FIG 1.

DR1 acts as a positive regulator of IAV replication. A549 cells were transduced with the indicated lentiviruses for 10 days to generate stable cell lines and then infected with IAV for 6 h at an MOI of 1 for qRT-PCR assay or Western blotting. (A and B) The stable cells harboring shDR1-1, shDR1-2, or the shLacZ control were infected with IAV, harvested, and then subjected to qRT-PCR assays or Western blotting by using the indicated antibodies. Cellular DR1 mRNA and NP vRNA were measured and normalized to GAPDH mRNA, and the value for the control cells was set to 1. Data represent means ± standard deviations (SD) (n ≥ 3; *, P ≤ 0.05 by Student's t test). (C) For overexpression assay, overexpressed Myc-tagged DR1 (DR1-Myc OE) or control (vector only) cells at 8 dpt were infected with IAV, harvested, and then subjected to Western blotting. The band intensities of NP and actin were quantified, and the NP/actin ratios are shown below the blots. (D) The stable DR1 KD and shLacZ control A549 cells were infected with IAV at an MOI of 0.01. At 24 or 48 hpi, the supernatants were titrated by plaque assay to determine the virus titers. Data represent means ± SD (n ≥ 3; *, P ≤ 0.05 by Student's t test). (E) For the wobble mutant rescue experiment, the DR1 KD (shDR1-1) and shLacZ control cells were further transduced with a lentivirus overexpressing a DR1 wobble mutant (DR1-Myc-wobble) or the control (vector only) for an additional 8 days before being infected with IAV, and the cell lysates were subjected to Western blotting.