Journal: PLoS Pathogens
Article Title: Host Cell Entry of Respiratory Syncytial Virus Involves Macropinocytosis Followed by Proteolytic Activation of the F Protein
Figure Lengend Snippet: RSV infection induces actin rearrangement. (A). RSV (moi ∼0.5) was bound to HeLa cells at 4°C, unbound virus was removed and cells warmed to 37°C, fixed at indicated times, and stained with phalloidin-AF488 (pseudocolored white) and anti-F-AF647 (red) antibody. Images represent Z-stack projections acquired with a confocal microscope. Arrowheads show actin blebs formed at the cell surface. (B). RSV (moi ∼30) was incubated with HeLa cells for 30 or 120 min at 37°C. Samples were processed according to the kit manufacturer's protocol (Cytoskeleton Inc.). Controls included mock-treated cells and cells either treated with F actin enhancer or F actin depolymerizing agent. (left) The F and G actin fractions were resolved by SDS-PAGE and western blots probed with anti-actin antibody. (right) Quantification of actin protein bands intensities by densitometry. (C–F). HeLa cells were pretreated with solvent (MOCK), cytochalasin D (CytoD), latrunculin A (LatA), jasplakinolide (Jas), nocodazole (Noc), taxol (Tax), NCS23766, pirl1, IPA-3, wiskostatin (Wisko), CK-869, CT04, Y24632 at indicated concentrations and each inhibitor was continuously present during following steps of the experiment: (C, E). Cells where infected with RSV (moi ∼3) or SFV-ZsGreen (moi ∼0.5) for up to 6 hours before FACS analysis of GFP expressing cells. (D, F). RSV (moi ∼3) was bound to the cells at 4°C followed by 1 h of internalization at 37°C. Cells were trypsinized, fixed and stained with anti-N-AF488 antibody, and the MFI of AF-488 measured by FACS.
Article Snippet: Inhibitors, antibodies and plasmids The inhibitors used included: PI-103 (Alexis Biochemicals), dynasore, dyngo-4a,dynol-31-2, pitstop-2 (Ascent Scientific), pirl1 (Chembridge), wiskostatin (Enzo), CAS 879127-07-8, CAS 371942-96-7, dec-RVKR-CMK, LY294002, NSC23766, staurosporine, taxol, wortmannin, Y27632, α-PDX (Calbiochem), bafilomycin A, blebbistatin, calphostin C, chlorpromazine, cytochalasin D, EIPA, genistein, IPA-3, jasplakinolide, latrunculin A, leupeptin, ML7, monensin, NH4Cl, nocodazole, and rottlerin (Sigma).
Techniques: Infection, Staining, Microscopy, Incubation, SDS Page, Western Blot, Indirect Immunoperoxidase Assay, FACS, Expressing