Inhibitors, antibodies and plasmids
AT-533 was systematically synthesized in a previously reported study [8], and 17-N-allylamino 17-demethoxygeldanamycin (17-AAG) (S1141) was purchased from Selleck. Cell Counting Kit-8(CCK-8)was obtained from Beyotime Biotechnology. Trizol Reagent was purchased from Invitrogen (Carlsbad, CA, USA). Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), and penicillin-streptomycin were bought from Gibco-BRL (Gland Island, NY, USA). Ubiquitin - proteasome inhibitor MG-132 was purchased from Selleck (S2619), and autophagy inhibitor chloroquine (CQ) was purchased from SigmaAldrich (C6628). These compounds were dissolved into designated concentration in dimethylsulfoxide (DMSO) ,whose final concentrations were less than 0.1%. The primary antibodies used in this study included mouse monoclonal antibodies (MAb) against HSV-1 ICP8 (Santa Cruz,11E2), HSV-1 pUL42 (Abcam, ab19311), LC3B (Cell Siganling technology, 2775) and rabbit monoclonal antibodies (Mab) against Total Hsp90 (Abcam,ab13492), Ubiquitin-P4D1 (Cell Siganling technology, 3936), Akt (Cell Siganling technology, 13038S), GAPDH (GeneTex, GTX100118), Flag-tag (Cell Siganling technology, 14793S), HA-tag (Cell Siganling technology, 3724), GFP-tag (Beyotime Biotechnology, AF0159). The secondary antibodies mainly included anti-rabbit IgG light chain(Abbkine, A25022) and anti-mouse IgG light chain(Abbkine, A25012). All the eukaryotic expression plasmids, including pLVX-EGFP-C1-UL30, pCMV-HA-UL42, p3xFLAG-UL5, pCMV-HA-UL8, pLVX-EGFP-UL52 were generated in our laboratory. cDNA was reverse transcripted using RNA obtained from HSV-1-infected cells as a template to amplify the virus coding sequence. All constructed plasmids were verified by DNA sequencing (TSINGKE Biological Technology). For information on the primers and vectors used to construct the plasmids, see Additional file 1: Table S1.
Cells and viruses
Human foreskin fibroblast(HFF, ATCC SCRC1041), Human embryonic kidney cells (HEK293T,ATCC CRL1573), African green monkey kidney cells (Vero, ATCC CCL181) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) complemented with 10% fetal bovine serum (FBS, Gibco). The maintenance medium used for the dilutions of virus and reagents was DMEM complemented with 2% FBS. HSV-1/F (ATCC VR-733) was obtained from Hong Kong University. ACV-resistant clinical HSV-1 strain (HSV-1/153), a TK-mutant derived from HSV-1 (KOS)-HSV-1/Blue[8] were a kind present from Tao Peng (Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences). These above viruses were propagated in Vero cells and preserved at -80℃ until use.
CCK-8 assay
The CCK-8 assay was manipulated according to the suppliers protocol (Beyotime Biotechnology, C0037). Briefly, HFF cells were cultured in 96-wells plates and reached at 85% cell confluence. Various concentrations of compound were added to the plate, with each concentration having three replicates. After 48 h of incubation, 10 µL CCK-8 solution (5 mg/mL) was added to each well, and the plate was incubated for 1h protected form light. Then, the plates incubated for 10 min at room temperature with gently shaking. The optical density (OD) at 450 nm was measured for each well with an enzyme-labeled reader (Bio-Rad, Hercules, CA, USA). The 50% cytotoxicity concentration (CC50) was defined as the concentration to reduce 50% cell viability.
Viral titer determination
Vero cells were cultured in 96-wells plates. And the next day, Vero cell monolayers were incubated with eleven-fold serial dilutions of 100 µl HSV-1 treatment and incubated at 37°C and 5% CO2 for 72h. Morphological changes of the cells (cytotoxic effects, CPEs) were observed every day under inverted microscope and cell viability was calculated based on CPEs. Control were cells treated only with maintenance medium DMEM. The plaque assay was carried out in triplicate. Virus was quantified by serial dilution and titration assay. The TCID50 (50% tissue culture infectious dose) was calculated using the formula of Reed and Muench method
Log10 50% end point dilutions = log10 of dilution showing a mortality next above 50% - (difference of logarithms × logarithm of dilution factor). Difference of logarithms = [(mortality at dilution next above 50%)-50%] / [(mortality next above 50%) - (mortality next below 50%)][9].
Antiviral effect of GFP-HSV-1
HFF cells were cultured in 96-wells plates, and the second day cell monolayers were incubated with eleven-fold serial dilutions of 100 µl Hsp90 inhibitor treatment and infected MOI = 1 GFP-HSV-1 at 37°C and 5% CO2 for 72h[10]. The plaque assay was carried out in triplicate. GFP fluorescence was quantified using a Synergy Neo HTS Multi-Mode Reader (Biotek Instruments). The relative fluorescence intensity was calculated using the formula as follows: Relative fluorescence intensity= (Hsp90 inhibitors treatment fluorescence intensity) / (GFP-HSV-1 control fluorescence intensity)×100%.
