Hsp70 increases the stability of viral proteins in the replicase complex.

(A) 293T cells were transfected with sh-NC or sh-Hsp70. After 24 h, cells were transfected with JEV replicon RNA. Cell lysates were harvested 36 h after transfection and immunoprecipitated with an anti-NS5 antibody. The precipitates were subjected to Western blotting with an anti-NS3, anti-NS5, and anti-Hsp70 antibodies. The protein levels were quantified by immunoblot scanning and normalized with respect to the amount of GAPDH (right panel). The results shown are from three independent assays, with the error bars representing the standard deviations (***, P < 0.0001, **, P < 0.01, student’s t-test). (B) 293T cells containing sh-NC or sh-Hsp70 were transfected with Flag-NS3 or Flag-NS5 plasmid or replicon RNA. Cell lysates were harvested 36 h after transfection and immunoprecipitated with anti-NS3 or anti-NS5 antibody. The precipitates were subjected to Western blotting with anti-NS3, anti-NS5, and anti-K48-ubiquitin antibodies. (C) 293T cells containing sh-NC or sh-Hsp70 were transfected with JEV replicon RNA. In one set of experiment, 20nM MG132 was added at 24 h post-transfection to inhibit proteasomal degradation. Cell lysates were prepared at 12 h after treatment and analyzed by Western blotting with anti-NS3, anti-NS5, and anti-Hsp70 antibodies. The protein levels were quantified by immunoblot scanning and normalized with respect to the amount of GAPDH (right panel) (**, P < 0.01, student’s t-test).