Figure 6

Effect of temperature on viral polymerase activity. 293T cells were infected with PR8 virus and incubated at 37°C or 41°C for 4 h. At this point 50 μg/ml of cycloheximide (+) or the same volume of DMSO (-) was added to the cells, temperature was shifted up (lanes 7, 8) or down (lanes 9, 10) and infections left to proceed for another 4 h. Total RNA was isolated and primer extension was carried out to detect segment 7-specific RNAs as labelled. Infected cells incubated at 37°C or 41°C for 4 h (lanes 1, 2) or 8 h (lanes 3, 4 and 5, 6 respectively) served as positive controls. Mock-infected cells incubated at 37°C for 8 h served as a negative control (lanes 11, 12).