Figure 5. Actin and actin regulatory proteins are important for ZEBOV infection.

(A) Suppression of Pak1 by siRNA blocks ZEBOV infection. HEK293 cells were transfected with siRNA targeting Pak1 (two distinct siRNA, i and ii, used) or non-targeting siRNA. Expression of Pak1 was evaluated by Western blot using an appropriate antibody (Cell Signaling Technology, MA) and relative peak intensity determined by densitometry using a Typhoon scanner and associated software (GE Biosciences, NJ). The impact of Pak1 suppression on gfpZEBOV infection was then determined and expressed relative to untransfected controls. (B) DN Pak1 reduces ZEBOV infection. HEK293T cells were transfected with plasmids encoding β-galactosidase (β-gal) or myc/GST-tagged forms of wt Pak1 or DN-Pak1. 36 h later, cells were infected with gfpZEBOV and after 24 h were fixed and stained for myc or GST tags using appropriate primary and secondary antibodies. Cells were then imaged and analyzed as in the methods. The proportion of cells that were expressing each tagged protein and infected by ZEBOV was calculated as a fraction of the total cell population and expressed relative to the infection seen for cells transfected with plasmid encoding β-galactosidase. (C) Suppression of CtBP/BARS by siRNA blocks ZEBOV infection. HEK293 cells were transfected with siRNA targeting CtBP/BARS (two used, i and ii) or non-targeting or firefly luciferase (luc) targeting siRNA. Expression levels were determined by evaluating immunofluorescent staining intensity of CtBP/BARS in nuclei of each cell (CtBP/BARS is predominantly localized to cell nucleus) and normalizing to the nuclear stain, DAPI and untransfected controls. The left panel shows portion of microscope image with cell nuclei stained with DAPI or CtBP/BARS antibody and center panel shows quantitation of staining from 20,000 cells. Right panel shows impact on infection by ZEBOV-GFP. (D) ZEBOV induces Arp2-nucleation. Vero cells were incubated in medium without virus or replication-competent infectious ZEBOV (MOI  = 5) for the indicated time. Subsequently cells were washed, fixed, permeabilized and stained for Arp2 protein using a specific antibody. The number and apparent size of Arp2 complexes was analyzed using the Analyze particles function of ImageJ software (http://rsbweb.nih.gov/ij/). While total number of Arp2 clusters did not change, the size distribution was altered by ZEBOV incubation with cells. This was expressed as the number of Arp2 complexes of the size ranges indicated (area occupied in image) relative to the total number of complexes (*-P<0.05, **-P<0.01). (E) Images showing Arp2 nucleation. Arp2 (red), DAPI stained nuclei (blue). Images were taken by confocal microscopy using a 100× oil immersion objective lens. (F) ZEBO-VLPs associate with Arp2 complexes. Vero cells were incubated with gfpZEBO-VLPs (green) for 30 min and then fixed, permeabilized and stained for Arp2 (red) using appropriate antibodies. (G) ZEBO-VLPs associate with actin foci and (H) VASP protein during cell entry. Vero cells were incubated with fluorescently-labeled ZEBO-VLPs or VSV-VLPs (green). After 30 min, cells were washed, fixed and permeabilized. For actin staining, cells were incubated with medium containing fluorescently-labeled phalloidin (red). For VASP staining, cells were incubated with anti-phospho-VASP antibody, followed by fluorescently-labeled secondary antibody (red). Arrowheads indicate representative examples of VLP colocalization with actin or VASP. All Images were taken by confocal microscopy using a 100× objective lens.