Fig 2: H7N9 PA determines the sensitivity of viral polymerase activity and growth to changes in human ANP32A expression. (A) GST pulldown assay to determine the interaction between human ANP32A and PG/S1421 (H7N9) polymerase complexes bearing different PAs. (B) ANP32A overexpression in HEK293T cells transfected with pCAGGS-ANP32A. (C) Luciferase assay in ANP32A-overexpressing cells (as shown in panel B) that were transfected with PG/S1421-PB2, -PB1, -NP, and different PAs, together with luciferase reporter constructs. Data shown are the ratio of the luciferase activity of the ANP32A-overexpressing group normalized to the pCAGGS control group. (D) siRNA knockdown of ANP32A in HEK293T cells. (E) Luciferase assay in ANP32A siRNA-treated HEK293T cells, as described for panel C. Data shown are the ratio of the luciferase activity of the ANP32A siRNA-treated group normalized to the scrambled siRNA-treated group. (F) Generation of Anp32a-KO HEK293T cells by using the CRISPR/Cas9 system. (G) Luciferase assay in Anp32a-KO HEK293T cells, as described for panel C. Data shown are the ratio of the luciferase activity of the Anp32a-KO HEK293T cells normalized to the HEK293T control group. (H) Generation of Anp32a-KO A549 cells by using the CRISPR/Cas9 system. (I) Anp32a-KO or control A549 cells were infected with the indicated PG/S1421(H7N9) viruses bearing different PAs. Data shown are the percentage of virus titers in Anp32a-KO cells normalized to the control A549 cells. CK/5PA1-191 stands for the construct PG/S1421-CK/5PA1–191; PG/S1421-PAmut stands for the construct PG/S1421-PA142R-147V-171V-182L; data are representative of three (C, E, and G) or two (I) independent experiments (means ± SDs); P values were determined by using a two-tailed unpaired Student's t test compared to the RNP complex bearing PG/S1421 PA (C, E, and G) and by using multiple t tests compared to viral titers in control A549 cells (I). NS, not significant.