Human-like PB2 627K influenza virus polymerase activity is regulated by importin-α1 and -α7

Abstract

Influenza A viruses may cross species barriers and transmit to humans with the potential to cause pandemics. Interplay of human- (PB2 627K) and avian-like (PB2 627E) influenza polymerase complexes with unknown host factors have been postulated to play a key role in interspecies transmission. Here, we have identified human importin-α isoforms (α1 and α7) as positive regulators of human- but not avian-like polymerase activity. Human-like polymerase activity correlated with efficient recruitment of α1 and α7 to viral ribonucleoprotein complexes (vRNPs) without affecting subcellular localization. We also observed that human-like influenza virus growth was impaired in α1 and α7 downregulated human lung cells. Mice lacking α7 were less susceptible to human- but not avian-like influenza virus infection. Thus, α1 and α7 are positive regulators of human-like polymerase activity and pathogenicity beyond their role in nuclear transport.

Figure 1. Importin-α1 and -α7 are required for human-like polymerase activity in human cells.

(A and B) Polymerase activity of WSN-PB2 627K (A) or WSN-PB2 627E (B) containing vRNPs in importin-α siRNA silenced human 293T cells. Activity of vRNPs in negative siRNA silenced cells (Ctrl) was set 100%. As a background control, vRNPs were transfected omitting the PB2 subunit in negative siRNA silenced cells (Ctrl). Means of at least three independent experiments +/− standard deviations (SD) are shown (*p<0.05, **p<0.01, ***p<0.001, by students t-test). (C) Polymerase activity of WSN-PB2 627K or WSN-PB2 627E containing vRNPs in human 293T (left) and avian DF1 (right) cells. Mock transfected cells were used a control (Mock). (D) Confirmation of importin-α silencing in human 293T cells by Western blot analysis. GAPDH was used as a loading control.

Figure 2. PB2 627K and PB2 627E monomers display similar importin-α binding affinities.

(A) PB2 binding to endogenous importins. 293T cells were transfected with expression plasmids for PB2-627K-FLAG or PB2-627E-FLAG. Mock transfected cells served as a control (Mock). Cells were harvested 48 h after transfection and subjected to immunoprecipitation of FLAG-tagged PB2 proteins. The amount of co-immunoprecipitated endogenous importins (α1, α3, α4, α5, α7 and β1) was analyzed by Western blot. (B) Quantification of PB2 binding to endogenous importins in (A) using a Bioimager as described in Experimental Procedures. Precipitated amounts of importins were normalized against precipitated PB2 levels. Data shown are derived from at least three independent experiments and represent mean +/− SD. (C and D) PB2 binding to overexpressed importins. 293T cells were co-transfected with expression plasmids for FLAG-tagged importin-α proteins (α1, α3, α4, α5 or α7) and untagged PB2 627K (C) or PB2 627E (D). PB2-only transfected cells served as a control (Mock). Cells were lysed 48h after transfection and immunoprecipitated using the FLAG-tag. The amount of co-immunoprecipitated PB2 and importin-β1 was determined by Western blot analysis.

Figure 3. NP mediates increased binding of importins to PB2-627K containing vRNPs.

(A) vRNP binding to endogenous importins. 293T cells were co-transfected with plasmids encoding PB2-627K-FLAG or PB2-627E-FLAG, PB1, PA, NP as well as the pPol-I-NP-Luc construct. Mock transfected cells were used a control (Mock). Cells were lysed 48 h after transfection and immunoprecipitation was performed using the FLAG-tag. The amount of co-immunoprecipitated PA, NP, importin-α1, -α3, -α4, -α5, -α7 and -β1 was analyzed by Western blotting. (B) Quantification of vRNP binding to endogenous importins in (A) using a Bioimager as described in Experimental Procedures. Amounts of Co-IP products were normalized against precipitated PB2 levels. Data shown represent the mean +/− SD of at least three independent experiments. (C) Trimeric polymerase binding to endogenous importins. Co-immunoprecipitation was performed as in (A), except NP expressing plasmid was omitted. (D) Quantification of trimeric polymerase binding to endogenous importins in (C) using a Bioimager as described in Experimental Procedures. Amounts of Co-IP products were normalized against precipitated PB2 levels. Data shown derived from four to six independent experiments and represent the mean +/− SD.

Figure 4. NP binding affinity to individual importin-α isoforms.

(A) NP binding to endogenous importins. 293T cells were either Mock transfected or with plasmids encoding NP-FLAG and lysed 48 h after transfection. Immunoprecipitation was performed using the FLAG-tag. Amount of co-immunoprecipitated importin-α1, -α3, -α4, -α5, -α7 and -β1 was determined by Western blot analysis. (B) NP binding to overexpressed importins. 293T cells were co-transfected with plasmids encoding FLAG-tagged importins (α1, α3, α4, α5 or α7) and NP. NP-only transfected cells served as a control (Mock). Cells were lysed 48 h after transfection and immunoprecipitated using the FLAG-tag. The amount of co-immunoprecipitated NP and importin-β1 was determined by Western blot analysis.

Figure 5. Importin-α silencing does not affect subcellular localization of PB2 or NP.

(A–D) vRNP complexes containing either WSN-PB2-627K-FLAG or WSN-PB2-627E-FLAG were expressed in unsilenced controls (A), importin-α1 (B), -α3 (C), or -α7 (D) silenced 293T cells. Subcellular distribution of PB2 and NP was analyzed by specific staining against PB2-FLAG and NP protein. Mock transfected cells were used as negative controls.

Figure 6. Human- but not avian-like virus growth depends on importin-α1 and -α7.

(A–D) Virus growth in importin silenced human cells. A549 cells were silenced using siRNA for importin-α1, -α3 or -α7 and infected at MOI of 0.001 with WSN-PB2_627K (A and B) or WSN-PB2_627E (C and D). Unsilenced (Mock) and control siRNA transfected cells (Ctrl) were used as a control. Virus titres were determined by plaque assay 72 and 96 hours post infection. As a further control negative siRNA silenced cells were infected (Ctrl). Data shown represent means of three to six independent experiments +/− standard deviations (SD) (*p<0.05, **p<0.01, ***p<0.001, by students t-test). (E) Confirmation of importin-α silencing in human A549 cells by Western blot analysis. GAPDH was used as a loading control.

Figure 7. Importin-α7^−/− mice are less susceptible to human- but not avian-like virus infection.

(A–F) Pathogenicity of human- and avian-like virus in wildtype and importin-α7^−/− mice. Wildtype (n = 16) or importin-α7^−/− (n = 16) mice were infected with 10⁵ p.f.u. (∼20-fold MLD₅₀) of WSN-PB2_627K (A, C and E) or 5×10⁶ p.f.u. (∼10-fold MLD₅₀) of WSN-PB2_627E (B, D and F). Survival (A and B) and weight loss (C and D) were monitored for 14 days. Mice receiving PBS were used as controls. Data shown representative average values per group. (E and F) Lungs were removed on days 3 (n = 5) and 6 (n = 5) post infection (p.i.). Virus titres were determined by plaque assay.