Entry of Challenge Virus Standard (CVS) -11 into N2a cells via a clathrin-mediated, cholesterol-, dynamin-, pH-dependent endocytic pathway
Rabies virus (RABV), a member of Lyssavirus of Rhabdoviridae family, is a kind of negative-strand RNA virus. The zoonosis caused by RABV leads to high mortality in animals and humans. Though with the extensive investigation, the mechanisms of RABV entry into cells have not been well characterized.
Chemical inhibitors and RNA interference (RNAi) were used to analysis RABV internalization pathway. The expression level of viral N protein was examined by quantitative PCR and western blot, and the virus infection in the cells was visualized by fluorescence microscopy.
We firstly examined the endocytosis pathway of the challenge virus standard (CVS) -11 strain in N2a cells. Chlorpromazine treatment and knockdown of clathrin heavy chain (CHC) significantly reduced viral entry, which proved clathrin was required. Meanwhile neither nystatin nor knocking down caveolin-1 (Cav1) in N2a cells had an effect on CVS-11 infection, suggesting that caveolae was independent for CVS-11 internalization. And when cholesterol of cell membrane was extracted by MβCD, viral infection was strongly impacted. Additionally by using the specific inhibitor dynasore and ammonium chloride, we verified that dynamin and a low-pH environment were crucial for RABV infection, which was confirmed by confocal microscopy.
Our results demonstrated that CVS-11 entered N2a cells through a clathrin-mediated, cholesterol-, pH-, dynamin-required, and caveolae-independent endocytic pathway.
KeywordsRABV N2a Clathrin Caveolin-1 Endocytosis
Australian bat lyssavirus
bovine ephemeral fever virus
Baby hamster kidney cells
Chicken embryo-related cells
Clathrin heavy chain
Challenge virus standard
Fetal bovine serum
Hour post infection
Infectious hematopoietic necrosis virus
Metabotropic glutamate receptor subtype 2
Multiplicity of infection
Nicotinic acetylcholine receptor
Neural cell adhesion molecule
p75 neurotrophin receptor
Radioimmunoprecipitation assay lysis buffer
Reverse transcription-quantitative Polymerase Chain Reaction
Small interfering RNA
Vesicular stomatitis virus
Rabies virus (RABV), a member of the genus Lyssavirus within the family Rhabdoviridae, which is a neurotropic pathogen, causes encephalomyelitis and high mortality in animals and humans. The virion is a bullet-shaped cylinder range from 100 to 430 nm in length and 45 to 100 nm in diameter that made up of five proteins: nucleocapsid (N) protein, large (L) protein, phosphoprotein (P), glycoprotein (G) and matrix (M) protein. Among them, N, P, L proteins form the ribonucleoprotein complex (RNP) along with the viral RNA genome, which is surrounded by G and M protein . N protein binds to genomic RNA tightly protecting RNA from degradation. M protein beneath the envelop associates with both envelop and RNP, and contributes to viral assembly . The neurotropism of RABV is decided by trans-membrane protein G because G is capable of recognizing receptors that exist in cell surface and plays an important role in fusion events between virus and vesicle membranes .
Attachment to membrane mediated by recognition of receptors initiates the infection process, before internalization follows. Till now, multiple receptors like nicotinic acetylcholine receptor (nAChR), neural cell adhesion molecule (NCAM), p75 neurotrophin receptor (p75NTR), and metabotropic glutamate receptor subtype 2 (mGluR2) are identified to be utilized by RABV [4, 5]. The endocytic pathways that viruses enter the host cells include clathrin-mediated, caveolae-mediated, macropinocytosis/phagocytosis and other mechanisms [6, 7]. Most members of Rhabdoviridae family such as Vesicular stomatitis virus (VSV) , Australian bat lyssavirus (ABLV) , infectious hematopoietic necrosis virus (IHNV)  or Bovine ephemeral fever virus (BEFV) internalize host cells through clathrin-mediated endocytosis . Although the study of rabies virus endocytic pathway is not in-depth, several researches on the precise mechanisms of RABV uptake into different kinds of cells have been conducted. Using a recombinant VSV of which the endogenous glycoprotein was replaced with that of RABV (rVSV RABV G), previous studies have found that RABV internalized African green monkey kidney cell line (BS-C-1)  and peripheral neurons  through clathrin-mediated endocytic pathway by pharmacological perturbations or protein abundance, while G protein is the key factor to facilitate endocytosis. Additionally virus particles were observed by electron micrographs in coated pits in chicken embryo-related (CER) cells  and hippocampal neurons . Nonetheless the mechanisms by which RABV enters cells are not well characterized.
