Cells and viruses
Neuro-2a cells (N2a) were grown in Dulbecco’s modified Eagle medium (DMEM; CCS30015.03 MRC) supplemented with 10% fetal bovine serum (FBS; A6806–45 NQBB) and maintained in a humidified incubator at 37 °C and 5% CO2. Baby Hamster Syrian Kidney (BHK) cells were cultured in DMEM with 5% FBS. The challenge virus standard strain (CVS) -11 of rabies virus was stored in our laboratory. Virus was propagated in BHK-21 cells. To generate virus stocks, BHK cells were grown in monolayers of T75 flask at 90% confluence and infected with CVS-11 at a multiplicity of infection (MOI) of 0.8, then harvested after 72 h. Virions were collected through three freeze-thaw cycles and centrifugation. Viral titers were determined by calculating the 50% tissue culture infectious dose (TCID50) on N2a cells using the Karber method.
Cell viability assay
Potential cytotoxic effects of drugs on N2a cells are evaluated by MTT reagent (M5655, Sigma). Briefly, subconfluent cell cultures grown in 96-well plates were incubated with various concentrations of drugs. After incubation for 48 h at 37 °C, 10 μl of the MTT (5 mg/ml) reagent was added to cells. Then after incubation at 37 °C for another 4 h, supernatant was extracted and DMSO (V900090, Sigma) was added, then absorbance at the wave-length of 490 nm was measured by using a plate reader (Tecan) after 15 min.
Drug treatments and cell infection
For investigating the entry mechanisms of RABV, we used chlorpromazine (C2481, TCI), MβCD (C4555, Sigma), nystatin (N9150, Sigma), dynasore (D7693, Sigma) and ammonium chloride (A9434, Sigma) to treat cells. N2a cell monolayers were seeded into 6-well plates or 24-well plates and pretreated with drugs as listed before for 1 h at 37 °C. After pretreatment, cells were washed with PBS and incubated with CVS at MOI of 0.1 for 1 h at 37 °C. At 3 h and 24 h postinfection (hpi), the viral RNA level was quantitated by using a reverse transcription-quantitative real-time PCR (RT-qPCR) assay and percentage of infection was observed by fluorescence microscopy. At 48 h postinfection (hpi), western blot was performed.
Real-time qPCR analysis
RNA was extracted from cells using Trizol reagent (9109, TaKaRa). First-strand cDNA was synthesized using a PrimeScript™ RT reagent Kit with gDNA Eraser (RR047A, TaKaRa). RT-qPCR was performed on the 7500 real-time PCR system (Applied Biosystems) according to the manufacturer’s instructions (Invitrogen; Life Technologies Corp, Carlsbad CA, USA) using SYBR green real-time PCR Master Mix (4,913,914,001, Roche). The cycling conditions were as follow: 40 cycles for 95 °C 10 min, 95 °C 15 s, and 60 °C 1 min. The RT-PCR primer sequences are as follow: virus nucleoprotein genome forward primer 5′-GGTTATTGCTCGATGTGCTCCT-3′ and reverse primer 5′-GCCGCCTCGTATTCTTGAAGTT-3′; CHC forward primer 5′-GAACAGAATCAGCGGAGAA-3′ and reverse primer 5′-TCAGAGCCAAGTCAGGAT-3′; caveolin-1 forward primer 5′-AAGGAGAAGATGGAGAAGGAC-3′ and reverse primer 5′-CTTGACGTGGAAGGTGAA-3′; GAPDH forward primer 5′-AGGTCGGTGTGAACGGATTTG-3′ and reverse primer 5′-TGTAGACCATGTAGTTGAGGTCA-3′.
siRNA transfection
For small interfering RNA (siRNA) analysis, the siCHC for the clathrin heavy chain (CHC) (GGGCCUGCUGCAGCGUGCAUUAGAA) and siCav1 for caveolin-1 (UCCAUACCUUCUGCGAUCCACUCUU) were synthesized by Invitrogen. Stocks (20 μM) were prepared of each siRNA, which were aliquotted and stored at − 20 °C. N2a cells were seeded at 4 × 105 cells/well in 6 well plates and incubated at 37 °C. After adhered to the plastic, the cells were transfected with 25 pmol siRNA. Normal control-siRNA was setup for comparison with the results from the experimental group. The transfection reagents Lipofectamine RNAiMAX (13,778,150, Invitrogen) was used according to the manufacturers’ instructions. After 24 h incubation at 37 °C, the N2a cells were infected with CVS-11 at MOI of 0.1. Cells were harvested and analyzed by qPCR at 3 h and 24 h p.i., western blot at 48 h p.i..
Western blot
N2a cells were washed with PBS and lysed in a modified radioimmunoprecipitation assay (RIPA) lysis buffer (#9806, Cell Signaling Technology) with 1 mM phenylmethylsulfonyl fluoride (PMSF). Protein concentrations were determined with a BCA Protein Assay kit (#23227, Thermo). An equal amount of protein lysate was separated by 8% or 10% SDS-polyacrylamide gels and transferred to PVDF membranes (3,010,040,001, Roche). Membranes were blocked in TBST containing 5% non-fat dried milk and incubated with primary antibodies overnight at 4 °C. The membranes were washed with TBST and incubated with secondary antibody (1:2000 dilutions in 5% non-fat dried milk) for 2 h at room temperature (RT). Bound antibodies were visualized by chemiluminescent HRP substrate (#32209, Thermo). The mean densities of protein bands were measured by Image J software. The primary antibodies used are as follows: anti-rabies Virus (5B12) (NB110–7542, Novus) (1:1000), GAPDH (1A6) mAb (MB001, Bioworld) (1:5000), Clathrin Heavy Chain (P1663) Antibody (#2410, Cell Signaling Technology) (1:500), Caveolin-1 Antibody (#3238, Cell Signaling Technology) (1:500).
Immunofluorescence analysis
N2a cells were washed with PBS in the 24-well plate. The cells were fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.1% Triton X-100 for 10 min. After blocked with PBS 0.1% with 5% goat serum for 2 h, the cells were incubated with fluorescein isothiocyanate (FITC) -anti-Rabies Monoclonal antibody (1:200) (800–092, FUJIREBIO) and Evans Blue (1:200) (E2129, Sigma) for 2 h at 37 °C. Fluorescence images were acquired using Olympus confocal (Olympus FV1000 confocal laser scanning microscope, Japan). Images were analyzed using Olympus, Image J and Photoshop software.
Statistical analysis
All data were presented as the mean standard deviations (SD). Student’s t-test was used to evaluate the statistical significance of pairs of treated or untreated groups. P < 0.05 represented a statistically significant difference. All statistical analyses and calculations were performed by using GraphPad Prism 5.