Background
Hand-foot-and-mouth disease (HFMD) is a common, usually self-limiting illness affecting large numbers of infants and young children worldwide [
1]. The clinical symptoms include fever, vesicular rashes on hands, feet, buttocks and the mouth. However, a small proportion of infected children may suffer severe complications including meningitis, acute flaccid paralysis, neurorespiratory syndrome and even fatal illness. The most important causative agents for HFMD are the enterovirus 71 (EV71) and coxackievirus A16 (CV-A16). The severe outcomes are often seen in EV71 infections but unusual with CV-A16. Recently, HFMD associated with other enteroviruses also emerged, including the coxackievirus A6 (CV-A6) and coxackievirus A10 (CV-A10) [
1].
EV71 is a member of the species
Enterovirus A, genus
Enterovirus within the family
Picornaviridae. The structure of the EV71 virion has been resolved recently, which showed an icosahedral shell with typical features of enteroviruses [
2,
3]. The EV71 mature virion has a canyon around the five-fold axes, probably as the binding site for EV71 specific receptor. Several receptors have been reported, however, only human scavenger receptor class B, member 2 (SCARB2) was subsequently shown to induce uncoating of EV71 in vitro [
4]. Recent studies suggested that the head region of SCARB2 comprising a pH-sensing three α-helix bundle might bind to the canyon and expel of the pocket factor from the EV71 virion, hence destabilizing the capsid and triggering the uncoating process [
5]. In addition to SCARB2, P-selectin glycoprotein ligand-1 (PSGL1), Annexin2, heparan sulfate, sialylated glycans, and nucleolin were also identified as EV71 binding receptors [
6‐
10]. However, these molecules cannot induce uncoating, so their roles may be related to capture EV71 on the cell surface and deliver it to SCARB2 in the endosomes.
Investigation of endocytic mechanisms revealed that clathrin mediated endocytosis (CME) was responsible for EV71 entry into human rhabdomyosarcoma cells (RD cells). Knockdown of the components in CME by small interfering RNAs (siRNAs) significantly inhibited EV71 infection. The dominant negative mutants of CME pathway, inhibitors of CME pathway and neutralizers of endosome acidification also reduced EV71 entry into RD cells [
11]. In addition, overexpression of human SCARB2 facilitated EV71 infection in a murine cell line, NIH/3 T3 cells. However, blocking CME by siRNA inhibited EV71 infection in the SCARB2-expressing NIH/3 T3 cells [
12]. Overall, these data suggest that EV71 may enter various cells via the CME dependent pathway and uncoat in an acidified endocytic compartment. Another study showed that EV71 could enter Jurkat cells (human T leukemia cell line) and PSGL-1-expressing L929 cells via the caveolar-dependent endocytic pathway (CDE) when binding to PSGL-1 [
13]. Both CME and CDE entry pathways require the dynamin to segregate the endocytic vesicles from the cell surface membrane. Thus, endocytosis inhibitors, including chlorpromazine [
14] and dynamin [
15] have been proposed or attempted to prevent or treat microbial infections.
Despite of the importance of SCARB2 in EV71 infection, several evidences indicate that the SCARB2-independent EV71 entry may exist as well. Normally, SCARB2 mainly localizes in the endosomal/lysosomal membrane, responsible for delivery of β-glucocerebrosidase from the endoplasmic reticulum to lysosomes, where the enzyme degrades glucocerebroside and its gene mutation causes defective metabolism of glucocerebrosides and Gaucher disease [
16,
17]. Only small amount of SCARB2 may shuttle to the plasma membrane as EV71 receptor. Thus, the EV71 binding and early entry very likely depend on other non-SCARB2 receptors. Second, SCARB2 transgenic mice study showed that not all tissues supported viral replication despite of their SCARB2 expression, suggesting some SCARB2-independent mechanisms contribute to EV71 susceptibility or permissiveness [
18]. Thirdly, one report found that knockdown of SCARB2 prevented EV71 infection in RD cells but not in Vero cells and concluded that other unidentified molecules functioned as EV71 receptors [
12]. Finally, although CV-A16 utilizes SCARB2 as its receptors, other major HFMD associated enteroviruses, CV-A6 and CV-A10 did not [
19]. Therefore, EV71 may enter host cells using other non-SCARB2 receptors and their endocytic pathways are unclear yet.
