Japanese encephalitis virus infects porcine kidney epithelial PK15 cells via clathrin- and cholesterol-dependent endocytosis
© Yang et al.; licensee BioMed Central Ltd. 2013
Received: 27 April 2013
Accepted: 9 August 2013
Published: 12 August 2013
Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes acute viral encephalitis in humans. Pigs are important amplifiers of JEV. The entry mechanism of JEV into porcine cells remains largely unknown. In this study, we present a study of the internalization mechanism of JEV in porcine kidney epithelial PK15 cells.
We demonstrated that the disruption of the lipid raft by cholesterol depletion with methyl-β-cyclodextrin (MβCD) reduced JEV infection. We also found that the knockdown of clathrin by small interfering RNA (siRNA) significantly reduced JEV-infected cells and JEV E-glycoprotein levels, suggesting that JEV utilizes clathrin-dependent endocytosis. In contrast, the knockdown of caveolin-1, a principal component of caveolae, had only a small (although statistically significant) effect on JEV infection, however, JEV entry was not affected by genistein. These results suggested that JEV entry was independent of caveolae.
Taken together, our results demonstrate that JEV enters porcine kidney epithelial PK15 cells through cholesterol- and clathrin-mediated endocytosis.
KeywordsJEV PK15 Cholesterol Caveolin-1 Clathrin Infection
Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that belongs to the family Flaviviridae. JEV is one of the most important endemic encephalitides and can cause acute viral encephalitis, of which there are approximately 50,000 cases in humans annually . JEV can infect a wide range of cells of different origins. Pigs act as amplifying hosts of JEV; therefore, the domestic pig was considered to be a risk factor in the transmission of the disease to humans [2, 3]. JEV is also an important pathogen in swine and causes considerable economic losses in pork production. The primary symptoms of pigs infected with JEV are fetal abortion and stillbirth in infected sows and aspermia in boars [4, 5]. JEV has a single-stranded positive-sense RNA genome of approximately 11 kb. The viral RNA encodes a single large polyprotein that is cleaved into three structural proteins, capsid (C), precursor membrane (prM) and envelope (E); and seven non-structural (NS) proteins, NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5. The JEV E protein is the major structural protein exposed on the surface of the virus particle and mediates binding and fusion during virus entry [6, 7].
Viruses enter cells through binding cellular receptors. The interactions between the viruses and receptors are highly specific, determining which cell types and species can be infected. Additionally, the entrance of viruses into the host cells involves several endocytic pathways, including clathrin-mediated, caveolae-mediated, cholesterol-dependent endocytosis, macropinocytosis/phagocytosis and other mechanisms [8, 9]. Clathrin-mediated endocytosis (CME) is the best characterized of the endocytic mechanisms, and most viruses utilize this type of endocytosis to enter cells. Recent studies have shown that JEV infects neuronal cells through a clathrin-independent, dynamin- and caveolae-mediated endocytosis pathway [10, 11]. Previous studies have found that JEV enters Vero and Huh7 cells through a clathrin-dependent pathway [12, 13]. In addition, JEV internalisation into neural stem cells occurs by clathrin-mediated, caveolae independent endocytosis . Persistent JEV infection has been demonstrated in porcine kidney cells  and numerous studies on JEV have been conducted in porcine kidney cells [16–20]. Moreover, vimentin has been identified as mediating the entry of JEV into porcine kidney cells . However, the precise entry mechanism for JEV internalization into porcine cells remains unclear.
In this study, we define the role of cholesterol in JEV infection through cholesterol depletion, which significantly decreased JEV infection. In addition, we used RNA interference (RNAi) to examine the roles of clathrin and caveolin-1 in the JEV entry process; the results indicated that knockdown of clathrin reduced JEV infection, however, knockdown of caveolin-1 showed only a small effect on JEV infection and JEV entry was not affected by genistein. These results indicate that JEV endocytosis in PK15 cells is dependent on cholesterol and clathrin but not on caveolae.
JEV infection is inhibited by the depletion of cholesterol
Entry of JEV into PK15 cells is caveolae independent
Cell viability tests were determined by the CCK-8 kit, minimal cellular cytotoxicity was observed in cells treated by 200 μM genistein compared with control (Figure 3C). These concentrations of genistein were previously reported to significantly inhibit JEV infection in rat neuroblastoma B104 cells . These results suggest that, although caveolae-dependent processes may play some role in JEV infection, caveolae-mediated endocytosis is not the major route by which JEV enters the cell.
