2.1. Cell Lines and Plasmids
Human embryonic kidney (HEK) 293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher, Waltham, MA, USA; Cat#10566016) containing 10% foetal calf serum (FBS; Gibco, Waltham, MA, USA; Cat#10099141), 2 mM L-glutamine (Life Technologies, Waltham, MA, USA; Cat#25030081), and 1% penicillin- streptomycin (Life Technologies; Cat#10378016) at 37°C with 5% CO2. Plasmids pCAGGS-VP30, pCAGGS-VP35, pCAGGS-NP, pCAGGS-L, p4cis-vRNA-RLuc, pCAGGS-Tim1, and pCAGGS-T7 were kindly provided by Drs. Heinz Feldmann and Thomas Hoenen, Rocky Mountain Laboratories, National Institute of Health. GFP-LC3 (Addgene, Watertown, MA, USA; Cat# 11546) and RFP-LAMP1 (Addgene, Cat# 1817) were purchased from Addgene company.
2.2. Production of trVLPs
Producer 293T cells (p0) in six-well plates were transfected with plasmids encoding each EBOV structural protein (75 ng pCAGGS-VP30, 125 ng pCAGGS-VP35, 125 ng pCAGGS-NP, 1,000 ng pCAGGS-L, and 250 ng p4cis-vRNA-RLuc), and pCAGGS-T7 (250 ng) encoding a Renilla luciferase reporter [21]. Cellular supernatants (200 mL) from 10 six-well plates containing released trVLPs were harvested at 72 h post-transfection, and the cell debris was pelleted by centrifugation at 175 × g. The supernatant was then used to infect target 293T cells (p1) previously transfected with ribonucleoprotein (RNP) components (125 ng pCAGGS-NP, 125 ng pCAGGS-VP35, 75 ng pCAGGS-VP30, 1,000 ng pCAGGS-L, and 250 ng pCAGGS-Tim1) [20]. Target 293T cells (p2 − p5) were treated in a similar manner. Cleared supernatants (33 mL) in 2 mL of 20% sucrose from the bottom of each tube were centrifuged at 125,000 × g in a SW-28 rotor for 3 h at 4°C. The resulting pellets were resuspended in 100 µL ice-cold NTE buffer (10 mM Tris pH 7.5, 100 mM NaCl, and 1 mM EDTA) by tapping gently approximately 100 times, and trVLPs were stored on ice or in a refrigerator at 4°C until further use.
2.3. Imaging of the DiI-labeled trVLPs internalization in live 293T cells
The trVLPs were suspended in 1 ml of NTE buffer, and 1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindocarbocyanine Perchlorate (DiI, final concentration:10 µM, Invitrogen, Waltham, MA, USA, Cat# D282) was added and thoroughly mixed. The mixture was gently shaken at room temperature for 1 h and then filtered through a strainer (0.22 µm pore size; Millipore, Darmstadt, Germany, Cat#SLGP033RB). 293T cells cultured in 8-well Chamber Slides with removable wells were incubated with the DiI-labeled trVLPs at 4°C for 10 min, washed with PBS, and warmed at 37°C. Then, 4',6-diamidino-2-phenylindole (DAPI, diluted by PBS in 10 µg/ml, Invitrogen, Waltham, MA, USA, Cat#P36931) was added to stain the nucleus. Later, the well was set on the stage of a fluorescence microscope, the internalization process of DiI-labeled trVLPs in 293T cells was imaged at different time points.
2.3. TCID50 and MOI of trVLPs
TrVLPs infectivity titre was titrated using 50% tissue culture infective dose (TCID50). TrVLPs were diluted at 10-fold serially with DMEM from 10− 1 to 10− 10 concentrations. The attenuated trVLPs (100 µl) were added to wells in each row of a 96-well plate, and 293T cell suspension (100 µl) was added to each well to a final cell density of 2×105 cells/ml. 293T cells without HP001 infection were included as controls. Cytopathic effects (CPEs) were observed by microscopy and recorded each day for 7 days, the results were used to calculate TCID50 by the Reed-Muench method[22]. The MOI value was calculated according to the equation MOI = 0.7×TCID50/ cell numbers (Table 1).
2.4. Electron Microscopy (EM) Analysis of trVLPs
trVLPs were pipetted onto a 300-mesh copper grid coated with carbon film, incubated for 5 min at room temperature, and the grids were washed twice with distilled water and negatively stained for 15 s with 1% uranyl acetate. The excess liquid was removed with a filter paper and trVLPs were visualized under a Hitachi H7000 transmission electron microscope.
2.5. Immunofluorescence
The 293T cells cultured in 24-wells plates were divided into groups, transfected with plasmids GFP-LC3 (800 ng/well), or GFP-LC3 plus LAMP1-RFP (400 ng/well each) respectively. 24 h post-transfection, the cells were treated with trVLPs (MOI = 1.5), or rapamycin (0.5 nM), or trVLPs (MOI = 1.5) plus 3-MA (0.5 nM) respectively. At 48 h after trVLPs infection or drug treatment, the GFP-LC3 and LAMP1-RFP dot formations were detected under a fluorescence microscope (DMi8-M, Leica, German). The cells containing ≥ 5 GFP-LC3 dot formation were defined as autophagy-positive cells [23].