Here, to further examine the features of EV71 infection, several cell lines were examined in a pilot study. Although EV71 could establish infection in most of them, the viral kinetics and spreading patterns in different cells were clearly distinct. Importantly, we found that the known EV71 entry inhibitors, including both cholopromazin and dynasore, unexpectedly led to enhanced EV71 infection in A549 cells, indicating the existence of a clathrin and dynamin-independent endocytic pathway for EV71 in human lung epithelial A549 cells.
Methods
Cells and Viruses.
Human lung epithelial A549 cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS, Gibco, Waltham, MA, USA), 100 U/ml penicillin, and 100 μg/ml streptomycin. Human rhabdomyosarcoma (RD) cells, human embryonic kidney 293 T (HEK293T) cells, human lung fibroblast MRC-5, African green monkey kidney Vero cells, human cervical epithelial Hela cells, human hepatoma epithelial cells Huh-7 and HepG2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. All the cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). EV71 strain (FJ08089) [
20] belonging to genotype C4 was originally isolated by Centers for Disease Control and Prevention in Fujian province and propagated in RD cells. The culture supernatants containing the virus were harvested when all cells were dying. After centrifugation, the virus was filtered, aliquoted and stored at −80 °C. Viral titers were determined in RD cells by end-point dilution assay.
Antibodies and Reagents.
Mouse monoclonal antibody against EV71 capsid protein VP-1 was purchased from Abcam (Cambridge, UK). Rabbit monoclonal antibody to AP2M1 (Abcam), Rabbit polyclonal antibodies to FCHO2 (ProSci, Poway, CA, USA), eIF4G (Cell Signaling Technology, Danvers, MA, USA) and Enterovirus 71 2C and 3C (GeneTex, Irvine, CA, USA), Mouse monoclonal antibodies against clathrin (CLTC, Santa Cruz Biotechnology, Dallas, Texas, USA) and dynamin (Santa Cruz Biotechnology) were used in Western-blot detection of the respective proteins. The secondary antibody conjugated to Allophycocyanin (APC) was from BD Biosciences (Franklin Lakes, CA, USA). AlexaFluor594-Conjugated AffiniPure Goat Anti-Mouse IgG (H + L) was from ZSGB-BIO (Beijing, China). Fluoroshield Mounting Medium with DAPI was from Abcam. AlexaFluor 488 conjugated transferrin from human serum and AlexaFluor 594 conjugated cholera toxin subunit B were from ThermoFisher scientific (Waltham,
MA, USA). Chloropromazin and dynasore were purchased from Selleckchem (Shanghai, China). Guanidine hydrochloride (GnHCl) was from MedChem Express (Monmouth Junction, NJ, USA). All drugs were prepared according to the manufacturer’s instructions under sterile conditions.
Cell viability assay.
Cell viability upon siRNA transfection and drug treatment was assessed by employing the cell counting kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan). Briefly, cells were seeded in 96-well cell culture plates and subsequently treated with drugs for 12 h before incubation with 10 μl CCK-8 for 20 min at 37 °C. After this, the data of absorbance at a wavelength of 450 nm was collected to analyze cell viability.
