Fig. 4: The adaptive mutations in PB2 arising from H5N1 virus transmission in cattle and other mammals facilitate the virus polymerase to utilize both cow and human ANP32 proteins.

A Phylogenetic analysis of ANP32 proteins derived from different hosts. B Dose-dependent effects of different ANP32 proteins on the cowOH12-H5N1 polymerase activity in the firefly luciferase reporter assay in 293T ANP32A, ANP32B, and ANP32E triple-knockout cells (293T TKO). Data are presented as mean values ± SD from n = 3 independent biological replicate experiments. Statistical significance was determined by two-way ANOVA with Dunnett’s multiple comparisons test. C Structure of the influenza viral polymerase complexed with the ANP32 protein (PDB: 8R1J). Mutations which affected polymerase activities are highlighted in red. D Electrostatic isosurface visualization of the viral polymerase showing the electrical changes (red negative, blue positive) due to cow-adaptive mutations and mammal-adaptive mutations, with a red dashed line indicating the putative path of the ANP32 LCAR domain. Firefly luciferase reporter assay was implemented to compare the effects of key PB2 substitutions on the cowOH12-H5N1 polymerase activity in 293T TKO cells complemented with chicken ANP32A (E), cow ANP32A (F), and human ANP32A (G). The relative polymerase activity was normalized to the wild-type (WT) cowPB2 group. Data are presented as mean values ± SD from n = 3 independent biological replicate experiments. Statistical significance was determined by one-way ANOVA with Dunnett’s post hoc test. Source Data are provided as a Source Data file.