Journal: Molecular Therapy

Article Title: Proteolytic Disassembly Is a Critical Determinant for Reovirus Oncolysis

doi: 10.1038/sj.mt.6300207

Figure Lengend Snippet: Infection of susceptible and resistant cell lines by proteolytically processed reovirus particles (infectious subviral particle or ISVPs) . ( a ) Cells with or without treatment with the protease inhibitor E64 (100 μmol/l) 1 hour before reovirus exposure were pulse-labeled with [ 35 S]methionine for 6 hours at 18 hours post infection (h.p.i.) with an MOI of 20 of reovirus or ISVPs (reovirions processed in 200 μg/ml of chymotrypsin for 30 minutes at 37°C). Cells were lysed; then reovirus proteins were immunoprecipitated from part of the lysate using rabbit polyclonal anti-reovirus antibodies and analyzed by SDS-PAGE and autoradiography. ( b ) Evaluation of reovirus infection using the same infection procedure as described previously but measured at 24 h.p.i. by immunofluorescence using a rabbit anti-reovirus type-3 antibody followed by FITC–conjugated goat anti-rabbit IgG to detect positive reovirus infection (green fluorescing cells). ( c ) Viral progeny production 48 h.p.i. with the foregoing treatments. Cells grown in 6-well plates were infected with reovirus at an MOI of 20; they were then assayed for progeny virus production by plaque titration on L929 cells following three rounds of freeze-thaw and serial dilution of the supernatants. All titration experiments were repeated in triplicate. ( d ) Cells grown to 50% confluence were infected with reovirus or ISVP at an MOI of 20. Cell viability was measured at 48 h.p.i. by MTT assay, and metabolically active cells were quantified by scanning the plates at the 595-nm reference wavelength in a microtiter plate reader. A MTT reagent without cells was used as background control. The experiments were performed in triplicate. ( e ) Evaluation of ISVP infection of other reovirus resistant cells—the glioma U343, the Burkitt lymphoma cell line Daudi, and the normal human foreskin fibroblast HS68—as measured either by immunofluorescence (left panel), metabolic SDS-PAGE as in a (middle panel), or MTT/WST-1 for viability (bottom panel) and proteolytic reovirus processing (right panel).

Article Snippet: For metabolic labeling, [ 35 S]-methionine was added to the culture medium at 18 h.p.i. for a period of 6 hours; then viral protein synthesis was assessed as previously described., For immunofluorescent studies, cells were grown on slides and processed as described but then incubated with rabbit polyclonal anti-reovirus type-3 serum (diluted 1/5,000 in PBS) for 1 hour at room temperature, washed, and incubated with secondary antibody (FITC–conjugated goat anti-rabbit IgG diluted 1/250 in PBS) (Cedarlane, Hornby, Canada) for 1 hour at room temperature.

Techniques: Infection, Protease Inhibitor, Labeling, Immunoprecipitation, SDS Page, Autoradiography, Immunofluorescence, Titration, Serial Dilution, MTT Assay, Metabolic Labelling