JEV infects PK15 cells by clathrin-mediated endocytosis
Lipid rafts are important for a number of cellular processes and play vital roles in virus entry . Cholesterol is a prominent component of lipid rafts. Lipid rafts could be disrupted by depletion of cholesterol using chemical drugs, such as MβCD, nystatin and filipin [29–31], and some evidence suggests that cholesterol is involved in virus entry [24, 32–34]. The inhibition of viral infection could be rescued by the addition of exogenous cholesterol. However, excess amounts of cholesterol block flaviviral infection . Previous studies found that JEV could enter various cell types through a cholesterol-dependent pathway [11, 13, 14, 23]. In the present work, PK15 cells were used to investigate the role of membrane cholesterol in JEV entry. Similar results showed that membrane cholesterol was an absolute requirement for JEV infection. Cellular entry is initiated when flaviviral gpE binds to cell surface receptors , and JEV internalisation occurs in a lipid-raft dependent manner . Therefore, the cellular receptors for JEV may located within the lipid raft domains.
Viruses enter host cells through several endocytic pathways. Recent studies have found that JEV enters rat neuroblastoma cells via caveolae-mediated endocytosis . Membrane cholesterol is required for caveolae formation, and caveolae-mediated endocytosis could be blocked by the depletion of cholesterol using MβCD . The present study shows that JEV entry is inhibited after cells were treated with MβCD; thus, we speculated that caveolae was involved in JEV internalization. Therefore, drug treatment and small interfering RNA technology were used to validate whether JEV entry into PK15 cells was caveolae-dependent. Although the flow cytometry results showed that the cells transfected with caveolin-1 siRNA inhibited JEV infection by 15% (statistically significant), suggesting that caveolae-mediated pathway may play some role in the JEV entry into PK15 cells, JEV infection was not affected by genistein. We conclude that JEV entry into PK15 cells was caveolae-independent. In addition, the receptor for JEV on PK15 cells is still uncharacterized. It is possible that a very small part of receptors was localized in caveolae, thus, the knockdown of caveolin-1 had a small effect on JEV infection.
Furthermore, MβCD, which destroys lipid raft structure by depleting cholesterol, also inhibits clathrin-coated pit budding . Therefore, we next examined the possibility of a clathrin-mediated pathway as an initial step in JEV entry into PK15 cells. Classic clathrin-mediated endocytosis is commonly used in virus internalization, and JEV entry occurs via a classic clathrin-mediated pathway in neural stem cells and Vero cells [13, 14]. In this study, JEV was also found to enter PK15 cells through the clathrin-dependent endocytic pathway. Based on published data and the results reported here, we assume that JEV is likely to infect different cell types by distinct pathways. The cellular receptors of JEV may be different in different cell types. However, a correlation between the cell receptors used and their entry pathway was unknown. Several potential cellular receptors of JEV were identified [12, 39, 40], but the genuine receptors of various cell types need to be identified to better understand the JEV pathogenic mechanism and to search for new therapeutic targets.
In the present study, we examined JEV infection using a chemical inhibitor and siRNA to disrupt different endocytic pathways in PK15 cells. Our results demonstrated that JEV entry was mediated by cholesterol- and clathrin-dependent, though not caveolin-1 dependent, pathways in PK15 cells. The mechanism of JEV entry in porcine cells was identified, and this research represents a new beginning for the development of new therapeutic targets in pigs.
Materials and methods
Cells and virus
PK15 and baby hamster kidney (BHK-21) cell lines (obtained from the American Type Culture Collection, Manassas, VA) were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Thermo Scientific HyClone, Beijing, China) supplemented with 10% fetal bovine serum and maintained in a humidified incubator at 37°C and 5% CO2. The JEV attenuated strain SA14-14-2 (GenBank accession: AF315119.1) was used in this study. Virus was propagated in BHK-21 cells. To generate virus stocks, BHK-21 cells were grown in monolayers of 75-cm2 flasks, and the cells were infected with JEV until 90% confluence was reached. The cells were harvested when the cytopathic effect was extensive (48 h). Virions were collected through three freeze-thaw cycles and centrifugation. Virus titers were determined by the 50% tissue culture infectious dose (TCID50) , and the virus suspensions at a multiplicity of infection (MOI) of 1 were utilized for all infection experiments.
Antibodies and reagents
Mouse anti-JEV E antibody was used for immunofluorescence assays and western blot analysis. Polyclonal rabbit anti β-actin, caveolin-1 and clathrin heavy chain antibodies were purchased from Cell Signaling Technology, Inc. (3 Trash Lane, Danvers, MA 01923, US). The Cell Counting Kit 8 (CCK-8), DAPI and genistein were purchased from Beyotime Biotechnology, Inc. (Jiangsu, China). Alexa Fluor 488-labeled goat anti-mouse IgG and Alexa Fluor 555-conjugated Cholera toxin B (CTxB) were purchased from Molecular Probes (Eugene, OR). HRP-conjugated goat-anti-rabbit and goat-anti-mouse secondary antibodies were purchased from Proteintech Group, Inc (Wuhan, China). Methyl-β-cyclodextrin (MβCD) was purchased from Sigma-Aldrich (St. Louis, MO) and dissolved in water.
