Fig. 1. NS2 enhances H9N2 vPol activity in human cells.

(A) vPol reconstitution assay in TKO cells to compare the effect of each ANP32-Flag construct on avian H9N2 vPol activity. The accompanying WBs show expression of Flag-tagged ANP32 constructs. Vec, empty vector control; hu, human; ca, canine; ch, chicken. (B) A549 cells, (C) HEK293T cells, or (D) MDCK cells were infected with H9N2 virus, and the supernatant was collected to detect the H9N2 growth kinetics using TCID₅₀ assays. Data are shown as means ± SD (n = 3) and are representative of three independent experiments. MOI, multiplicity of infection. (E) vPol reconstitution assay to compare the effect of different influenza viral proteins on H9N2 vPol activity in HEK293T cells. (F) vPol reconstitution assay in HEK293T cells to compare the effect of increasing doses of H9N2-NS2 on H9N2 vPol activity. WB analysis shown right (RNP proteins of H9N2 and endogenous huANP32A/B). ACTB indicates β-Actin. In (A), (E), and (F), data shown are firefly activity normalized to Renilla, plotted as means ± SD (n = 3), and are one representative of three independent experiments. P values were determined using one-way analysis of variance (ANOVA) followed by a Dunnett’s multiple comparisons test. NS, not significant; *P < 0.05 and ****P < 0.0001.