Genome-informed structural analysis of polymerase and glycoprotein adaptation in H5N1 clade 2.3.4.4b.
IMPORTANCE Understanding the molecular mechanisms driving H5N1 clade 2.3.4.4b is critical for pandemic preparedness. OBJECTIVE To characterize the molecular drivers of viral fitness and mammalian adaptability in recent H5N1 viruses by integrating evolutionary dynamics with structural simulations. METHODS This study analyzed 2,398 H5Nx genomes (2000-2024) through phylogenetic and selective pressure analyses. HA/NA structures were predicted with AlphaFold 3 and evaluated by AutoDock4 docking, whereas polymerase-ANP32A/B complexes were modeled using template-based methods and their binding free energies were estimated using MM/GBSA. Polymerase-ANP32E complexes were predicted with AlphaFold 3 and similarly evaluated by MM/GBSA. The binding affinities (ΔG) for the sialic acid (SA) receptors and human ANP32 proteins were quantified through molecular mechanics/generalized born surface area calculations. RESULTS Clade 2.3.4.4b showed significant antigenic drift in the HA receptor binding site, reducing affinity for α2,3-SA and α2,6-SA receptors. On the other hand, the emergence of a full-length stalk N1 NA with second sialic acid-binding site mutations (e.g., N366S) compensated for reduced HA affinity by enhancing the NA binding stability. In the polymerase complex, both the PB2-627E/631L variant (-144.00 kcal/mol; unadjusted p = 0.0058) and the known mammalian-adaptive 627K/631M variant (-144.67 kcal/mol; unadjusted p = 0.0165) showed more favorable predicted human ANP32B binding free energies than the ancestral 627E/631M state (-136.46 kcal/mol). CONCLUSIONS AND RELEVANCE The co-occurrence of HA, NA, PB1, and PB2 signatures was associated with clade expansion and produced structural predictions consistent with altered receptor or ANP32 interactions; experimental validation is required before inferring effects on fitness or zoonotic risk.