Naturally Occurring PB2 Paired Substitutions Confer Host-Dependent Polymerase Phenotypes in H7N9 Avian Influenza Viruses with Differential ANP32 Support

H7N9 avian influenza viruses have caused repeated human infections since 2013, but the effects of naturally occurring PB2 substitution combinations on polymerase activity remain poorly understood. We analyzed 899 avian-derived and 1087 human-derived H7N9 PB2 sequences collected between 2013 and 2025. Six PB2 positions (109, 122, 191, 340, 559, and 640) showed distinct frequency changes across epidemic waves and host origins. Pairwise analysis identified five representative combinations with different temporal patterns. In minigenome assays, four paired substitutions reduced polymerase activity in human 293T cells at both 33 °C and 37 °C, whereas none of the five significantly altered activity in chicken DF1 cells at 37 °C or 41 °C. Several paired substitutions showed polymerase activity profiles different from those of the corresponding single substitutions in the tested cellular assays. ANP32 overexpression experiments showed different polymerase activity responses across the tested cellular conditions. Under chicken ANP32A expression, none of the paired-substitution polymerases differed significantly from the wild type, whereas human ANP32A and ANP32B overexpression did not enhance their activity in DF1 cells. Co-immunoprecipitation confirmed detectable interactions between PB2 variants and human or chicken ANP32 proteins. Structural modeling further revealed differences in predicted PB2–ANP32A interfaces among the tested variants. Together, these results show that the tested PB2 substitution combinations produced different polymerase activity profiles in human 293T and chicken DF1 cells and exhibited different responses to ANP32 overexpression under the examined conditions. The findings provide insight into the functional consequences of PB2 evolution during H7N9 circulation.