Small RNA interference and gene knockdown.
siRNA for clathrin heavy chain (CLTC, Gene ID: 1213), dynamin 2 (DNM2, Gene ID: 1785), adaptor related protein complex 2 mu 1 subunit (AP2M1, Gene ID: 1173), FCH domain only 2 (FCHO2, Gene ID: 115,548) and scrambled siRNA, which were used in RNA interference experiments, were purchased from Gene Pharma (Shanghai, China), and the sequences of siRNA are listed in Additional file:
1 Table S1. All transfections were performed in a 12-well plate format. 5 × 10
4 RD or 1 × 10
5 A549 cells per well were plated overnight. Then 2 μl transfection reagent and 2.5 μl siRNA (20 μM) were prepared in Opti-MEM Reduced Serum Media (200 μl/well, Gibco) and incubated at RT for 10 min to form siRNA-lipid complexes. Subsequently, 800 μl Opti-MEM and 200 μl siRNA-lipid complexes were added to the cells. The final concentration of pooled siRNAs was 50 nM. The gene knockdown efficiency was determined by real-time qRT-PCR at 48 h and by Western blotting at 96 h. The experiments of EV71 infection were conducted at day 4 after siRNA transfection.
Monitoring viral infectivity by Flow cytometry.
EV71 or CV-A16 infected cells were trypsinized with 0.25% trypsin-EDTA and then collected for intracellular staining. Harvested cells were washed with 1% FBS-PBS, fixed and permeabilized with Fixation/Permeabilization solution (BD Biosciences) for 20 min, then washed with 1 × BD Perm/Wash buffer, incubated with the mouse anti-EV71 VP-1 antibody (diluted by 1: 1000, Abcam) at RT for 40 min, then washed and stained in 0.1 ml of APC-conjugated rat anti-mouse IgG1 (diluted by 1: 200, BD Biosciences) for 30 min, washed and resuspended in Perm/Wash buffer, finally analyzed by BD Accuri C6 (BD Biosciences).
EV71 single round infection system.
The plasmids of EV71 subgenomic replicon and EV71 capsid were kindly provided by Prof. Wenhui Li from National Institute of Biological Sciences, Beijing (NIBS). EV71 replicon construct contains all the gene segments of EV71 except the capsid portion which is replaced by the firefly luciferase. This EV71 replicon construct was led by a T7 promoter in the plasmid, and the replicon RNA was obtained from the linearized plasmid by in vitro transcription. The vector information and detailed experimental procedures can be found in the published method [
21]. In brief, the EV71 pseudotype viruses were produced by sequential transfection of capsid plasmid and EV71 replicon RNA into HEK293T cells. Capsid plasmid was firstly transfected into HEK293T cells at 60–80% confluence; 24 h later, replicon RNA was then transfected using lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). EV71 pseudotype was harvested 24 h post-RNA transfection with two rounds of freeze-thaw cycle.
50 μl of EV71 pseudotype was mixed with 450 μl of fresh DMEM and applied to cells in 24-well plate. 12 h later, 20 μM CPZ or DMSO was added. In addition, 0.5 μg/well EV71 replicon RNA was transfected into cells in 24-well plate. 12 h post-transfection, 20 μM CPZ or DMSO was applied. Firefly luciferase activity was measured following the Luciferase Assay System manual (Promega, Madison, WI, USA) at 24 h post-infection or post-transfection.
Virus binding assay.
Cells were seeded in 12-well plates (3 × 105 cells/well). The next day, cells were incubated with EV71 at an MOI of 50 and CPZ (20 μM) or DMSO for 2 h at 4 °C. Then cells were washed with cold PBS to remove unbound viruses. Total RNA was then extracted using the RNA purification kit (TIANGEN, Beijing, China). Reverse transcriptions were then carried out using the HiScript II first strand cDNA synthesis kit (Vazyme, Nanjing, China). SYRB Green-based qPCR was performed on the Eppendorf Realplex Mastercycler (Eppendorf, Hamburg, Germany) using GoTaq qPCR Master Mix (Promega). The primers for EV71 and h18 s quantification were as follows: EV71-F: 5′- GCAGCCCAAAACAACTTCAC-3′, EV71-R: 5′- AATTTCAGCAGCTTGGAGTGC-3′; h18 s-F: 5’-GTAACCCGTTGAACCCCATT-3′, h18 s-R: 5’-CCATCCAATCGGTAGTAGCG-3′.
