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

Fig. 3. FluPol replication activity is enhanced in the presence of CTD and ANP32A.

Fig. 3

WSN FluPol binding to huANP32A (A) and WSN FluPol oligomerisation (B) in the presence of increasing amounts of pS5 CTD, as assessed using split-luciferase-based complementation assays. Plasmids encoding mCherry or mCherry fused to CTD-WT (dark grey bars) or CTD-S5A (serine five residues replaced with alanines, light grey bars) were co-transfected in increasing amounts (mean ± SD, n = 3, **p < 0.002, ***p < 0.001, two-way ANOVA; Sidak’s multiple comparisons test). Luminescence signals are represented as fold-changes compared to untagged mCherry co-expression. In (B), cell lysates were analysed by western blot using the indicated antibodies (n = 1). C 51-mer vRNA template (v51_mut_S) derived from segment 4 of A/Zhejiang/DUID-ZJU01/2013(H7N9)/KJ633805 and used in in vitro replication activity assays. The vRNA 5’end (1–30) and 3′ end (31–51) are coloured in pink and gold, respectively, with introduced mutations in red. The theoretical de novo full-length replication product is coloured in light blue (1–33) and dark blue (35–51), with U34 in red. The expected stalled elongation state is schematically represented. de novo replication activity assays of Zhejiang-H7N9 FluPol using v51_mut_S, in the presence of either 3 NTPs (AUG) or 4 NTPs (AUGC), with or without pS5 CTD(6mer) and huANP32A full-length (D, n = 3) or deletion mutants (E, n = 3). Tentative full-length and stalled replication products are indicated by an arrow. LRR leucine-rich repeat, LCAR low complexity acidic region, NLS nuclear localisation signal. Nts: molecular weight marker. F WSN and Anhui-H7N9 vRNPs were reconstituted in ANP32AB KO cells with a model Fluc-vRNA, and co-expressed with the indicated huANP32A proteins (V5-tagged and fused to SV40-NLS). Luminescence signals are represented as a percentage of huANP32A FL. (mean±SD, n = 3, ***p < 0.001, two-way ANOVA; Sidak’s multiple comparisons test). G Lysates of ANP32AB KO cells transiently expressing the indicated huANP32A proteins were analysed by western blot using an anti-V5 antibody (n = 1). H, I RNA-sequencing of Zhejiang-H7N9-4M FluPol de novo replication products in the presence of all NTPs, the pS5 CTD(6mer) and huANP32A. H The number of reads is plotted according to the recurrence of the exact 5’ cRNA motifs indicated on the left. Reads that do not encompass these motifs are plotted as ‘Other’. I The percentages of full-length replication products are plotted according to the 5’ terminal nucleotide indicated on the left. Source data are provided as a Source data file.