Effect of temperature on viral RNA and protein synthesis. 293T cells were infected with PR8 virus or mock-infected (lanes 17–20) and incubated at 31°C, 37°C, 39°C or 41°C. A. Total cellular RNA was isolated at the indicated times post-infection and subjected to primer extension analysis for segment 5 transcripts. Products were separated on a 6% polyacrylamide gel and visualised by autoradiography. Black arrows indicate products derived from viral RNAs (as labelled). The white arrow indicates a cellular derived background product. Note the generally lower levels of product in lane 5, due to a loss of that particular sample. B. Segment 1, 2, 5, 7 and 8-specific RNA from cells incubated at 37°C and 41°C was quantified at 5 h.p.i. by densitometry of exposed X-ray film. The amounts at 41°C are expressed as the percentage of the corresponding 37°C values. Mean % ± SD from 3 (segment 8), 4 (segment 1) or 5 (segments 2, 5 and 7) independent experiments are plotted. C, D. Mock-infected (M) or infected 293T cells, incubated at either 37°C or 41°C, were pulse radiolabelled with 60 nCi/μl 35S-Methionine for 2 h periods ending at the indicated times p.i. before analysis by SDS-PAGE and autoradiography or (D) immunoprecipitation using rabbit antiserum against PB1 (α1), PB2(α2) or PA (αA). Black arrows indicate viral proteins (as labelled).