Drug treatments and virus infection
PK15 cells were plated on 6-well plates and grown to approximately 80% confluence at 37°C in a CO2 incubator. The cells were washed three times with phosphate-buffered saline (PBS) and then pre-treated with MβCD or genistein for 1 h at 37°C at various concentrations. After treatment, the cells were infected with JEV for 1 h at 37°C in the presence of the chemical. The cells were washed to remove excess virus and drug, then further incubated in cell culture medium containing 2% FBS at 37°C with 5% CO2. Western blot and immunofluorescence staining analysis for JEV E protein expression were performed 36 h post-infection.
RNA interference experiments
For small interfering RNA (siRNA) analysis, siRNA oligonucleotides with specificity for porcine CAV1 and clathrin heavy chain (CHC) and non-targeting siRNA (siCtrl) were synthesized from GenePharma (Shanghai, China). The target sequence of siCAV1 was as follow: siCAV1-1 5’- CACACAGUUUCGAUGGCAUCUTT-3’. Sequences of siCHC-1 (equal mixture of the two RNA duplex oligonucleotides) were as follows: 5’-AAGCUGGGAAAACUCUUCAGA-3’ , 5’-UAAUCCAAUUCGAAGACCAAU-3’ , siCHC-2: 5’-GGCCCAGGUGGUAAUUAUUUU-3’. PK15 cells were transfected with 50 nM siRNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Subsequent experiments were performed 48 h post-transfection. Knockdown efficiencies were quantified by western blot analysis.
Western blot analysis
Cells were washed with PBS three times and lysed in a modified radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride [PMSF]). Protein concentrations were determined with a BCA Protein Assay kit (Solarbio, China). An equal amount of protein lysate was separated by 12% SDS-polyacrylamide gels and transferred to PVDF membranes (Millipore, Bedford, MA). The membranes were blocked with 5% nonfat milk in tris-buffered saline containing 0.1% tween-20 (TBST) and then incubated with primary antibodies overnight at 4°C. The membranes were washed three times with TBST (each wash was 10 min), and HRP-conjugated goat anti-rabbit or goat-anti-mouse secondary antibody (1:3000 dilutions in blocking buffer) was added. Bound antibodies were visualized by SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL). The mean densities of protein bands were measured by ImageJ software (National Institutes of Health, Bethesda, Maryland).
The cells were fixed with 4% paraformaldehyde for 20 min at room temperature (RT) and permeabilized with 0.2% Triton X-100 for 15 min. The cells were then incubated with a blocking buffer (PBS containing 5% bovine serum albumin [BSA]) at 37°C for 30 min. After three washes with PBS, the cells were stained with anti-JEV E mouse antibody at room temperature for 1 h. After being washed with PBS, the cells were incubated with Alexa Fluor 488 conjugated goat anti-mouse antibody (1:200 dilutions in blocking buffer) IgG. The nuclei were stained with DAPI.
PK15 cells infected with JEV were washed one time with PBS, detached and transferred to 1.5 ml centrifuge tubes. The cells were centrifuged at 1000 rpm/min for 10 min and fixed with 4% paraformaldehyde for 15 min at room temperature. After being permeabilized with Triton X-100, the cells were incubated with anti-JEV E mouse antibody overnight at 4°C. The cells were washed 3 times with PBS then incubated with Alexa Fluor 488 goat anti-mouse IgG at 1:200 for 1 h at room temperature. The cells were washed with PBS, resuspended in 500 μl PBS and analyzed using FACScan flow cytometer with CellQuest pro software (BD Biosciences, San Jose, CA). The cells were counted as infected if their fluorescence densities were greater than the intensity of the uninfected cells. The amount of infected cells relative to the untreated or siCtrl-transfected controls was given as percent infection. At least 10,000 cells were analyzed per sample.
The results were presented as the mean ± standard deviation (SD). Statistical significance was assessed by Student’s t-test, and statistical significance was ascribed when p<0.05.
We thank Professor Shengbo Cao (Huazhong Agriculture University, Wuhan, China) for generously providing the mouse anti-JEV E antibody and JEV attenuated strain SA14-14-2. This work was supported by the National High Technology Plan (863, 2013AA102502), the National Science Foundation of China (31101693, 31072009), the Fundamental Research Funds for the Central Universities (2010PY008).
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