Viral entry assay by immunofluorescence microscopy.
A549 cells and RD cells were seeded in 12-well plates (3 × 105 cells/well) with coverslips. The next day, A549 cells and RD cells were pretreated with 2.5 mM GnHCl for 30 min at 37 °C, and then incubated with EV71 at an MOI of 50 (A549 cells) or 10 (RD cells) at 4 °C for 2 h respectively. Then, A549 and RD cells were shifted to 37 °C and treated by 20 μM CPZ or DMSO for another 6 h. Thereafter, cells were washed with cold PBS, fixed with 4% paraformaldehyde (PFA, Sigma, St Louis, MO, USA) for 15 min, permeabilized with 0.05% Triton X-100 in 2% FBS/PBS, and then stained with mouse anti-EV71 VP-1 antibody. Three washes with PBS were followed by a 30 min-incubation in the dark with the secondary antibodies conjugated to AlexaFluor 594. After three washes with PBS, coverslips were applied to mounting medium with DAPI. Digital confocal imaging was performed on EVOS® FL Color Imaging Systems (Life technology, Grand Island, NY, USA).
Western blotting.
Cells were first harvested and then lysed in Radioimmunoprecipitation assay buffer (RIPA buffer) with a mixture of protease inhibitors. The concentration of proteins was quantified by bicinchoninic acid (BCA) protein assay and equal amounts of proteins were loaded and separated by SDS-PAGE, then transferred to a nitrocellulose membrane. The membrane was blocked with 0.1% Tween-20 in PBS containing 5% BSA and then was incubated overnight with primary antibodies at 4 °C. The membrane was then washed three times in 0.1% Tween-20/PBS and incubated with anti-rabbit IgG conjugated to AlexaFluor680 (Jackson ImmunoResearch, West Grove, PA, USA) for 2 h at room temperature. The immunoblots were visualized using an Odyssey Fc Imager (Lincoln, NE, USA).
Transferrin and Cholera Toxin B Uptake assay.
Transferrin is a common marker for clathrin-mediated endocytosis (CME) while Cholera Toxin B is the marker for caveolar-dependent endocytosis (CDE). To verify the inhibitory effects of CPZ and DNS, the inhibitors of CME and dynamin respectively, the uptake assay of fluorescence-labeled transferrin (through CME) and Cholera Toxin B (through CDE) were performed. Cells were seeded in 12-well plates with coverslips and then pretreated with 20 μM CPZ, 80 μM DNS or DMSO for 2 h. After treatment, cells were incubated on ice for 10 min and washed. Then, cells were incubated with 25 μg/ml AlexaFluor488 labeled human Transferrin or 10 μg/ml AlexaFluor594 labeled Cholera Toxin B for 15 min at 37 °C. Thereafter, cells were washed with PBS, fixed with 4% PFA for 15 min, and permeabilized with 0.05% Triton X-100 in 2%FBS/PBS. After wash, coverslips were applied to mounting medium with DAPI. Digital confocal imaging was performed on EVOS® FL Color Imaging Systems.
Statistical analysis
Significance of differences was evaluated using paired Student’s t test. Statistical analysis was performed with GraphPad Prism version 6.0 (La Jolla, CA, USA).
Discussion
EV71 is a prototype of
Enterovirus A species which consist more than 20 serotypes, causing HFMD, herpangina, and other diseases in infants and young children [
24]. Unlike
Enterovirus B and
Enterovirus C species which have been extensively investigated, the study of
Enterovirus A is relatively few. Most of the knowledge about
Enterovirus A came from the studies of EV71. Several molecules had been identified as potential EV71 receptors, however, only SCARB2 is widely distributed, capable of viral binding, viral internalization, and triggering uncoating [
25]. Two dynamin-dependent endocytic pathways, the CME and CDE, were discovered to be utilized for EV71 entry into SCARB2 and PSGL-1 expressing cells respectively [
4]. Here, expanding the study of EV71 in various cell lines, we surprisingly found that CME and dynamin inhibitors even enhanced EV71 infection in A549 cells, indicating an unknown dynamin-independent endocytic pathway by EV71. Clearly, this data increased our knowledge of
Enterovirus A.