CPEs Cell viability assay
HFF cells were cultures in 96-well plates. And the next day, HFF cell monolayers were incubated with eleven-fold serial dilutions of 100 µL HSV-1 treated with or without Hsp90 inhibitors at 37°C and 5% CO2. After 72h, 10 µL CCK-8 solution was added to each well, and incubated for 1h in dark. Then, the plates incubated for 10 min at room temperature with gently shaking. The optical density (OD) at 450 nm was measured for each well with an enzyme-labeled reader (Bio-Rad, Hercules, CA, USA). The CPEs inhibition ratio was calculated by the formula as follows. The CPEs inhibition ratio= [(Absorption of treatment groups) - (Absorption of cell control)] / [(Absorption of virus control) - (Absorption of cell control)] ×100% [11].
The detection of DNA synthesis
HFF cells were cultured in 12-well plates. And the second day cells infected with HSV-1 (MOI = 20) were incubated with or without AT-533 or 17-AAG for 12 h. Viral DNA was extracted using GeneJET Viral DNA and RNA Purification Kit (Thermo). Real-time PCR assay was used to quantify the viral DNA. Then the HSV-1 DNA copy numbers were expressed relative to the virus control groups. The primer pairs are seen at Additional file 1: Table S2.
Real-time PCR (Q-PCR)
HFF cells were cultured in 12-well plates. And the second day cells infected with HSV-1 (MOI = 20) were incubated with or without AT-533 or 17-AAG for 2, 3, 4 h, respectively. Total RNA was isolated using Trizol (Invitrogen) and subjected to cDNA synthesis using a PrimeScript RT reagent kit (Takara). Real-time PCR (RT-PCR) was conducted to determine the expression levels of UL9, UL30, UL42 of HSV-1/F and at 2, 3 and 4h.p.i, respectively. The primer pairs were the same as described above.
Protein docking stimulation
ZDock (http://zdock.umassmed.edu/) was manipulated to predict the structures of protein-protein interaction and symmetric multimers. Prediction of protein-protein interaction was in three steps as follows[12]. Firstly input ligand and receptor protein structures and choose the ZDock version options; Secondly select the blocking/contacting residues; Thirdly view docking results online or download in Pymol. Binding affinity (∆G) and dissociation constant (Kd) predicted values for the protein-protein interactions was obtained from docking results.
Molecular docking stimulation
LeDock (http://www.lephar.com/index.htm) was manipulated to stimulate the docking between small molecular compounds and proteins. Docking binding affinity (∆G) ༜0 kJ/mol indicated ligand molecules can spontaneously bind to receptor proteins, while (∆G)༜5 kJ/mol indicated both of them can bind stably[13]. The target proteins in all networks were obtained 3D structures from the RCSB PDB database (http://www.rcsb.org/)[14]. 2D structures of all compounds was obtained from ZINC (http://zinc.docking.org/)[15]. Top10 docking complexes were docked between each protein and ligand, and chosen the complexes with the smallest binding energy.
Co-immunoprecipitation (co-IP)
HFF cells were infected with HSV-1 for 12h in 100 mm2 flask dish, which were collected and lysed in 100 µL SDS Lysis Buffer (Beyotime, China) containing 1% PMSF and centrifuged at 12,000 g. The supernatant was divided into two parts, one for input and other was incubated with IgG (normal mouse or rabbit IgG, primary antibody) at 4℃ overnight. Then the mixture was treated with 35µL of volume of Protein A/G magnetic beads at 4℃for 2 h. Next, the immune-precipitates were collected, washed three time with PBS, and re-suspended in 30mL 1*SDS-PAGE buffer (Beyotime, China). Finally, the samples were boiled for 10 min and analysed by western blotting.
Plasmid transfection
HEK293T cells seed in 12-well plates, and the second day the mentioned plasmids were performed with Lipofectamin-3000 transfection reagent according to the manufacturer’s instructions (Invitrogen). Briefly, 500ng of the corresponding plasmids and Lipofectamine 3000 reagent were diluted in 100µL Opti-MEM I reduced serum medium (Invitrogen) and the diluted plasmids were then added to the Lipofectamine 3000 (1:1 ratio), mixed, and incubated at room temperature for 10 min. The transfection mixture was then added to cells at 60–70%, and was replaced with flesh 2% FBS DMEM after transfection 6h.
Western blotting
HFF cells were seeded in 12-well plates with the density of 1.5 × 106 cells/ well. Until 85% cell confluence, cells were infected with HSV-1 (MOI = 20) at 37°C for 4 h. Therefore, DMEM maintenance medium with or without AT-533 or 17-AAG was added. At 12h post infection, the cells were washed three times with PBS, and were lysed with 1*SDS-PAGE buffer (Beyotime). The equal amount (40 µg/sample) proteins were subjected to Western Blot analysis.
Immunofluorescence assay
HFF cells were cultured in 12-wells plates, and second day cells infected with HSV-1 (MOI = 20) at 37°C for 4h for viral DNA replication. Cells were transferred into maintain medium with or without AT-533 or 17-AAG and incubated for 8h. Cells were fixed for 15 min with 4% paraformaldehyde (PFA) and permeabilized with 0.01% Triton X-100 for 5min, and subsequently incubated with anti-UL42 antibody (Abcam) at 4℃ overnight and Alexa Fluor 488 (1:1000) secondary antibody (Invitrogen) at room temperature for 1h. After each step the slides were washed triplicate with PBS, and which finally were preserved with PBS. Finally, the nuclear staining with 4,6-diamidino-2-phenylindole (DAPI, Molecular Probes) was performed for 15min. Fluorescence was recorded in a fluorescence microscopy (Canon).
Statistical analysis
Research data were calculated as the mean ± SD, and statistical significance were determined by the student’s t test. The statistical significance were determined by P values (P < 0.05). More than three times was performed at each time.