Fig 2: 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 3: Identification of key domains involved in the interaction of ANP32 and KPNA6. (A) Cartoon showing the wild type of human ANP32 proteins and the mutants (ANP32A-NLSm) with destroyed nuclear location signal by mutating KRKR into AAAA. The NLS area of ANP32A is 236 to 239, and the NLS of ANP32B is 239 to 242. (B) ANP32A-Flag and ANP32A-NLSm-Flag were cotransfected with KPNA6-HA into HEK293T cells. Following anti-Flag precipitation 24 h after transfection, the immunoprecipitated samples were subjected to Western blotting (left panel). Experiments were performed in the left panel, except that ANP32B-Flag and ANP32B-NLSm-Flag were used for transfection (right panel). (C and D) Immunofluorescence analysis of the indicated Flag constructs in HEK293T cells. (E) Cartoon showing the structure of the different KPNA6 truncations and the position of different key domains in KPNA6, which includes an N-terminal importin ß-binding (IBB) domain, armadillo (Arm) repeats that function as internal cargo NLS-binding sites, and a C-terminal region (CAS). (F) Different HA-tagged KPNA6 truncations and Flag-tagged ANP32A (left panel) or Flag-tagged ANP32B (right panel) were transfected into HEK293T cells. Following anti-Flag precipitation 24 h after transfection, the immunoprecipitated samples were subjected to Western blotting.

Fig 4: Overexpression of KPNA6 inhibits the polymerase activity of influenza virus. (A) Flag-tagged ANP32A constructs or the low-complexity acidic region (LCAR 162 to 249) truncations of ANP32A (ANP32A-?LCAR), together with PB1, PB2, and PA expression plasmids of H7N9AH13(PB2-627K), were transfected into HEK293T cells. The immunoprecipitated samples were subjected to SDS-PAGE analysis with Coomassie blue staining following anti-Flag precipitation. The corresponding proteins are marked with an arrow (A, left); high-confidence candidates were identified by mass spectrometry (MS) analysis with the immunoprecipitated samples and are shown as a table (A, right). (B and C) Minigenome assay of H7N9AH13(PB2-627K) (B) and IBV-Yamagata (C) in human HEK293T cells with coexpressed empty vector and different Flag-tagged constructs. Error bars represent the standard deviation (SD) of the replicates within one representative experiment. NS, not significant; *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. (D and E) The mRNA level of endogenous KPNA6 in A549 (D) and HEK293T (E) cells in response to IAV (H1N1WSN) infection (MOI = 0.01). The cells were collected at 0, 12, 24, 36, and 48 h postinfection, followed by quantitative PCR to measure the mRNA level of KPNA6. Error bars represent the SD of the replicates within one representative experiment. NS, not significant; *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. (F) The protein level of endogenous KPNA6 in HEK293T cells in response to IAV (H1N1WSN) infection (MOI = 0.01). The cells were collected at 0, 12, and 24 h postinfection, followed by Western blotting of KPNA6 and influenza polymerase subunit PA. The bar represents the levels of KPNA6 and PA in the left panel, which were normalized to ACTB. NS, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig 5: KPNA6 inhibits ANP32-vRNP interaction in a dose-dependent manner. (A) The effect of the increased amount of KPNA6 overexpression on the 10 or 320 ng of ANP32A and ANP32B supported H7N9AH13(PB2-627K) vPol activity in DKO cells. Error bars represent the SD of the replicates within one representative experiment. NS, not significant; *, P < 0.05; **, P < 0.01; ****, P < 0.0001. (B) DKO cells were transfected with the H7N9AH13(PB2-627K) minigenome reporting system and 200 ng of KPNA6 and increased ANP32A. Luciferase activity was measured 24 h after transfection. Error bars represent the SD of the replicates within one representative experiment. NS, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (C) The effect of the increased amount of KPNA6 overexpression on the 10 ng or 320 ng of ANP32B supported H7N9AH13(PB2-627K) vPol activity in DKO cells. Error bars represent the SD of the replicates within one representative experiment. NS, not significant; **, P < 0.01; ***, P < 0.001. (D) DKO cells were transfected with the H7N9AH13(PB2-627K) minigenome reporting system and 200 ng of KPNA6 and increased ANP32B. Luciferase activity was measured 24 h after transfection. Error bars represent the SD of the replicates within one representative experiment. NS, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. (E) The impact of the increased amount of ectopic expression of KPNA6 on the ANP32A-vRNP interactions. An experiment was performed as in Fig. 3D. (F) The impact of the increased amount of ectopic expression of KPNA6 on the ANP32B-vRNP interactions. An experiment was performed as in Fig. 3D. (G) Experiment performed as in panel E, except the PB1 was omitted when transfected to prove that ANP32A binds only to vRNP. (H) Experiment performed as in panel F, except the PB1 is omitted when transfected to prove that ANP32B binds only to vRNP.