Figure 1

NP NLS2 contributes to nuclear import during influenza A virus infection. (A) Schematic diagram of the location of NLS1 and NLS2 on influenza A NP. The amino acid sequences is based on influenza A virus, strain X-31, A/Aichi/68 (H3N2). Numbers indicate amino acids positions. (B) HeLa cells expressing 5GFP, 5GFP-NLS1, or 5GFP-NLS2 were infected with influenza A virus (X-31). At 24 h post-infection, the supernatant was collected and the viral titer was determined by infecting MDCK cells and counting plaques 3 days post-infection. Bar graphs show the mean ± standard error of the mean from three independent experiments (***p < 0.001, one-way ANOVA followed by Tukey’s tests). (C) Competition of nuclear import of NP by NLS1 and NLS2 during an infection. HeLa cells expressing 5GFP, 5GFP-NLS1, or 5GFP-NLS2 were infected with influenza A virus for 2 or 10 h and the subcellular localization of 5GFP and NP were analyzed by confocal fluorescence microscope after immunolabeling NP (red) using a specific antibody. DAPI (blue) was used to observe the nucleus. Scale bars, 10 µm. (D) Quantification of the ratio of nuclear to cytoplasmic fluorescence (Fn/c) of NP, corrected for background fluorescence, for experiments shown in C. Shown are the means ± standard error of the means scored from three independent experiments. (***p < 0.001, one-way ANOVA followed by Tukey’s tests). The levels of expression of 5GFP, 5GFP-NLS1, and 5GFP-NLS2 for experiments in Fig. 1B and C are shown in Fig. S2.