuraminidase (NA) gene affected protein function to mimic the biological features of a swine-origin NA gene. This mutation improved the activity and stability of the human-origin protein in swine, which is likely to improve replication and infectivity of human IAV in the swine host. This study demonstrates how specific protein features influence how human IAV adapts to pigs and how novel influenza A viruses may evolve to become established in the swine population.

Technical Abstract: 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.