Figure 1.
Subcellular localization of unconventional NLS mutants.
(A) Schematic diagram and subcellular localization of the N-terminal 110-amino acid region (NP110aa) NLS mutants. The unconventional NLS (aa 3-TKGTKRSYEQM-13), overlapping bipartite NLS (aa 90-KKTGGPIYRRVDGKWRRELILYDKEEIRRIWR-121) or bipartite NLS (aa 198-KRGINDRNFWRGENGRKTR-216) are indicated in gray in the schematic showing of full length NP and NP110aa. A549 cells were transfected with plasmids expressing WT and mutant NP110aas. The nuclear and cytoplasmic localization of WT and mutant NP110aas were determined by monitoring mRFP fluorescence. Over five hundred mRFP-expressing cells were counted and classified as nuclear or cytoplasmic localization. (B) Fluorescence microscope images showing mRFP, WT NP110aa and a mutant lacking the N-terminal 13-amino acid tail region (NP14-110aa) (upper panel). Nucleus is stained with DAPI (lower panel). (C) Fluorescence microscope images showing WT and mutant NP110aas. The localization of NP was consistent with that observed using mRFP fluorescence.
Figure 2.
Production of recombinant viruses expressing unconventional NLS mutants.
HEK-293T/MDCK cells were transfected with plasmids expressing four viral proteins and eight vRNAs. To generate the R8A NP and S9A NP mutant viruses, plasmids expressing mutant NP proteins and genomes were used as a substitute for the WT plasmid. After 72 h of transfection, supernatants were harvested and subjected to plaque assays using MDCK cells. Values represent the mean ± SD of measurements taken from three samples in each of four independent experiments.
Figure 3.
Effect of unconventional NLS mutants on viral replication and vRNA transcription.
(A) A549 cells were infected with the S9A NP mutant and WT NP viruses at a multiplicity of infection of 1. At 24 h post-infection, the supernatants were harvested and the virus titer was determined using plaque assays. Data represent the relative growth of the S9A mutant compared with that of the WT. (B) Mini-genome assay using the S9A NP mutant. The effect of the S9A mutation on viral transcription was determined using a mini-genome assay with the plasmid encoding the S9A NP mutant. Luciferase activity was measured 48 h after the transfection of viral protein- (PB2, PB1, PA or NP) and vNP-luc-expressing plasmids. To analyze the effect of the S9A mutation on vRNA transcription, luciferase activity was compared with that generated by the WT. Data represent the mean ± SD of measurements from three samples in each of two independent experiments (*p<0.05, by students t-test).
Table 1.
Conservation of 8 and 9 amino acid residue in human and avian influenza A virus.
Figure 4.
(A) Purification of mRFP-Flag-tagged WT and mutant NP110aas from COS-7 cells and GST-tagged importin α isoforms, Rch1, Qip1 and NPI-1 from Escherichia coli. For purification of NP110aa, COS-7 cells were transfected with pCAGGS encoding mRFP-Flag-tagged WT and mutant NP110aas, lysed and proteins were purified using ANTI-FLAG M2 Agarose beads and the 3× Flag peptide. For purification of importin α proteins, Escherichia coli expressing GST-tagged Rch1, Qip1 and NPI-1 were lysed and proteins were purified using glutathione-Sepharose 4B. Purified proteins were subjected to 10% or 15% SDS-PAGE and stained by Coomassie brilliant blue. (B) Binding of WT and mutant NP110aas to the three importin α isoforms. Both GST beads bound to Rch1, Qip1, NPI-1 and non-bound GST beads were incubated with the WT, 14-110aa, R8A or S9A NP110aa proteins. Binding of NP110aa to importin α was detected by Western blotting with anti-FLAG M2 monoclonal antibody (mAb). (C) Quantification of band intensity. Different concentrations of Rch1, Qip1 and NPI-1 proteins (0, 5, 25 and 50 µM) were subjected to 10% SDS-PAGE and Western blotting with anti-Rch1, -Qip1 and -NPI-1 mAbs. Band intensities were measured by Image J and the standard curves for the amount of importin α isoforms were constructed. (D) Competition binding assay of importin α to WT and S9A NP110aa-binding beads. Purified importin α isoforms, Rch1, Qip1 and NPI-1 from Escherichia coli were subjected to 10% SDS-PAGE and stained with Coomassie brilliant blue. All three importin α proteins (50 µM) were incubated with WT and S9A NP110aa-binding Flag beads at 4°C for 1 day. After washing with buffer, the binding of importin α to WT and S9A NP110aa was detected by Western blotting with anti-Rch1, -Qip1 and -NPI-1 mAbs. Empty Flag beads were used as the negative control. The amount of importin α proteins which bound to WT NP110aa and S9A NP110aa were calculated by analyzing the standard curves as shown in C right panel. (E) Binding of WT full length NP, WT and S9A NP110aa to the three importin α isoforms in HEK-293T cells. HEK-293T cells were transfected with pCAGGS encoding mRFP-Flag-tagged WT full length NP, WT or S9A NP110aa, cells were lysed and proteins were purified using ANTI-FLAG M2 Agarose beads and the 3× Flag peptide. The bound importin αs to NP was detected by Western blotting with anti-Rch1, -Qip1 and -NPI-1 mAbs. The same amount of lysate was used for control. The amounts of importin α proteins which bound to WT NP, WT NP110aa and S9A NP110aa were calculated by analyzing the standard curves as shown in C right panel.
Figure 5.
Viral transcription and growth in Qip1-silenced cells.
(A) HEK-293 cells were transfected with Qip1 siRNA for 24 h. Silencing of Qip1 was confirmed by Western blotting with an antibody specific for Qip1. (B) Qip1-silenced HEK-293 cells were transfected with plasmids encoding PB2, PB1, PA, WT or S9A NP and vNP-luc, and luciferase activity was measured after 48 h. Columns and error bars represent the mean ± SD of measurements from three samples in each of three independent experiments (*p<0.05, by students t-test). (C) A549 cells were transfected with Qip1 siRNA for 48 h. Silencing of Qip1 was confirmed by Western blotting with an antibody specific against Qip1. (D) Qip1-silenced A549 cells were transfected with pCAGGS encoding mRFP-Flag-tagged WT or S9A NP110aa. The localization of NP was consistent with that observed using mRFP fluorescence. (E) The Qip1-silenced A549 cells were infected with A/WSN/33 virus containing WT or S9A NP at an MOI of 0.01. The viral titer in supernatant of non-targeting siRNA transfected (triangle) or Qip1 siRNA transfected (square) cells was determined using a plaque assay. Data represent the mean ± SD of measurements from three samples in each of three independent experiments (*p<0.05, by students t-test).