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

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 7 — A 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), control pCAGGS-Renilla luciferase (0.04 µg) and either -ANP32, muANP32A-FLAG, muANP32B-FLAG or muANP32E-FLAG (0.04 µg). Data presented are 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 one-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Tky WT; -ANP32 vs +muANP32A p = 0.923, -ANP32 vs +muANP32B p > 0.0001, -ANP32 vs +muANP32E p = 0.923, Tky PB1-K577E + PA-Q556R; -ANP32 vs +muANP32A p < 0.0001, -ANP32 vs +muANP32B p < 0.0001, -ANP32 vs +muANP32E p < 0.0001, Tky PB1-K577E; -ANP32 vs +muANP32A p = 0.0003, -ANP32 vs +muANP32B p < 0.0001, -ANP32 vs +muANP32E p = 0.0135, Tky PA-Q556R; -ANP32 vs +muANP32A p < 0.9999, -ANP32 vs +muANP32B p < 0.0001, -ANP32 vs +muANP32E p > 0.9999). Accompanying western blot showing expression of vinculin, PB2 and ANP32-FLAG. B Six to eight-week-old female BALB/c mice were mock infected (10 mice) or infected intranasally with 10⁵ PFU of Tky05 WT (10 mice) or PB1 K577E + PA Q556R virus (10 mice). 5 mice per group were culled at day 2 and 5 mice culled at day 7. Weight loss was measured each day. One mouse was excluded from the Tky05 WT group for weight loss calculation because it did not become infected after inoculation. Data are presented as mean values +/− SD. Statistical significance was determined by two-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (displayed is a comparison between WT and PB1-K577E + PA-Q556R; day 1 p > 0.3258, day 2 p < 0.0001, day 3 p < 0.0001, day 4 p < 0.0001, day 5 p < 0.0001, day 6 p < 0.0001, day 7 p < 0.0001). C Viral titres from the homogenized lung tissue on day 2 p.i. (n = 5 murine lungs per group). Data are presented as mean values +/− SD. Statistical significance was determined by an unpaired t-test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (WT vs PB1-K577E + PA-Q556R p = 0.0058). D Symmetric dimer of the influenza A polymerase (PDB: 6QX8) with mouse-adaptive mutations mapping to the interface. Highlighted residues are PA N291, L295, L336, A343, D347, E349, K353, K356, L550, T552, I554, Q556 and PB1 K577. Source data are provided as a Source Data file.