FIG 6.

Amino acid residues responsible for the enhanced support of polymerase activity of swine ANP32A also mediate increased binding to influenza trimeric polymerase. (A) Split-luciferase assays showing the relative binding of different ANP32 proteins to trimeric polymerase from human pH1N1 or avian H5N1 viruses. PB1 was tagged with the N-terminal part of Gaussia luciferase, while ANP32 proteins were tagged with the C-terminal part. NLR, normalized luminescence ratio, calculated from the ratio between tagged and untagged ANP32/PB1 pairs. Assay performed in 293T cells. Data indicate triplicate repeats plotted as mean with standard deviation, repeated across two separate experiments with representative data shown. Statistical significance was determined by one-way ANOVA with multiple comparisons between the swA and huA wild types and mutants. ***, 0.001 ≥ P > 0.0001; ****, P ≤ 0.0001. (B) Minigenome assays with reconstituted polymerases from 3 different influenza viruses, performed in human eHAP cells with ANP32A and ANP32B knocked out and complemented with wild-type swine ANP32A or B or N129I mutants thereof. Data indicate triplicate repeats plotted as mean with standard deviation, repeated across two separate experiments with representative data shown. Data normalized to each polymerase with wild-type swine ANP32A. ****, P ≤ 0.0001. (C) Western blot assay showing protein expression levels of FLAG-tagged swine ANP32 wild type or N129I proteins during a minigenome assay. (D) Phylogenetic tree of mammalian ANP32A proteins. Species that contain the highly proviral 156S shown in red; species with 156P shown in black. Phylogenetic trees made using the neighbor-joining method based on amino acid sequence. Statistical significance was determined by one-way analysis of variance (ANOVA) with multiple comparisons against an empty vector.