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. 2014 Oct 16;10(10):e1004463. doi: 10.1371/journal.ppat.1004463

Figure 9. IQGAP1 is co-localized with inclusions and nucleocapsids and supports MARV release.

Figure 9

(A) Co-localization of Tsg101 and IQGAP1 in NP inclusions. Huh-7 cells were co-transfected with plasmids encoding NPwt or NPPSAPmut and VP40, mCherry-Tsg101 and IQGAP1-YFP. Cells were fixed 24 h p.tr., stained with NP-specific antibody and subjected to immunofluorescence analysis. White arrow shows co-localization of mCherry-Tsg101 (red), IQGAP1-YFP (green) with NP inclusions (blue) in the merge picture. In NPPSAPmut transfected cells only mCherry-Tsg101 and IQGAP1-YFP are co-localized (arrowhead). (B) Co-localization of Tsg101 and IQGAP1 in MARV infected cell. Huh-7 cells were infected with rMARVwt or rMARVPSAPmut and co-transfected with mCherry-Tsg101 and IQGAP1-YFP encoding plasmids. Cells were fixed 24 h p.i. stained with NP-specific antibody and analyzed by CLSM. Lower panels show higher magnification of boxed area and white arrow indicates co-localization of nucleocapsid with mCherry-Tsg101 and IQGAP1-YFP in wild type infected cells whereas mutant nucleocapsids did not show any co-localization. (C) IQGAP1 depletion of infected cells. MARV-infected Huh-7 cells (MOI of 1) were transfected with IQGAP1-specific siRNA or control siRNA at 1 h p.i. Cells and culture supernatants were harvested at 48 h and 72 h p.i. Lysates and supernatants collected at 72 h p.i. were subjected to Western Blot analysis. (D) Virus titers in the supernatants of MARV infected cells transfected with IQGAP1-specific or control siRNA were determined by TCID50 titration, p-value (*, P≤0.05). (E) Phenotype of IQGAP1-knockdown in MARV-infected cells at 48 h p.i. Huh-7 cells grown on cover slips were infected with MARVwt and treated with IQGAP1 specific or control siRNA and subjected to immunofluorescence analysis using NP-specific antibody. Grey boxes indicate marginal region of cells. Lower panels show higher magnification of boxed area, arrow indicates accumulation of nucleocapsids upon IQGAP1 knockdown at cell periphery marked with dashed line. Bars, 10 µm.