Cell lines, viruses, and compounds
The cell lines utilized in the study encompassed Vero (African green monkey kidney) cells, HeLa (Henrietta Lacks cells), Vero (African green monkey kidney) cells, and RD (Human rhabdomyosarcoma) cells, which were provided by Professor Jianguo Wu. The cell lines were cultivated in high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) supplied by HyClone (UT, USA), enriched with 10% fetal bovine serum (FBS; HyClone, UT, USA) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA) in a 37 °C, 5% CO2 incubator. The propagation of the Coxsackievirus B3 (CVB3), B4-5 (CVB4-5), and B4-7 (CVB4-7) strains was conducted in HeLa cells. The EV71 strain (GenBank accession no. JN230523.1) was amplified in RD cells. The Pilaralisib (XL147) was procured from Selleck Chemicals (S7645, Houston, TX, USA).
Plaque assay
The Vero cells (1 × 106 cells/well) were plated in 6-well plates (Cat: 41396, Beaver, China) and incubated at 37 °C with 5% CO2 for 20 h. When the monolayer confluence reached approximately 80%, the RD cells were exposed to EV71 (MOI = 0.1) in a medium for 2 h at 37 °C. Following the removal of the supernatant, serial five-fold dilutions of Pilaralisib (0.006 µM, 0.032 µM, 0.16 µM, 0.8 µM, 4 µM, and 20 µM) were added to the wells and incubated for an additional 48 h at 37 °C. The infectious virus was released by subjecting the RD cells to three freeze/thaw cycles to prepare the virus stock. Approximately 1 × 106 Vero cells per well were plated in a 6-well plate (Cat: 40106, Beaver, China) and cultured for 20 h to reach monolayer confluence. Thereafter, the diluted EV71 virus (1 mL, 1:105 of virus stock) was added to each well of the 6-well Vero plates for 2 h. Then, the supplement was replaced with DMEM containing low melting-point agarose (0.8%) (Cat: 2276GR005, neoFroxx GmbH, Einhausen, Germany) and FBS (2%) for 72 h. Following fixation with 4% formalin for 4 h at 25 °C and staining with crystal violet (0.5%) (Cat: 21070906, Biosharp, China) for 30 min, the plaques were enumerated.
Western blot analysis
The cells were lysed using western blot lysis buffer (Cat: BL509A, Biosharp, China) supplemented with the addition of a phosphatase inhibitor cocktail (CAS: 56-25-7, Roche, Switzerland). Following protein denaturation, the proteins were separated using a Bio-Rad electrophoresis system and transferred onto hybridized nitrocellulose (NC) membranes (Cat: HATF00010, Millipore, Billerica, MA, USA). Subsequent to blocking with 5% milk (Cat: 1172GR100; neoFroxx GmbH), the NC membrane was incubated at 4 °C overnight with primary antibodies (dilution of 1:1000), followed by incubation with secondary antibodies (dilution of 1:5000) for 2 h at 25 °C. Protein visualization was achieved using the Super Signal West Pico chemiluminescent substrate (CLiN, Shenzhen, China). The following antibodies were utilized in accordance with the manufacturer’s recommendations: anti-phospho-AKT (CST, cat: 4060 S), anti-total AKT (CST, cat: 4691 S), and anti-total VP1 antibody (CST, cat: 9167), goat anti-rabbit IgG (Cat: SA00001-2, Proteintech, China), goat anti-mouse IgG (Cat: SA00001-1, Proteintech, China), β-actin (Cat:60008-1, Proteintech, China), and GAPDH (Cat: 10494-1, Proteintech, China).
