FIG 2.

Hsp90 activity is required for MuV RNA synthesis after the secondary transcription phase. (A) Schematic of the experimental procedures showing the times of RNA collection and 17-AAG treatment after virus infection. (B and C) Vero cells were infected with MuV at an MOI of 5.0. At the indicated times postinfection, total cellular RNA was extracted and subjected to an RT reaction using oligo(dT) and strand-specific primers. The levels of N mRNA and genomic RNA were determined by qPCR. The values were normalized to the level of the control gene HPRT1 and are shown as the log fold change. Three independent experiments were performed, and the most representative data are shown. (D and E) Vero cells were infected with MuV at an MOI of 5.0 and cultured with or without 50 μg/ml of CHX and 0.5 μM 17-AAG or DMSO. At 0 and 24 hpi, total cellular RNA was extracted and subjected to an RT reaction using oligo(dT) and strand-specific primers. The levels of N mRNA and genomic RNA were determined by qPCR. The values were normalized to the value for the control gene HPRT1. (F) BHK/T7-9 cells were treated with the indicated concentrations of 17-AAG for 48 h, and then cell viability was determined and calculated as a percentage of the viability of cells treated with DMSO. (G) BHK/T7-9 cells were transfected with the minigenome plasmid (pFL-MuV-MG) and three support plasmids (pCR-N, -P, and -L). At 4 h posttransfection, the medium was replaced with fresh medium containing 17-AAG, as indicated in the figure. pCR-L was omitted from the transfection mixture to establish a negative control. The luciferase activity in DMSO-treated cells was set to 100%. Error bars indicate the standard deviations of triplicates. The significance of differences between the means was determined by Student's t test. *, P < 0.05; **, P < 0.01. ND, no significant difference.