Are reovirus particles lacking genomic dsRNA capable of inducing apoptosis?

METHODS

1. Purified dsRNA⁺ and dsRNA^- virions and ISVPs were incubated with calf erythrocytes in U-bottomed microtiter plates and serially diluted.
   
   
2. A partial or complete shield of erythocytes on the bottom of the wells was interpreted as a positive hemagglutination (HA) result
   
   

   Particle type	Particle/PFU ratioa	HA titerb
   dsRNA+ virions	108	1.25 x 108
   dsRNA- virions	18,182	2.5 x 108
   dsRNA+ ISVPs	23	1.25 x 108
   dsRNA- ISVPs	6,250	2.5 x 108

   ^a Particle/PFU ratios were determined by dividing the number of viral paricles per ml by the number of PFUs per ml.
   ^b HA titer is defined as the minimum number of viral particles required to agglutinate bovine erythrocytes.
   
   Why does an HA titer have to be determined?
   ANSWER The HA titer was used to quantify particle, since dsRNA- particles cannot produce PFUs.

   
   

3. HeLa cells were infected with either dsRNA⁺ virions, dsRNA^- virions, dsRNA⁺ ISVPs, or dsRNA^- ISVPs at an MOI of 5 x 10^-4 HA units per cell.
   
   
4. Apoptosis was quantified 24 h after infection using acridine orange staining.
   
   
5. HeLa cells were also infected with dsRNA^- ISVPs at different MOIs (10^-4 HA units per cell) and apoptosis was assessed 24 h after infection using acridine orange staining.

Why were reovirus particles lacking dsRNA used?

ANSWER To demonstate that events after penetration are are dispensable for reovirus-induced apoptosis, since dsRNA- virions cannot complete the replication cycle.

RESULTS

BACK TO EXPERIMENTAL MENU

BACK TO INTRODUCTION