FIG. 6.

Minus-strand synthesis by the nsP3 mutants. CEF cells, infected with an MOI of 100 of each virus at 30°C, were maintained at 30°C (A) (dashed lines) or were shifted up to 40°C when about 15% of the maximal rate of RNA synthesis had been achieved at 30°C or between 2 and 3 h p.i. depending on the virus. Duplicate cultures shifted to 40°C were incubated in the presence (B) or absence (A) (solid lines) of cycloheximide beginning at the time of shift. Minus-strand synthesis was determined by pulse-labeling cells with 200 μCi of [³H]uridine/ml for 15-min periods over the first 1 h of shift and analysis of the purified viral RF RNA for radiolabeled minus-strand RNA, as described in Materials and Methods. Incorporation into newly made minus strands is expressed as the percentage of the total labeled RF RNA that was in minus-strand RNA. Symbols: •, SIN HR; ○, ts4; ▴, ts138; ▪, Toto:ts138AS recombinant; ▾, ts7B5.