Fig 2: Schematic model of Hsp90a regulation of HSV-1 a genes in the context of Hsp90 inhibition or Hsp90a knockdown. Hsp90 inhibition or Hsp90a siRNA induces VP16 autophagosome degradation, leading to reduced a gene transcription, and then suppressed HSV-1 replication in vitro and in vivo. Specifically, Hsp90 inhibition disrupts the interaction between VP16 and Hsp90a and then induces the degradation in an autophagy-dependent manner. Notably, Hsp90 inhibition inhibits the mTOR pathway and then activates autophagy

Fig 3: Hsp90a is involved in modulating the transcription of HSV-1 a genes. a, b The promoter activity of HSV-1 a genes in the presence of Hsp90 inhibitors. Vero cells were cotransfected with reporter plasmids as described in section 2.4 of the Materials and Methods. Cells were then treated with 17AAG (0.5 µM) or AT533 (2 µM) for 2 h. Cell lysates were subjected to luciferase activity assays (a and b, upper) and analyzed by western blotting to detect VP16 overexpression efficiency (A and B, lower). Bar graph represents the result of DLRs from 3 independent experiments expressed as means ± SEM; (c, d) The promoter activity of a0 and a4 in the context of Hsp90a or Hsp90ß downregulation. Vero cells were transfected with reporter plasmids together with siHsp90a or siHsp90ß (100 nM) as indicated for as described in section 2.4 of the Materials and Methods. Cell lysates were then harvested to test luciferase activity. Bar graph represents the result of DLRs from 3 independent experiments expressed as means ± SEM; (e) Effects of Hsp90a or Hsp90ß knockdown on the RNA levels of a0 and a4. Vero cells were transfected with siHsp90a-2, siHsp90a-3, and siHsp90ß (100 nM) for 24 h and then infected with HSV-1 (MOI 50). Total RNA was extracted at 2 hpi and then subjected to the analysis of RNA levels of a0 and a4 using qRT-PCR. f Hsp90a restored the suppressed promoter activity of a0 and a4 genes induced by Hsp90a knockdown. Vero cells were cotransfected with siHsp90a-2 (100 nM) and the indicated luciferase reporter plasmids as described in section 2.4 of the Materials and Methods. The cell lysates were subjected to luciferase activity assays. Bar graph represents the result of DLRs from three independent experiments expressed as means ± SEM; (g) Plaque formation assays showing that HSV-1 infection was affected by Hsp90a knockdown. Cells were infected with HSV-1 for 2 h after transfecting siHsp90a or siHsp90ß (100 nM) knockdown and then subjected to plaque assays (left). Quantitative histograms were prepared (right). h Virus yield assay in the context of Hsp90 knockdown and Hsp90 inhibition. Cells were infected with HSV-1(MOI 1) for 24 h after transfecting siHsp90a (100 nM) and then subjected to determine the yield of virus; the group of acyclovir was also included as a control. i Hsp90 inhibition reduced the fluorescenceintensity of GFP-HSV-1 infected SH-SY5Y cells; SH-SY5Y cells were infected with GFP-HSV-1 (MOI 1) for 24 h in the presence of 17AAG (0.5 µM) or AT533 (2 µM). The cells were observed with fluorescence microscope. Scale bars, 200 µm

Fig 4: VP16 interacts with Hsp90a via its conserved core domain. a, b Hsp90 inhibition led to VP16 degradation in FLAG-VP16 plasmid-transfected cells. SH-SY5Y cells (a) and Vero cells (b) were transfected with FLAG-VP16 plasmid (3 µg) for 48 h and then treated with AT533 (2 µM) for 2 h. Proteins were extracted and subjected to western blot analysis; P3*FLAG-CMV was included as an empty vector control. c Hsp90a knockdown contributed to VP16 degradation in VP16-expressing cells. SH-SY5Y cells were cotransfected with siHsp90a-2 (100 nM) and FLAG-VP16 plasmid (3 µg) for 48 h. The cell lysates were then analyzed by western blotting. d SH-SY5Y cells were infected with HSV-1 (MOI 50) for 2 h in the presence of AT533 (2 µM), immunoprecipitated with anti-VP16 antibodies, and subjected to western blot analysis using anti-Hsp90 antibodies. Oct-1 was used as a positive indicator that interacts with VP16. e VP16 interacted with Hsp90a in 293 T cells. 293 T cells were cotransfected with FLAG-VP16 (5 µg) and HA-Hsp90a (5 µg) plasmids for 48 h. The cells were then lysed, subjected to immunoprecipitation using anti-FLAG antibodies, and then analyzed by western blotting. f Diagrammatic sketch of VP16 truncated mutant construction. g VP16 interacted with Hsp90a via its conserved core domain. 293 T cells were cotransfected with different truncated mutant plasmids of VP16 (5 µg), including WT, V1, V2, and V3, together with HA-Hsp90a (5 µg) plasmid for 48 h. The cells were lysed and subjected to immunoprecipitation using anti-FLAG antibodies and then analyzed by western blotting

Fig 5: Effect of maneb and paraquat on autophagic pathway.Striata from saline, maneb, paraquat, and maneb+paraquat injected mice were homogenized in a modified RIPA buffer containing 1.0% sodium cholate, and separated into cholate-soluble and cholate-insoluble fractions and analyzed by Western blots, as described under “Materials and Methods”. The blots show representative gels while the bar graphs are composites summarized from all animals (n = 5–6). (A) Autophagy pathway proteins in the soluble and (B) insoluble supernatant were probed using antibodies specific for mTOR, Beclin1, and Atg12, and expressed relative to β-actin used as loading control. (C) LC3 I (upper band) and LC3 II (lower band in longer exposure blot image) were probed in cholate soluble fractions using LCB antibodies, and the ratio of LC3 II to LC3 I was assessed to measure autophagic flux. (D) Chaperone proteins in the cholate soluble and (E) cholate insoluble fractions were probed using antibodies specific for Hsc70, Hsp70, and Hsp90, and expressed relative to β-actin used as loading control. Values are mean ± SEM as compared to saline injected control. Asterisks (*, **, ***) indicate values significantly different from saline controls (p<0.05, p<0.01, p<0.001,respectively). Student's t-test was performed to compare control and treatment groups for all data.