FIG 6.

Early DR1 KD affects viral RNA transcription and replication. (A) DR1 KD and shLacZ control A549 cells at 4 dpt were infected with IAV at an MOI of 5 (at 37°C) for 15 or 25 min, harvested, and then subjected to subcellular fractionation of total RNA. The NP vRNA in different fractions was measured by qRT-PCR and normalized to U6 snRNA or GAPDH mRNA for the nuclear fraction or the cytoplasmic fraction, respectively. The percentage of vRNA was the ratio of the amount of vRNA from each fraction to the amount of vRNA from whole cells. Data represent the means for two independent experiments. (B and C) DR1 KD and shLacZ control A549 cells at 4 dpt were infected with IAV at an MOI of 1 for the indicated times, harvested, and then subjected to qRT-PCR assays. Data represent means ± SD (n ≥ 3; *, P ≤ 0.05 by Student's t test). (D) DR1 KD and shLacZ control HEK293T cells at 4 dpt were transfected with PB1, PB2, PA, and NP mRNAs, RLuc-mRNA, and FLuc-vRNA (3P/NP), or only with FLuc-vRNA and RLuc-mRNA, as negative controls (mock). At 24 h posttransfection, the total cell lysates were subjected to luciferase activity assay. The value for the control cells transfected with 3P/NP was set to 1. Data represent means ± SD (n ≥ 3; *, P ≤ 0.05 bt Student's t test).