Virus, cells, and antibodies
The DF-1 cell line, an immortalized cell line of chicken embryo fibroblasts that is susceptible to many isolated avian viruses, was provided by Dr. Lucy Lee, USDA. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 5% fetal bovine serum (FBS) at 37 °C in a 5% CO2 atmosphere. A specific monoclonal antibody 2H11 against IBDV was prepared in our previous work . The IBDV Q strain is an IBDV isolated from the infected chickens. This strain proliferated in DF-1 cells with a multiplicity of infection (MOI) of 1 and was cultured at 37 °C in DMEM (Gibco, China) with 1% FBS. After the cytopathic effect (CPE) completely appeared, approximately 36 h, the cells and supernatants were collected. Viruses were obtained by 3 cycles of freezing and thawing, and titers were determined using the Reed-Muench method.
Cell protein extraction
DF-1 cells transfected with the plasmid pcDNA-VP2 or with the pcDNA vector were separately cultured in DMEM containing 5% FBS at 37 °C in 5% CO2. After 72 h, the cells were washed 3 times with precooled phosphate-buffered saline (PBS) and harvested using a disposable cell scraper. The cells were centrifuged (13,200 rpm, 5 min), and lysis buffer (containing a cocktail of protease inhibitors, 150 mM NaCl, 1% NP-40, 25 mM Tris, and 5% glycerol) was added. The cell pellets were dispersed by repeat pipetting, placed on ice for 30 min, and centrifuged at 13,200 rpm/min for 25 min. Protein A + G agarose or control resin was added to the supernatants and the mixture was softly shaken at 37 °C for 3.5 h. The samples were centrifuged at 1000×g for 5 min, the supernatants were collected.
Coimmunoprecipitation assays were performed using a coimmunoprecipitation crosslinking kit (Thermo Fisher Scientific, Pierce Biotechnology, IL, USA) according to the manufacturer’s instructions. The kit enables the isolation of native protein complexes from a lysate or other complex mixture by directly immobilizing purified antibodies onto an agarose support. In this study, supernatants containing cell protein extracts were incubated with the monoclonal antibody 2H11, which is specific for the IBDV VP2 protein. Native proteins isolated using the kit were resuspended in 5 × SDS sample buffer, boiled for 10 min, and subjected to 10% SDS-PAGE. After electrophoresis, the gels were stained with a silver staining kit (Thermo Fisher Scientific, Pierce Biotechnology, IL, USA). The differentially abundant protein bands compared to those in the negative control were excised and identified by mass spectrometry.
Mass spectrometric analysis
As indicated above, differentially abundant proteins were identified by comparison of the protein bands of the experimental and the control groups. The differential proteins were excised and sent to Shanghai Zhongke New Life Biotechnology Co., Ltd. for mass spectrometry analysis.
The gel samples were added to approximately 200-400 μL of ACN/100 mM NH4HCO3, washed and decolored to transparency, and freeze dried after removal of the supernatants. The samples were combined with DTT and incubated at 56 °C for 30 min, after which the DTT solution was replaced with 200 mM IAA prior to incubation in the dark for 20 min. The supernatants were removed, and 100 mM NH4HCO3 was added to the samples followed by incubation at room temperature for 15 min. The NH4HCO3 solution was replaced with 100% ACN, and the samples were incubated for 5 min, absorbed and freeze dried. Trypsin solution (2.5–10 ng/μL) was added to the mixture and incubated at 37 °C for approximately 20 h. The original solution was transferred to a new Eppendorf tube, and 100 μL of extraction solution (60% ACN/0.1% TFA) was added to the gel. After ultrasonication for 15 min, the samples were combined with the enzymatic hydrolysate and lyophilized. A solution of 0.1% formic acid was added to the samples for resolving, and the samples were collected by filtration through a 0.22-μm membrane.
The mass-charge ratios of the polypeptide fragments were determined using a full scan method each time. Bioworks Browser 3.3 software was employed to retrieve the corresponding database for the mass spectrometry test raw file to obtain the protein identification results. The retrieval parameters were as follows: database: uniprot; taxonomy: Gallus gallus; enzyme: trypsin; dynamical modifications: oxidation (M); fixed modifications: carbamidomethyl (C); max missed cleavages:2; peptide charge state: 1 + , 2 + , and 3+; proteomics tools: 3.1.6. Filter by Delta CN:charge =1 Delta CN ≥ 0.1; charge =2 Delta CN ≥0.1; charge =3Delta CN ≥0.1; Filter by Xcorr:charge =1 Xcorr ≥1.9; charge =2 Xcorr≥2.2; charge =3 Xcorr≥3.75.
