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. 2017 Feb 14;91(5):e01606-16. doi: 10.1128/JVI.01606-16

FIG 3.

FIG 3

IRAV associates with the DENV replication complex. (A) HEK293 cells stably expressing IRAV or a negative control were infected with DENV at an MOI of 0.1, and samples were collected at time zero and 72 h postinfection, followed by titration using the plaque assay method. The inset represents expression of IRAV in transfected HEK293 cells compared to the negative control, as determined by Western blot analysis. GAPDH was used as a loading control. (B) HEK293 cells stably expressing IRAV or negative-control (NC) cells were treated with either negative-control siRNA or an siRNA specific to IRAV (IRAV_I). The cells were then infected with DENV for 72 h, followed by titration on Vero cells. (C) IRAV coimmunoprecipitates with DENV proteins. HEK293 cells were either left uninfected (−) or infected with DENV (+) for 48 h, followed by transfection with a plasmid expressing GFP-IRAV (IRAV) or GFP-CAT (Control) for an additional 48 h. The cell lysates were then collected, and co-IP experiments were performed using antibodies to GFP. Input and IP samples were then analyzed by Western blotting for the presence of DENV envelope (DENV-E), DENV NS3, or DENV NS4A protein. (D) Confocal microscopy of IRAV colocalized with replication complexes in DENV-infected or mock-infected A549 cells. Green, IRAV; red, DENV-NS3. The nucleus was stained with DAPI (blue). Colocalization between IRAV and DENV-NS3 is shown in white. Low Mag, a lower-magnification field showing a cluster of infected cells. (E) Confocal microscopy of IRAV colocalized with replication complexes in DENV-infected or mock-infected A549 cells. Green, IRAV; red, DENV NS4A. The nucleus was stained with DAPI (blue). Colocalization between IRAV and DENV NS4A is shown in white. (F) Colocalization coefficients of IRAV and DENV NS3 (NS3) or DENV NS4A (NS4A) in DENV-infected A549 cells as determined by Pearson's linear correlation coefficient. (G) Confocal microscopy of IRAV colocalized with replication complexes in DENV-infected or mock-infected monocyte-derived macrophages. Green, IRAV; red, DENV-NS3. The nucleus was stained with DAPI (blue). (H) Confocal microscopy of IRAV colocalized with replication complexes in DENV-infected or mock-infected monocyte-derived macrophages. Green, IRAV; red, DENV NS4A. The nucleus was stained with DAPI (blue). The error bars represent standard deviations.