Figure 4

Effect of chemical inhibition of heat shock responses on viral RNA synthesis. A, B. Inhibition of hsp70 synthesis by quercetin. MDCK cells were infected (I) or mock-infected (M) with PR8 virus, treated (+) or mock-treated (-) with 30 μM quercetin and incubated at 37°C or 41°C for 5 h. A. Cell lysates were analysed by SDS-PAGE and Western blotting to detect hsp70/hsc70 and, as a loading control, β-actin. B. Total RNA was isolated and primer extension analysis was carried out to detect segment 5 and 2 vRNA. C, D. Inhibition of hsp90 chaperone function by geldanamycin. 293T cells were infected (I) or mock-infected (M) with PR8 virus, treated with 0, 20 or 30 ng/ml of geldanamycin (as indicated) and incubated at 37°C or 41°C for 5 h. C. Cell lysates were analysed by Western blotting to detect PB2 and, as a loading control, LAP-2 proteins. D. Total RNA was analysed by primer extension to detect segment 2-specific RNA.