Indirect immunofluorescence assay (IFA) and confocal microscopy
DF-1 cells were cultured on glass cover slips, fixed on glass with 3% paraformaldehyde for 20 min at room temperature, and washed 3 times with PBS. The cells were then incubated with a membrane disrupting solution containing 0.25% Triton X-100 at room temperature for 5 min. These samples were blocked with 2% bovine serum albumin (BSA) at 37 °C and incubated for 45 min. An anti-HSC70 antibody or normal immunoglobulin G (IgG) was diluted to 1:100 as the primary antibody, and FITC-conjugated goat anti-mouse IgG was used as the secondary antibody. The samples were incubated with the two antibodies for 45 min and then observed using a laser scanning confocal microscope.
Protein extracts of DF-1 cells transfected with pcDNA-VP2 or the pcDNA vector were separated by 10% SDS-PAGE. The proteins were then transferred to a nitrocellulose membrane, which was then blocked with 5% nonfat milk in Tris-buffered saline (TBS) buffer at 4 °C overnight and washed 2 times with TBS. The proteins were incubated with a primary antibody at 37 °C for 2 h and subsequently with a secondary antibody at 37 °C for 2 h. After washing 3 times, detection using chemiluminescence reagents was performed.
HSC70 inhibitor (VER-155008) inhibition assay
A VER-155008 stock liquor was prepared by dissolving VER-155008 (Sigma) in dimethyl sulfoxide (DMSO) and diluted with water into a series of concentrations. DF-1 cells were subcultured in 24-well plates for 18–24 h. The inhibitor (VER-155008) was added to the cell culture medium at a final concentration of 5 μmol/L, and the medium was added to the cells in the incubator at 37 °C for 6 h. The inhibitor was removed from the culture medium by washing twice with sterile PBS. DF-1 cells were cultured in fresh medium containing 1% FBS and infected with IBDV at an MOI of 0.1 for 2 h. After washing 3 times with PBS, medium containing 1% FBS was added to cells. Cell morphology tests, 50% tissue culture infectious dose (TCID50) determination, western blotting and IFA were performed at 24, 48, and 72 h, and a positive control was performed synchronously at the indicated time points. The inhibitory effects of different concentrations of the inhibitor were also assessed. DF-1 cells were cultured in 6-well plates for 18 h and incubated at 37 °C for 6 h with the cell culture medium containing different diluted concentrations of the inhibitor (5 μM, 0.5 μM, 0.05 μM and 0.005 μM), which was then replaced with fresh culture medium. DF-1 cells were inoculated with IBDV at an MOI of 0.1 and cultured for 72 h. The CPE, viral titer and protein expression level were measured.
The effect of inhibition was determined by detecting the viral titer (TCID50) of IBDV. The detailed steps are as follows. DF-1 cells were trypsinized and plated in 96-well plates (3 × 104 cells per well) and placed in a CO2 incubator at 37 °C for 18 h. The supernatants from antibody-blocking assays or HSC70 inhibitor assays were diluted 101- to 1010-fold with cell culture medium containing 1% FBS. The culture medium in 96-well plates was discarded and replaced by the fresh medium diluted described above. Every diluted concentration of the inhibitor was added to 8 duplicate wells for DF-1 cells infection. After incubation for 2 h at 37 °C, the medium of each well was replaced by fresh medium containing 1% FBS. After 1 week, the cells were observed using an inverted microscope, and a well with CPE was selected. The TCID50 was determined by the CPE and calculated by the method of Reed and Muench.
Antibody inhibition assay
DF-1 cells were cultured in 48-well plates, and each well was seeded with 1.3 × 105 cells. After incubation for 24 h, anti-HSC70 antibodies at different concentrations (6 μg/mL, 12.5 μg/mL, and 25 μg/mL) were added. A normal control IgG was used at a final concentration of 25 μg/mL. A positive control group without any antibody pretreatment was normally infected with IBDV. The DF-1 cells were incubated with anti-HSC70 antibodies at different concentrations at 37 °C for 2 h and subsequently challenged with IBDV at an MOI of 0.01 in fresh medium. At the same time, DF-1 cells were incubated with the normal control IgG at a concentration of 25 μg/mL at 37 °C for 2 h, and then infected with IBDV at an MOI of 0.01 in fresh medium. After incubation for 2 h, unbound viruses were removed by extensive washing with PBS. The infected cells were cultured in medium containing 1% FBS. All infected cells pretreated with the anti-HSC70 antibody were examined by western blotting, IFA, and viral titer (TCID50) determination after 48 h. Infected cells pretreated with normal control IgG and the positive control group were also analyzed.