Does treatment of cells with inhibitors of reovirus disassembly block apoptosis induced by virions?

Reovirus virions are converted to ISVPs infectious subvirion particles in the cellular endosome. This process requires acidic pH and the activity of cysteine-containing proteases enzymes that breakdown proteins . HeLa cells were incubated in the absense and presense of either ammonium chloride (AC), an inhibitor of endosomal acidification, or E64, an inhibitor of cysteine-containing proteases, to determine whether acid-dependent or protease-dependent steps in viral uncoating are required for reovirus induced apoptosis.

METHODS

1. HeLa cells (5 x 10⁴) were either mock infected or infected with T3SA+ virions a strain of reovirus at a MOI muliplicity of infection of 100 PFU per cell. Cells were also treated with 10 ng of TNF-a a known inducer of apoptosis per ml instead of T3SA⁺ virions as a positive control.
   
   
2. Cells were incubated in the absense or presence of 10 mM AC or 200 uM E64, concentrations that block reovirus disassembly and growth in HeLa cells.
   
   
3. Viral yields were calculated by dividing the viral titer at 48 h by the viral titer at 0 h.
   
   
4. The percent apoptotic cells was assessed by acridine orange fluorescent dye that stains DNA staining 48 h after infection. For each experiment, 200 to 300 cells were counted, and the percentage of cells exhibiting condensed chromatin a hallmark of apoptosis was determined by fluorescence microscopy.
   
   
5. Results are expressed as the mean viral yield or the mean percentage of cells undergoing apoptosis for three independent experiments. Error bars indicate standard deviation of the means.

RESULTS

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