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Figure 2 | Virology Journal

Figure 2

From: Do viruses require the cytoskeleton?

Figure 2

Analysis of SINV replication during cytoskeletal drug treatment. A and B. BHK cells infected at an MOI = 1 for 1 hour with SINV expressing an nsP3-GFP fusion protein (green), called SINV/NSP3-GFP, before treating the infected cells with the appropriate cytoskeletal drug for 5 hours. At 6 hours post-infection, the infected cells were stained for fluorescence microscopy with rabbit anti-microtubule antibodies (secondary antibody was donkey anti-rabbit AlexaFluor 595), the F-actin filaments with phalloidin AlexaFluor 568-conjugate (A), or staining for the structural proteins with rabbit antibodies raised against purified virus (visualized by donkey anti-rabbit Alexa Fluor 595-conjugate) (B). Insets in B (red), show localization of structural proteins. NT, no treatment. Nuclei were stained with Hoechst 33342 (blue). Bars represent 10 μm. White arrows point to juxtanuclear foci of structural proteins, blue arrow points to scattered structural protein foci. C. BHK cells infected with SINV (MOI = 1 pfu/cell) for 1 hour and then incubated with medium or medium containing 30 μM colchicine for an additional 5 hours were treated with a Golgi stain (wheat germ agglutinin Alexa Fluor 595 conjugate, red) along with antibodies against SINV structural proteins that were visualized with anti-rabbit FITC conjugate (green) to localize the structural proteins. Nuclei were stained with Hoechst 33342 (blue). Bars represent 20 μm. White arrows point to structural protein overlap with the Golgi. Yellow arrows point to fragmented, but overlapping, Golgi and structural proteins. D. Western blotting of SINV-infected (MOI = 1 pfu/cell) BHK cell lysates prepared as in A. Calnexin serves as a loading control. NSP2 was detected with rabbit anti-NSP2 antibody. The structural proteins, E1, pE2 and E2 were detected with rabbit polyclonal antibodies. pE2 and E2 proteins were detected with cdE2 antibodies. Protein bands were visualized with the species-specific alkaline phosphatase-conjugated antibodies and substrate NBT/BCIP.

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