FIG. 8.

MRV dsRNA synthesis can occur in the absence of ongoing protein synthesis. (A) Experimental design. (B) Infected cells were incubated at 37°C with 15 mM GuHCl for 19 h. At 19 h p.i., GuHCl-containing medium was aspirated and replaced with serum-free, phosphate-free medium containing 15 mM GuHCl. The cells were incubated for an additional 1 h at 37°C. At 20 h p.i., the medium was aspirated and replaced with serum-free, phosphate-free medium containing 30 μl of [³²P]orthophosphate (300 μCi/reaction) ± 20 μg of cycloheximide/ml. The cells were then incubated for an additional 30 min at 37°C. The cells were lysed, and the total RNA was extracted with TRIzol reagent. Labeled MRV dsRNA was separated by electrophoresis in 10% polyacrylamide gels and visualized by phosphorimaging. The positions of individual MRV dsRNA genome segments are indicated at right.