Fig. 3.

Hsp90 inhibition mediates autophagy-dependent degradation of VP16. a Hsp90 inhibitors induce the accumulation of endogenous ubiquitinated proteins. Vero cells were infected with HSV-1 (MOI 50) for 2 h in the presence of 17AAG (0.5 μM), SNX-2112 (0.8 μM), or AT533 (2 μM), and total protein was extracted for western blot analysis. b MG132 failed to rescue the loss of VP16 induced by Hsp90 inhibition. Vero cells were transfected with FLAG-VP16 plasmid (3 μg) for 48 h and treated with AT533 (2 μM) for 2 h in the presence of MG132 (5 μM). Total protein samples were extracted and subjected to western blot analysis (left). Notably, the accumulation of p21, a cellular protein degraded by the proteasome, was included as an indicator of proteasome activity inhibition. Quantify One densitometric analysis of VP16 band from FLAG-immunoblots (means ± SD of 3 independent experiments) (right). c Hsp90 inhibitors activated the macro-autophagy pathway in HSV-1-infected cells. Vero cells were infected with HSV-1 (MOI 50) for 2 h in the presence of SNX-2112 (0.8 μM) or AT533 (2 μM) and then analyzed by western blotting to determine the expression of mTOR pathway-associated proteins; P62 is an autophagy receptor that interacts directly with both the cargo to become degraded. d CQ rescued the loss of VP16 induced by Hsp90 inhibition. Vero cells were transfected with FLAG-VP16 plasmid (3 μg) for 48 h and treated with AT533 (2 μM) for 2 h in the presence of CQ (50 μM). Protein was extracted, and protein expression of VP16 was analyzed by western blotting (left). Quantify One densitometric analysis of VP16 band from FLAG-immunoblots (means ± SD of 3 independent experiments) (right). e 3-MA restored the loss of VP16 protein induced by Hsp90 inhibition. Vero cells were transfected with FLAG-VP16 plasmid (3 μg) for 48 h and then pretreated with 3-MA (5 mM) for 3 h before treatment with AT533 (2 μM) for 2 h, as indicated. The samples were then analyzed by western blotting. f The SMART diagram represents the major domains within VP16 that were obtained from UniProt (upper). Additional information regarding the predicted domains is also provided as a Table (lower). g, h 3-MA restored the suppressed promoter activity and downregulation of α0 and α4 genes in HSV-1-infected cells. g, h Vero cells were transfected with indicated reporter plasmids for 18 h, pretreated with 3-MA (5 mM) for 3 h, and then treated with AT533 (2 μM) for 2 h. Cell lysates were subjected to luciferase activity assays. Bar graph represents the result of DLRs from 3 independent experiments expressed as means ± SEM. h Vero cells were pretreated with 3-MA (5 mM) for 3 h and infected with HSV-1 (MOI 50) for 2 h in the presence of AT533 (2 μM). Total RNA was then extracted and subjected to qRT-PCR analysis