Fig 2: GA does not inhibit EV71 RNA transcription but accelerates the proteosomal degradation of de novo synthesized capsid proteins.RD cells were treated with 2 µM of GA by 30 min early of MOI= 0.01 of EV71 5746 infection and incubated for 16 hours in the presence of drug. Alternatively, cells were treated with GA at 4 or 8 hours post infection and then continued the incubation up to 16 hours. (A) Cell extracts were prepared to detect EV71 capsid or HSP90 proteins by immunoblotting with Mab979 antibody or anti-HSP90 antibody (N-17), respectively. Cellular ß-actin was detected by blotting the same membrane with monoclonal anti-ß-actin antibody. Data represent one of two independent experiments. (B) RNA were prepared at 4 and 16 hours post infection to quantify EV71 transcripts as described in the legend of Figure 4. Each ß-actin normalized 2Ct value from GA-treated cells was ratio to the value from the value of 2Ct obtained from the vehicle-treated cells. (C) RD cells were treated with vehicle, or 2 µM of GA, or 20 µM of MG132 alone, or GA plus MG132, at 4 hours post EV71 infection and continued the incubation for 12 hours. The extracts were prepared to detect viral capsid proteins by Western blotting using Mab979 antibody. Data represent one of two independent experiments.

Fig 3: Down-regulation of HSP90α/β inhibits EV71 infection.200 pmoles of siRNA specific to HSP90 or the same amounts of control siRNA were transfected to RD cells. After 24 hours of transfection, cells were following infected separately with MOI=0.01 of C2 and B4 genotypes of EV71 5746 and E59 strains. Cells were washed after one hour of infection and cultured for another 16 hours. (A) Cells were lysed and detected the content of HSP90α/β and EV71 capsid proteins by Western blot using anti-HSP90α/β and Mab979 antibodies, respectively. Cellular β-actin was detected by blotting the same membrane with monoclonal anti-β-actin antibody. Corresponding changes in HSP90α/β and EV71 capsid proteins levels compared to the expression levels in the cells with 200 pmoles control siRNA as 1.0 were quantified using Image-Pro Plus 6.0 software. (B) Cells were incubated with anti-HSP90α/β antibody (H-114) or control rabbit IgG (filled curve) at 4°C for another one hour. The cells were washed and fixed by 2% paraformaldehyde in PBS for 30 min. After washing, the treated cells were incubated with anti-rabbit IgG-TR antibody. Stained cells were run on a FACScan flow cytometer and analyzed by using CellQuest software. (C) Cytosolic fractions were prepared as described in the materials and methods and subjected to western blot to detect HSP90α/β as described in (A). Data represent one of two independent experiments. Unpaired student t test with Welch correction was used for statistical analysis (*p<=0.05, **p<=0.01).

Fig 4: Model illustrating how HSP90 interacted with EV71 and supported EV71 infection.In the infection, EV71 particles attach to the surface receptors (such as human SCARB2 or PSGL-1), HSP90 expressed on the surface may following bind to EV71 and help for virus entry. The newly synthesized viral capsid proteins may bind to HSP90 to avoid being degraded by proteosome that facilitates viral assembling. Targeting of HSP90 using the inhibitor geldanamycin (GA) and its analog, 17-allyamino-17-demethoxygeldanamycin (17-AAG), can efficiently control EV71 infection based on the blockage the described pathways that HSP90 involved.

Fig 5: HSP90 interacts with EV71 on the surface of infected cells.(A) RD cells were treated with 2 µM (gray curve) or 0 µM (0.1% DMSO; black curve) of GA for one hour and then washed with 1xPBS before incubated with anti-HSP90 antibody (N-17) or control goat IgG (filled curve) at 4°C for another one hour. The cells were washed and fixed by 2% paraformaldehyde in PBS for 30 min. After washing, the cells were stained with anti-goat IgG-TR antibody and analyzed by the FACScan flow cytometer. (B) Pretreatment of RD cells with 10 and 20 µg of anti-HSP90 antibody (H-114) or control rabbit IgG (Iso-Ig) at 4°C for one hour before MOI=0.1 of EV71 5746 infection was conducted. After one hour infection, cells were washed with medium and incubated for another 16 hours. Viral capsid proteins in the cells were detected by western blot using MAB979 antibody. The internal β-actin was detected by blotting the same membrane with monoclonal anti-β-actin antibody. Corresponding changes in EV71 VP0 levels compared to the expression levels in the cells without antibody treatment as 1.0 were quantified using Image-Pro Plus 6.0 software. (C) Performance of co-immunoprecipitation of EV71 5746 and recombinant HSP90β by anti-EV71 E1 antibody or non-immune mouse IgG (mIgG) following by immunoprecipitation with protein G-agarose was described in the Materials and Methods. Precipitates were washed and subjected to detect the associated EV71 capsid proteins and HSP90 by blotting with MAb979 and anti-HSP90 antibodies, respectively. (D) MOI =0.1 of EV71 5746 was pre-mixed with various amounts of recombinant HSP90β or bovine serum albumin (BSA) and incubated at 4°C for overnight. EV71-HSP90β or EV71-BSA mixtures were inoculated into 12-well plate of RD cells and incubated on ice for 90 min. Followed by washing the unbound EV71 away, total RNA were extracted to quantify EV71 transcripts as described in the legend of Figure 4. Each normalized 2Ct value from EV71-HSP90β mixtures-treated cells was ratio to the value from the value of 2Ct obtained from the only EV71-treated cells that represented as 100%. A schematic representation of the percentage of EV71 attachment was shown. Unpaired student t test with Welch correction was used for statistical analysis.