FIG 1.
huANP32A&B are indispensable for influenza A virus polymerase activity and viral replication. (A) Wild-type 293T cells and single-gene-knockout 293T cell lines, including BUB3, CLTC, CYC1, NIBP, ZC3H15, C14orf173, CTNNB1, ANP32A, ANP32B, SUPT5H, HTATSF1, and DDX17, were transfected with firefly minigenome reporter, Renilla expression control, and the polymerase of H1N1SC09. The luciferase activity was measured 24 h after transfection, and data indicate the firefly luciferase gene activity normalized to the Renilla luciferase gene activity. Statistical differences between samples are indicated, according to a one-way ANOVA, followed by a Dunnett’s test (NS, not significant; *, P < 0.05; **, P < 0.01; ****, P < 0.0001). Error bars represent the SEM within one representative experiment. (B) Wild-type 293T cells were transfected with pMJ920 vector (a plasmid expressing eGFP and Cas9) and gRNAs targeting huANP32A and huANP32B to generate huANP32A&B double-knockout cells (DKO). The endogenous proteins of different cells (293T cells, huANP32A knockout cells [AKO], huANP32B knockout cells [BKO], and DKO cells) were identified by Western blotting with antibodies against β-actin, huANP32A, and huANP32B as described in Materials and Methods. (C) Design scheme of sgRNAs for huANP32A and huANP32B. (D to F) Wild-type 293T cells, huANP32A knockout cells (AKO), huANP32B knockout cells (BKO), and huANP32A&B double-knockout cells (DKO) were transfected with firefly minigenome reporter, Renilla expression control, and either H1N1SC09 polymerase (D), H7N9AH13 polymerase (E), or WSN polymerase (F). The luciferase activity was measured at 24 h posttransfection. For panels D to F, the data are the firefly luciferase gene activity normalized to that of Renilla luciferase activity. Statistical differences between cells are indicated, following a one-way ANOVA and subsequent Dunnett’s test (NS, not significant; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Error bars represent the SEM of replicates within one representative experiment. The expression levels of H1N1SC09 polymerase proteins were assessed by Western blotting in panel D. (G) Wild-type 293T, AKO, BKO, and DKO cells were infected with WSN virus at an MOI of 0.01. The supernatants were sampled at 0, 12, 24, 36, and 48 h postinfection, and the virus titers were determined by endpoint titration in MDCK cells. Error bars indicate the SD from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.