Fig 2: KPNA6 without NLS domain (KPNA6-?147–239) can still inhibit polymerase activity. (A) HEK293T and KPNA6-KO cells were transfected with the H7N9AH13(PB2-627K) minigenome reporting system and different constructs, KPNA6, KPNA6-?147–239, KPNA6-?316–404. Luciferase activity was measured 24 h after transfection. Error bars represent the SD of the replicates within one representative experiment. NS, not significant; **, P < 0.01; ***, P < 0.001. (B) DKO cells were transfected with the H7N9AH13(PB2-627K) minigenome reporting system and ANP32B, KPNA6, KPNA6-?147–239. Luciferase activity was measured 24 h after transfection. The samples were subjected to Western blotting. Error bars represent the SD of the replicates within one representative experiment. ***, P < 0.001. (C) DKO cells were transfected with the H7N9AH13(PB2-627K) and different constructs, as indicated in the figure. Twenty-four hours after transfection, cell lysates were used for anti-Flag precipitation. The immunoprecipitated samples were subjected to Western blotting.

Fig 3: KPNA6 competitively binding vRNP with ANP32A to inhibit polymerase activity supported by ANP32 proteins. (A) DKO cells were transfected with H7N9AH13(PB2-627K) minigenome reporting system, with an increased amount of either ANP32A or ANP32A-NLSm. Luciferase activity was analyzed as mentioned above. Error bars represent the SD of the replicates within one representative experiment. NS, not significant. (B) DKO cells were transfected with H7N9AH13(PB2-627K) minigenome reporting system, with an increased amount of either ANP32B or ANP32B-NLSm. Luciferase activity was analyzed as mentioned above. Error bars represent the SD of the replicates within one representative experiment. NS, not significant. (C) The impact of an increased amount of KPNA6 overexpression on the H7N9AH13(PB2-627K) vPol activity supported by ANP32A or ANP32A-NLSm. Error bars represent the SD of the replicates within one representative experiment. **, P < 0.01. (D) DKO cells were transfected with the H7N9AH13(PB2-627K) minigenome reporting system together with 200 ng KPNA6 and an increased amount of ANP32A or ANP32A-NLSm. Luciferase activity was measured 24 h after transfection. Error bars represent the SD of the replicates within one representative experiment. *, P < 0.05; **, P < 0.01. (E and F) Experiments performed as in panels C and D, respectively, except that ANP32B and ANP32B-NLSm were used. (G) DKO cells were transfected with the H7N9AH13(PB2-627K) minigenome reporting system and different constructs as indicated in the figure. Twenty-four hours after transfection, cell lysates were used for anti-Flag precipitation and anti-HA precipitation. The immunoprecipitated samples were subjected to Western blotting.

Fig 4: ANP32A is required for IAV genome replication. (A) Human HEK-293T cells or (B) chicken DF-1 cells were infected with influenza A/WSN/33 (H1N1) virus carrying either a lysine (K; WSN) or glutamic acid (E; WSN-K627E) on residue 627 of the PB2 subunit for 6 h at an MOI of 1. The accumulation of viral RNA was analyzed by primer extension and 6% PAGE. The graph shows the relative mean intensity of viral RNA normalized to 5S rRNA from three independent biological replicates. Error bars represent the standard deviation of the means and asterisks represent a significant difference from the control group (one-way ANOVA with Dunnett’s post hoc test) as follows: ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (C-D) HEK-293T cells were transiently transfected with plasmids expressing GFP or chANP32A. At 24 h posttransfection, cells were infected with (C) WSN or (D) WSN-K627E virus at an MOI of 0.01. Infectious viral titers were determined by plaque assay at the indicated time points postinfection from three independent biological replicates. Error bars represent the standard deviation of the means and asterisks represent a significant difference from the control group (multiple paired t tests with Holm-Šídák correction for multiple comparisons) as follows: ns, P > 0.05; *, P < 0.05. (E) HEK-293T cells were transfected with plasmids expressing huANP32A (hu) or chANP32A (ch) or GFP as a negative control. At 24 h posttransfection, cells were infected with wild-type WSN or WSN-K627E at an MOI of 1 for 6 h. The accumulation of viral RNA was analyzed by primer extension and 6% PAGE. The graph shows the relative mean intensity of viral RNA normalized to 5S rRNA from three independent biological replicates. Error bars represent the standard deviation of the means and asterisks represent a significant difference from the control group (one-way ANOVA with Dunnett’s post hoc test) as follows: ns, P > 0.05; *, P < 0.05; **, P < 0.01; ****, P < 0.0001. (F) HEK-293T cells were transfected with plasmids expressing NP, PA, PB1, PB2 (627K or 627E), segment 6 vRNA and huANP32A, chANP32A or GFP. At 48h posttransfection, the accumulation of viral RNA was analyzed by primer extension and 6% PAGE. The graph shows the relative mean intensity of viral RNA normalized to 5S rRNA from four independent biological replicates. Error bars represent the standard deviation of the means and asterisks represent a significant difference from the control group (one-way ANOVA with Dunnett’s post hoc test) as follows: ns, P > 0.05; *, P < 0.05; ***, P < 0.001. (G) HEK-293T cells were infected with wild-type WSN or WSN-K627E at an MOI of 10 for 6 h in the presence of cycloheximide. The accumulation of viral RNA was analyzed by primer extension and 6% PAGE. The graph shows the relative mean intensity of viral RNA normalized to 5S rRNA from three independent biological replicates. Error bars represent the standard deviation of the means and asterisks represent a significant difference from the control group (one-sample two-tailed t test) as follows: ns, P > 0.05. (F-G) A549 cells were transfected with siRNA targeting human ANP32A and ANP32B. At 48 h posttransfection cells were either (F) lysed for Western blot analysis for ANP32A and ANP32B expression or (G) infected with WSN at an MOI of 1 for 12 h. The accumulation of viral RNA was analyzed by primer extension and 6% PAGE.

Fig 5: KPNA6 affects ANP32-vRNP interaction. (A) (left) The dose-dependent effect of KPNA6 expression on the polymerase activity of H7N9AH13(PB2-627K) polymerase activity assays was conducted by transfection with indicated plasmids in HEK293T cells, (middle) Western blot of 293T cell lysates from the left panel, and (right) Western blot of polymerase protein expression in WT and KPNA6 knockout cell lines. Error bars represent the SD of the replicates within one representative experiment. *, P < 0.05; ****, P < 0.0001. (B and C) 293T cells were transfected with either empty control or the indicated Flag-tagged constructs, together with HA-tagged KPNA6. Following anti-Flag precipitation (B) and anti-HA precipitation (C), the indicated proteins were detected by Western blotting. (D) 293T cells were transfected with either empty control or HA-tagged KPNA6, together with the viral trimeric polymerase, NP, and a minigenome reporter pHH21-huPolI-vLuc. Following anti-huANP32B precipitation, the indicated proteins were detected by Western blotting. The bar graph represents the relative amount of PB1 and PB2 immunoprecipitated by the ANP32B. Error bars represent the SD of the replicates from three independent experiments. **, P < 0.01; ***, P < 0.001. (E) HEK293T cells and KPNA6 knockout cell lines were transfected with the viral trimeric polymerase, NP, and a minigenome reporter pHH21-huPolI-vLuc. Following anti-huANP32B precipitation, the indicated proteins were detected by Western blotting. The bar graph represents the relative amount of PB1 and PB2 immunoprecipitated by the ANP32B. Error bars represent the SD of the results from three independent experiments. ***, P < 0.001.