FIG 1 .

Knockdown of CNOT4 inhibits IAV replication. (A) Immunofluorescence analysis of secondary RNAi screening. A549 cells were transduced with various shRNA-harboring lentiviruses that targeted the different genes identified in the primary pooled shRNA library screen (19). The transduced cells were then infected with WSN/33 virus at an MOI of 1. At 6 h p.i., the cells were subjected to immunofluorescence staining using NP antibody and DAPI. The intensity of NP-DAPI was used as an indicator and normalized to control shLacZ staining intensity to estimate the percentage of cells infected with IAV. (B and C) CNOT4 knockdown efficiency (B) and relative viral NP RNA levels (C) were determined by qRT-PCR, using RNA from the various CNOT4 knockdown A549 cells (shRNA clones CN1 to CN5) at 6 h p.i. (means ± SD, n = 4). *, P < 0.05; **, P < 0.01; ***, P < 0.001, based on one-way ANOVA with Dunnett’s multiple-comparison test. (D) Viral proteins were detected by immunoblotting at 6 h p.i. with an anti-NP antibody. Actin was used as an internal control. (E) Virus titers from progeny virus released into the medium were determined at 24 h p.i. via plaque assay on MDCK cells (means ± SD, n = 3). ***, P < 0.001, based on one-way ANOVA with Dunnett’s multiple-comparison test.