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. 2023 Oct 10;14:6135. doi: 10.1038/s41467-023-41308-4

Fig. 2. Tky05 PB1 K577E and PA Q556R mutations enhance influenza polymerase activity in the absence of human ANP32A/B.

Fig. 2

Minigenome assays were performed in 24-well plates of eHAP DKO cells transfected with pCAGGS Tky05 PB2 (0.04 µg), PB1 or K577E (0.04 µg), PA or Q556R (0.02 µg), NP (0.08 µg), reporter pPolI-luc (0.08 µg) and control pCAGGS-Renilla luciferase (0.04 µg) and (A) +/− huANP32B-FLAG (0.04 µg). Western blot showing expression of PB2, PB1, PA and tubulin. B Minigenome with 0, 0.02, 0.04 or 0.08 µg of huANP32B, chANP32B and huANP32E. Accompanying Western blot showing expression of vinculin, PB2 and ANP32-FLAG. C Minigenome with 0.08 µg of huANP32E. All data presented is representative of n = 3 biological repeats each conducted with n = 3 wells, presented as mean values +/− SD. Statistical significance was determined by multiple comparisons of a one-way ANOVA (A, C) or test for trend of a one-way ANOVA (B) *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (A; Tky WT -ANP32 vs Tky WT +huANP32B P < 0.0001, Tky WT -ANP32 vs Tky PB1-K577E + PA-Q556R -ANP32 p = 0.00015, B; trend for Tky PB1-K577E + PA-Q556R chANP32B titration P < 0.0001, trend for Tky PB1-K577E + PA-Q556R huANP32E titration P < 0.0001, C; Tky WT vs Tky PB1-K577E + PA-Q556R p < 0.0001, Tky WT vs PB1-K577E p = 0.0063, Tky WT vs PA-Q556R p = 0.878). Source data are provided as a Source Data file.