FIG 7.
Difference between antiviral effects of cIAV DsiRNAs in moDCs transfected before and after IAV infection. Human moDCs from four different blood donors were separately mock transfected (control, UV IAV, or no-siRNA bars) or pretransfected with the indicated siRNAs (10 nM) either 21 h before or 1 h after A/Udorn/307/72 (H3N2) virus infection. Cells were infected with live or UV-irradiated A/Udorn/307/72 IAV at an MOI of 1. At 24 h p.i. cells from four different blood donors were pooled, and IAV M1 RNA expression was determined by qRT-PCR from isolated total cellular RNA or by Western blotting from cell lysates. (A) Relative IAV M1 RNA expression in cells transfected 21 h before infection or 1 h after infection. The values were normalized against β-actin gene-specific mRNA, and relative IAV M1 RNA levels were calculated by the ΔΔCT method using untreated cells as a calibrator. The means (±SD) of three parallel determinations are shown. Data are representative of three individual experiments. Statistical significance was determined against results from samples of nontransfected cells (boxed bars). *, P < 0.05. (B) Western blot analysis for the expression of viral PB1, NP, M1, and NS1 proteins and GAPDH protein in siRNA/DsiRNA transfected moDCs after A/Udorn/72 IAV infection. Cells were collected at 24 h p.i., and whole-cell lysates were prepared. Cellular proteins (30 μg/lane) were separated by 10% SDS-PAGE, followed by Western blot analysis with the indicated antibodies. One representative experiment of three independent experiments is shown.