Immunofluorescence assay
RD cells were seeded in a 24-well plate (Cat: 702011, NEST) and infected with EV71at an MOI of 0.1 for 2 h. Various concentrations (0.006 µM, 0.032 µM, 0.16 µM, 0.8 µM, 4 µM, and 20 µM) of Pilaralisib were added and incubated for an additional 24 h. After washing and fixing at a temperature range of 20–30 °C, samples were permeabilized with Triton X-100 (0.5%) for 30 min. The samples were then blocked with 1% BSA in PBS for 1 h. Anti-dsRNA antibodies (1:500; J2-2021, Scicon, Hungary) were incubated overnight at 4 °C, followed by 50 min incubation with CY3-conjugated secondary antibodies (Cat: SA00009-1, Proteintech, Rosemont, IL, USA) at 25 °C and the plates were foil-wrapped to protect from light. Following the process of washing with PBST (0.05% Tween20 in PBS), DAPI (Cat: G1012, Biyuntian, China) incorporated for a duration of five minutes. Thereafter, images were obtained through utilization of the Operetta High-Content Analysis System, an apparatus manufactured by PerkinElmer, situated in Waltham MA USA.
Quantitative real-time PCR
The total cellular mRNA of EV71-infected cells was extracted using TRIZOL reagent (Cat: 15596-018, Invitrogen) according to the manufacturer’s instructions. Subsequently, cDNA synthesis was performed using random hexamer primers with reverse transcriptase (Cat. M1701, Promega, Madison, USA). For quantitative real-time PCR (qRT-PCR), a reaction mixture of 20 µL was prepared, containing 5 µL of cDNA, 5 µL of forward and reverse primers, and 10 µL of SYBR™ Green PCR Master Mix (Bio-Rad, USA). The qPCR reactions were subsequently conducted on a QuantStudio™ Real-Time PCR System (Thermo Fisher Scientific). The primer sequences utilized are detailed in the primer list provided in the supplementary materials (Supplementary Table S1).
Cytotoxicity assay
The compounds’ toxicity was evaluated using the MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay. In summary, RD cells were seeded at a density of 1 × 104 cells/well in 96-well plates and incubated for 16 h before being treated with five-fold serial dilution of the compounds. Control cells were treated with vehicle (DMSO) in a medium containing 2% FBS. Following a 48-hour drug administration period, the culture supernatants were removed and replaced with a medium containing MTT (CAS: 298-93-1, Biosharp, China) at a concentration of 0.5 mg/ml for an additional 4 h at 37 °C. The formazan product was then dissolved in DMSO and the resulting solution was measured for its optical density at a wavelength of 490 nm using a microplate analyzer (TECAN Infinite F50 Robot, China) with reference wavelength of 620 nm. The concentration that caused a 50% reduction in survival (CC50) compared to the control group was calculated using nonlinear regression analysis (Prism, GraphPad 8.0). It is noteworthy that all conditions were performed in triplicate for three independent experiments.
Mouse infection model
Three-day-old suckling mice were intraperitoneally injected with EV71 (8 × 106 plaque-forming units per mouse). Following a 2-hour incubation period, the mice were administered intraperitoneal injections of either 2.5 mg/kg or 1 mg/kg doses based on their weight. The control groups consisted of uninfected and infected animals treated with a placebo alone. Following a 2-day treatment period, randomly selected mice were euthanized by cervical dislocation, and tissues, including the brain, heart, liver, spleen, and limb, were collected for analysis. Quantitative polymerase chain reaction (qPCR) was employed to assess EV71 mRNA levels in these tissues. To evaluate pathological changes, hematoxylin-eosin (HE) staining was performed on tissue sections to evaluate pathological changes.
Ethics statement
Pregnant ICR mice (gestational age: 13–15 days) were procured from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and were housed in a pathogen-free facility throughout the study period. Standard housing and environmental conditions were maintained. All the animal experimental procedures and husbandry were in accordance with the guidelines approved by the Animal Care and Use Committee and the Wuhan Institute of Virology (approval number WIVA38202103).
Statistical analysis
The data obtained from the experiment are presented as the means ± standard deviations (SD) and were analyzed using GraphPad Prism 8 software. Statistical comparisons between groups were made using paired sample t-test, with a p-value less than 0.05 being considered statistically significant. To ensure the reliability of the experimental results, all experiments were independently repeated three times.