FIG 5.

Hsp90 is dispensable for stability of the L protein and the polymerase activity after an interaction with the P protein. (A and B) 293T cells were transfected with pCAGGS-HA-MuV-L alone or cotransfected with pCAGGS-HA-MuV-L and pCAGGS-P. At 23.5 h posttransfection, the cells were treated with 50 μg/ml CHX for 30 min. Then, 0.5 μM 17-AAG was added to the cells, and the cell lysates were collected at the indicated times and subjected to immunoblotting. The relative band intensities of L and P proteins were normalized to the β-actin level, and the relative expression levels of each viral protein based on the levels of cells collected at 0 h are shown in panel B. (C) Vero cells were infected with MuV at an MOI of 5.0. At 11.5 hpi, the cells were treated with 50 μg/ml of CHX for 30 min. Then, 0.5 μM 17-AAG was added to the cells, and total cellular RNA was extracted at the indicated times 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, and the values were normalized to the level of the control gene HPRT1 and shown as log fold changes. Error bars indicate the standard deviations of triplicates. The significance of differences between the means was determined by Student's t test. **, P < 0.01. ND, no significant difference.