2.6. TEM Analysis of Autophagy
At 48 h after infection or treatment, the cells were collected and fixed in 2.5% glutaraldehyde in 0.1 M PBS for at least 4 h or overnight at 4°C. Later, the specimens were treated with 0.1% OsO4 solution buffered in 0.1 M PBS for 2 h, dehydrated in a graded series of ethanol, and embedded in epoxy resin 812. Ultrathin sections of the specimens were collected on copper grids, double-stained with uranyl acetate and lead citrate, and examined by transmission electron microscopy (H-7000, Hitachi, Hitachinaka, Japan).
2.7. Western blot analysis
The total protein from 293T cells was extracted using RIPA extraction buffer (Beyotime, Shanghai, China, Cat# P0013K) with PMSF protease inhibitor (Solarbio, Beijing, China, Cat# P6730). The protein concentration was then quantified using the bicinchoninic acid (BCA) method (Sigma, Santa Clara, CA, USA, Cat# BCA1-1KT). The extract was eluted with 2× sodium dodecyl sulfate (SDS) loading buffer and resolved by SDS-polyacrylamide gel electrophoresis (PAGE). The proteins were transferred to polyvinylidene fluoride (PVDF) membranes, incubated with the sequential addition of the relevant primary and secondary antibodies, and detected with the Super Signal West Pico chemiluminescent substrate (Thermo Fisher; Cat# 34580). Moreover, to check siRNA interference with the expression of target proteins in a specific manner, an immunoblot analysis of the target host proteins was performed following siRNA transfection. GAPDH was used as the internal control. When immunoblot analysis was performed with lysosomal samples, the level of LAMP2 protein was used as an internal control.
2.8. Antibodies
Antibodies recognizing LC3B (Cat# 3868S), Phospho-mTOR(Cat# 5536S), Phospho-Akt (Cat# 4060S), HSPA1A (HSP70; Cat# 4873S), LAMP1 (Cat# 15665s), LAMP2 (Cat# 49067S), GAPDH (Cat# 2188), HRP-linked anti-rabbit IgG (Cat# 7074S), and HRP-linked anti-mouse IgG (Cat# 7076S) were purchased from Cell Signalling Technology (Danvers, MA, USA). Rapamycin (Cat# 1912) and 3-Methyladenine (3-MA, Cat# 3977) were purchased from R&D Systems (Minneapolis, MN, USA). HSC70 (HSPA8, Cat# NBP2-12880) and HSP90AB1 (Cat# NB110-61640) were purchased from Novus Biologicals (Littleton, CO, USA). PHLPP1 (Cat# MBS151247) was obtained from MyBioSource company (San Diego, CA, USA).
2.9. SiRNA knockdown
SiRNAs targeting HSC70, HSPA1A and HSP90AB1 were designed and synthesized with three siRNA sequences for each one, the most efficient sequence for RNA interference (RNAi) was evaluated by qPCR and were chosen for tests (Table 2). Meanwhile, cytotoxity of siRNAs were estimated by nuclei counting, the siRNA was classified as hypotoxicity if 75 or more nuclei within one vision of microscope (40×, 0.24mm2) (Supplementary Fig. 1). In 24-well plates, 100 µL of opti-MEM medium (Invitrogen; Cat# 31985070) containing 1.4 µL siRNA and 4.5 µL HiPerFect transfection reagent (Qiagen, Dusseldorf, Germany; Cat# 301705) was added to each well, and the plates were shaken gently for 1 min. After a 10 min incubation at room temperature, a cell suspension (400 µL) containing 1 × 105 cells were added to obtain a final siRNA concentration of 75 nM. The cells were incubated at 37°C and 5% CO2 for 48 h.
2.10. Lysosome Isolation
Lysosomes were purified from 293T cells using a lysosome isolation kit (Sigma, Cat# LYSISO1-1KT). According to the protocol, 1 × 108 293T cells were collected and the cell membranes were disrupted by ultrasound. The breakage was evaluated by staining with Trypan blue. The specimen was then serially centrifuged at 1,000 × g and 20,000 × g. The pellet was collected, and calcium chloride was later added to 8 mM, incubated for 15 min at room temperature, centrifuged at 5,000 × g, and the supernatants that contained lysosomal matrices were collected.
2.11. Statistical analysis
Data that exhibited a normal distribution were presented as the mean ± standard deviation. Student’s t-tests (two-tailed, paired or unpaired t-tests with Welch's correction) were performed where appropriate. All analyses were performed with GraphPad Prism 5 (GraphPad Software, UK) and differences between the group means with p < 0.05 were considered statistically significant.