FIG 2.
RHA knockdown reduced the viral RNA and protein levels. A549 cells were transfected with siNC or siRHAm for 48 h and applied for the following detections. (A) Entry assay. Cells were inoculated with DENV2 (MOI of 3), followed by incubation at 4°C or 37°C for 1 h. Cells were washed with PBS and harvested for RNA extraction and real-time PCR to measure viral RNA levels. (B) Cells were mock infected or infected with DENV2 and harvested at 6, 12, and 24 h p.i. The viral RNA level was measured by real-time PCR. (C and D) Cells were infected with DENV2 and harvested at 8, 16, and 24 h p.i. for Western blotting to detect NS3 (C) or at 24 h p.i. for IFA to detect prM (D). Actin served as an internal control. (E) At least 200 cells from each sample in three independent experiments were counted. (F) Effect of RHA knockdown on translation of the DENV2 reporter replicon. Cells were transfected with pDENrep-FH (16681) replicon (WT) and the NS5 GVD mutant (NS5GVD) RNAs. The cells were harvested at 6 h posttransfection for luciferase assay. Data are shown as means ± SD from at least three independent experiments. NS, no statistical significance; ***, P < 0.001 by unpaired, two-tailed Student's t test.