Is viral RNA synthesis required for reovirus-induced apoptosis?

 

 

METHODS

1. The concentration of ribavirin a guanosine analog that inhibits production of viral ss- and dsRNA required to block RNA synthesis was tested by infecting HeLa cells with T3SA⁺ reovirus at an MOI of 1,000 PFU per cell. Cell were incubated for 24 h in media containing 25 to 200µM of ribavirin and radiolabeled ³⁵S-methionine-cysteine.
   
   
2. Cells lysates were immunoprecipated using a polyclonal serum that recognizes all reovirus proteins except sigma 1.
   
   
3. The immunoprecipated proteins were run on an SDS-polyacrylamide gel and visualized by autoradiography or quantified using a phosphorimager.
   
   
4. To test for the inhibition of reovirus-induced apoptosis, HeLa cells were mock infected or infected with T3SA⁺ reovirus at an MOI of 1000 PFUs per cell and incubated for 24 h at various concentrations of ribavirin.
   
   
5. The amount of apoptosis induced at various ribavirin concentrations was quantified using acridine orange staining.
   

RESULTS

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