Figure 3.
Hsp90 associates with the capsid precursor P1 and is required for its processing to mature capsid proteins. (A) Association of 35S-labeled P1 with Hsp90 and its cochaperone p23 in the presence or absence of GA, measured by immunoprecipitation. (NI) Nonimmune control. (B) Pulse-chase analysis of poliovirus proteins from infected cells grown in the presence or absence of GA. Total cytoplasmic extracts separated by SDS-PAGE were visualized by autoradiography. P1-derived (labeled arrows) and P2- and P3-derived (arrowheads) proteins are indicated. (C) Relative band intensity of P1 and P1-derived capsid proteins in control and GA-treated cells, calculated from B as percent of P1 at 15-min chase time point. (D) GA treatment promotes P1 degradation by the proteasome. The effect of GA on degradation of 35S-labeled P1, expressed in cells by infection with a recombinant vaccinia virus (VV-P1) (Ansardi et al. 1991), was examined in the presence or absence of the proteasome inhibitors LC and ALLN, and the lysosomal protease inhibitor E64. (E) Processing of in vitro translated P1 into capsid proteins by purified 3Cpro is blocked by GA even in the absence of proteasomal function. (CHX) Cycloheximide. (F) Role of Hsp90 in picornavirus capsid maturation. Hsp90 binds newly translated P1, probably in cooperation with Hsp70 (see Discussion; Macejak and Sarnow 1992). Together with ATP and its cofactors, such as p23, Hsp90 folds P1 to a processing-competent conformation (P1*) and protects it from proteasomal degradation. Upon cleavage by 3CPro, the mature capsid proteins no longer interact with Hsp90.