Figure 4.

Molecular Defects of a Restricted Avian Polymerase Are Rescued by chANP32A₂₉ (A) chANP32A₂₉ enhances RNP formation for an avian-style polymerase. RNPs were reconstituted in human cells by co-expressing the indicated polymerase, NP, a genomic RNA, and chANP32A variants. NP was immunoprecipitated, and co-precipitating PB2 was measured as a proxy for RNP formation. RNP assembly was quantified from two independent assays, normalized to controls, and represented as the mean ± SD. chANP32A₂₉ functions redundantly with enhancing promoter mutations. Polymerase activity assays were conducted with the indicated ANP32A proteins and either WT or 3–8 mutant vRNA reporters. Proteins were detected by western blot. C, empty vector control. Data are shown as mean (column) of technical triplicates (dots) ± SD. *p < 0.05 (Student’s t test) between PB2 K627 and E627 at each condition. chANP32A increases the enzymatic activity of a restricted polymerase independent of RNP formation. NP-independent polymerase activity on a micro-gene vRNA template was measured by primer extension. chANP32A variants were co-expressed where indicated. mRNA and vRNA products were quantified by phosphorimaging from three independent experiments, normalized to PB2 K627 in the absence of ANP32A, and presented as mean ± SD. *p < 0.05, one-way ANOVA with post hoc Tukey HSD test. Tests were performed separately for vRNA and mRNA levels, and comparisons were made to PB2 K627 or E627 polymerase activity in the absence of ANP32A.