Functional redundancy in chicken ANP32A mediates species-specific support of avian influenza virus polymerase

Host restriction of avian influenza virus (AIV) polymerase in human cells is driven by species-specific differences in ANP32A/B proteins. While AIV polymerase relies on ANP32A/B containing a 33-amino-acid insert unique to avian species, the structural and/or mechanistic basis for this requirement remains poorly characterized. Here, we demonstrate that chicken ANP32A (chANP32A) displays three functional determinants enabling its species-specific support of AIV polymerase: (1) a SUMO-interacting motif (SIM), (2) SUMOylation at residues K68/K153, and (3) a 28-amino-acid segment within the avian-specific insertion. These determinants function synergistically and redundantly, requiring at least two for optimal activity, to enhance interactions between AIV viral ribonucleoprotein (vRNP) and chANP32A, thereby promoting AIV vRNP assembly. In contrast, human ANP32A/B—which lack the other two determinants and rely solely on SUMOylation—exhibit a limited capacity to support AIV polymerase activity. Our findings unveil a cooperative mechanism where SUMO-dependent processes and structural motifs in chANP32A enforce species-specific adaptation of AIV polymerase, shedding light on how ANP32A/B governs host restriction of AIV polymerase.