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. 2024 Feb 5;15:1064. doi: 10.1038/s41467-024-45205-2

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© The Author(s) 2024

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. 1 — A Model of actively replicating Zhejiang-H7N9 Flu_APol replicase-ANP32-encapsidase (FluPol_(R)-ANP32A-FluPol_(E)). Ribbon diagram representation with PA (green), PB1 (grey), PB2 (blue) and ANP32A (pink). The model was constructed by superposing Zhejiang-H7N9 elongating Flu_APol domains (PDB: 7QTL)⁴³ for the FluPol_(R) and apo-dimer Zhejiang-H7N9 Flu_APol (PDB: 7ZPL)⁴³ for the FluPol_(E), on those of Flu_CPol replication complex (PDB: 6XZQ)¹³. ANP32A was left unchanged. Key FluPol CTD-binding residues are highlighted on the FluPol_(R) (left) and FluPol_(E) (right). B WSN FluPol binding and activity assays in HEK-293T cells. Left Y-axis (linear scale): FluPol (PA WT or indicated PA CTD-binding mutants) binding to the CTD (grey bars), huANP32A (light blue bars) and chANP32A (dark blue bars) was assessed using split-luciferase-based complementation assays. Right Y-axis (logarithmic scale): FluPol activity (hatched bars) was assessed by vRNP reconstitution, using a model vRNA encoding the Firefly luciferase (Fluc-vRNA). The luminescence signals are represented as a percentage of PA WT (mean ± SD, n = 6, 4, 4, 3, **p < 0.002, ***p < 0.001 (one-way ANOVA; Dunnett’s multiple comparisons test). C WSN vRNPs were reconstituted in HEK-293T cells using the NA vRNA. Steady-state levels of NA mRNA, cRNA and vRNA were quantified by strand-specific RT-qPCR³⁵ and are represented as a percentage of PA WT (mean ± SD, n = 3, *p < 0.033, **p < 0.002, ***p < 0.001, one-way repeated measure ANOVA; Dunnett’s multiple comparisons test). D–F FluPol trans-complementation assays upon vRNP reconstitution in HEK-293T cells with a model Fluc-vRNA. D Trans-complementation of a transcription-defective (FluPol_(T-), PA D108A)⁸ and replication-defective (FluPol_(R-), PA K664M)³⁶ FluPol. Luminescence is represented as percentage of PA WT (mean ± SD, n = 3, **p < 0.002, one-way repeated measure ANOVA; Dunnett’s multiple comparisons test). WSN vRNPs were co-expressed with (E) a replication-defective (FluPol_(R-), PA K664M)³⁶ or (F) a transcription-defective (FluPol_(T-), PA D108A)⁸ FluPol. Luminescence signals are represented as the fold-change (FC) relative to the background, which is defined as the sum of signals measured when the FluPol_(CTD-) and FluPol_(R-/T-) were transfected alone (mean ± SD, n = 4, *p < 0.033, **p < 0.002, ***p < 0.001, two-way ANOVA; Sidak’s multiple comparisons test). Source data are provided as a Source data file.