Our goals are to discuss the pathway of RABV internalization in neuronal cells and clarify whether CVS-11 enters N2a cells via clathrin- or non-clathrin-mediated endocytosis. In this study we used chemical inhibitors and RNA interference (RNAi) to examine the roles of clathrin and caveolin-1 in the viral entry process. The results indicated that chlorpromazine and knockdown of clathrin heavy chains (CHC) reduced CVS-11 infection, however, CVS-11 entry was not affected by nystatin or knockdown of caveolin-1. In addition, we defined the involvement of cholesterol, dynamin, low-pH in CVS-11 infection through chemical approaches. Ultimately the results will promote our current recognization of Lyssavirus endocytosis mechanism and provide a novel target of antiviral drug development.
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′.
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..
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).
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.
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.
RABV entry is dependent on clathrin-mediated endocytic pathways
Cholesterol is required for RABV infection
RABV infection is caveolae independent
RABV entry is dependent on dynamin
RABV entry requires a low pH condition
Though rabies virus has been studied for hundreds of years, the researches about the mechanisms of the entry into infectious cell still remain few. In this study, we investigated for the first time the N2a cells entry process of CVS-11 particles through a clathrin-mediated, cholesterol-, dynamin-, pH-dependent endocytic pathway.
Viruses utilize different endocytic pathways to enter host cells. Among these, clathrin-mediated endocytosis is most frequently used by many viruses. Previous studies used a recombinant VSV of which the endogenous glycoprotein was replaced with that of RABV and found that clathrin-mediated pathway was utilized in African green monkey kidney cell line (BS-C-1) and peripheral neurons [12, 13]. To further examine the internalization of CVS-11 into N2a cells, we initially used chemical inhibitor (chloroquine) and siRNA targeting the heavy chain of clathrin (CHC) to disrupt the clathrin-dependent entry pathway. As demonstrated previously, virus infection was significantly inhibited, suggesting that CVS-11 entry needed clathrin involved. Cholesterol is a prominent component of lipid rafts and play vital roles in virus entry [23, 24, 25]. It was reported that the cholesterol depletion leaded to increase in RABV adsorption and infection in both BHK-21 and HEp-2 cells . In the present work, N2a cells were treated with chemical drug of MβCD to deplete cholesterol, but opposite results were shown that membrane cholesterol was an absolute requirement for CVS-11 infection, which was consistent with the cholesterol’s function in formation of clathrin-coated endocytic vesicles . Since membrane cholesterol is also required for caveolae formation , we next examined whether caveolae played any role in CVS-11 internalization. To specifically inhibit caveolae-mediated endocytosis, nystatin was added and siRNA was used to knockdown the expression of caveolin-1 (Cav1). CVS-11 infection was not affected, so we concluded that CVS-11 entry into N2a cells was caveolae independent. Dynamin as a GTPase mediates membrane fusion required for clathrin-mediated endocytosis . The essential role of dynamin in CVS-11 entry process was also determined from dynasore markedly decreasing CVS-11 infection in N2a cells. The low-pH dependence of CVS-11 infection could easily be speculated from the reduction of viral infection after ammonium chloride treatment.
In this study, we used chemical inhibitors and siRNA to dissect the internalization mechanism of CVS-11 in N2a cells for the first time. Evidences presented here demonstrated that CVS-11 entry was mediated by clathrin-, cholesterol-, dynamin- and pH-dependent, but not caveolin-1 dependent, pathway in N2a cells. Our studies have supplemented the deficiency of RABV entry-related researches and contributed to better understanding the RABV pathogenic mechanisms.
MZ designed the experiments. JG, XW, MZ, EL carried out the experiments. JG, XW performed the data and image analyses. MD, ZG participated in part of the data analysis. YG guided the analysis and wrote the paper. All authors read and approved the final manuscript.
This work was supported by the National Key Research and Development Program of China (Grant No. 216YFD0500402); the National Natural Science Foundation of China (Grant No. 31472208, 31702238) and the Jilin Scientific and Technological Development Program (Grant No. 20180520039JH).
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The authors declare that they have no competing interest.
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