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. 2017 Mar 13;16(5):728–742. doi: 10.1074/mcp.M116.065904

Fig. 8.

Fig. 8.

VprBP supports IAV replication. A, B, 293T cells were transiently transfected with the pHW expression plasmids coding for PB1, PA, PB2, NP and pPol-A/NS-Luc encoding a firefly luciferase cDNA of negative polarity flanked by the noncoding regions of the viral A/NS segment. The effect of VprBP was analyzed by co-transfection of 50 pmol Flexitube siRNA directed against VprBP (Qiagen, Hilden, Germany) (A) and by co-transfection of the expression construct (500 ng) (B). Luciferase activity of cells transfected with AllStars Control siRNA or empty plasmid was set to 100%. Cells not expressing PB1 were used as negative control. Data are means + S.E. (A: n = 3; B, n = 6; biological duplicates). p values were calculated using Wilcoxon signed rank test. C, A549 cells were transfected with 500 ng VprBP expression plasmid for 24 h. Then, cells were infected with Pan or Mal virus at an MOI of 1. At 48 h p.i. supernatants were collected and cell lysates were prepared in 1% SDS buffer. Titers were determined using standard plaque assay and protein content of infected cells was analyzed by immunoblot. Cells transfected with an empty plasmid were set to 100%. p values were determined by Mann Whitney U test. Data are means + S.E. (n = 4; biological duplicates). A–C: *, p < 0.05; **, p < 0.01; ***, p < 0.001. D: A549 cells were infected with Pan or Mal virus at an MOI of 0.5. At 4 h and 16 h p.i. cells were fixed, permeabilized, stained with specific primary antibody against NP, SAMHD1 and with DAPI. White arrows indicate NP positive cells with low SAMHD1 expression levels, yellow arrows mark infected cells with high levels of SAMHD1. Images were representative for three independent experiments. Scale bar: 10 μm. Infected cells (white NP signal) were classified according to their SAMHD1 expression levels. Cells with SAMHD1 signal below the threshold of 52 (green signal) were classified as SAMHD1 negative, while cells with SAMHD1 expression levels above the threshold of 52 were ranked as SAMHD1 positive (red signal). Data are means + S.E. (n = 3).