To our best knowledge, CPZ either inhibited or had no effect on viral entry in previous reports [
15,
26], so the observation of enhancement of EV71 infection by CPZ is somewhat unusual and surprising. We confirmed that the enhanced EV71 entry was replication competent, since viral activities significantly increased, indicated by higher levels of eIF4G cleavage, viral nonstructural proteins, viral RNA and titers. So it is quite perplexed how CPZ affects EV71 infection in opposite ways in different cells. CPZ is an amphipathic molecule and binds to lipid bilayers; it had been reported to induce fusion of human red blood cells and viral envelopes, so we wandered that CPZ might modify the membrane properties [
27,
28]. However, two facts disagreed with this hypothesis. First, CPZ was reported to affect membrane fluidity only at higher concentrations (mM) rather than μM range utilized here. Second, we did not observe that CPZ enhanced binding of virions to any target cells. CPZ also showed no direct effect on EV71 virions in pretreatment experiments. Another possibility is that CPZ may enhance other endocytic pathways due to a compensatory response after its inhibition of CME. Actually, the cross-regulation between different endocytic components were recently reported [
29]. In this regard, we reasoned that CPZ resulted CME inhibition in A549 cells might positively affect other uncharacterized CME-independent endocytic pathways which were responsible for both the increased transferrin uptake and increased EV71 entry. Characterization of the exact steps of viral life cycle affected by CPZ also confirmed that the early entry step and not other processes were enhanced. Thus, our data supported that the endocytic system functioned uniquely in different cell types and another CME-independent pathway was responsible for EV71’s entry in A549 cells. Interestingly, CPZ was recently reported to be a strong autophagy inducer [
30,
31]. As autophagy was found to be required for efficient EV71 replication [
32‐
34], could CPZ positively affect EV71 activity through inducing autophagy? Although without clear evidence, we found that CPZ inhibited the activity of transfected EV71-subgenomic replicon RNA, suggesting CPZ did not enhance viral translation and replication. Therefore, our data dispute the potential involvement of autophagy which mainly affects post-entry activities. These findings also have implications for applying CPZ and its derivatives in antiviral therapies. CPZ is an FDA-approved antipsychotic drug used to treat schizophrenia and bipolar disorder. Due to its safety and its potency in inhibiting viral endocytosis, it had been attempted before for treating John Cunningham virus infections [
14]. However, our data indicate that the effect of its application may be complicated since it may rather enhance viral infectivity at different tissues.
Our study reveals a novel clathrin and dynamin independent endocytic pathway for cellular entry of EV71 and CV-A16. Previous studies investigated limited number of cell lines, such as RD [
11], SCARB2 expressing murine NIH-3 T3 and L929 cells [
12], or Jurkat and PSGL-1-expressing L929 cells [
13], and concluded that CME or CDE were indispensable for SCARB2- or PSGL-1 mediated EV71 entry. However, EV71/CV-A16 can infect various organs and cause a variety of diseases. They enter the host through fecal-oral route, spreading from mucosa, crossing epithelial barrier to the circulation, and then reaching to multiple tissues [
35]. Therefore, a complete understanding of EV71 entry requires a detailed investigation of various cell types. Indeed, the closely related
Enterovirus B viruses were found to use alternative surface receptors and internalize in receptor-limited cell types [
36]. Another neurotropic virus, herpes simplex virus can use different endocytic route to infect Hela cells, cultured epiderm and neurons [
37,
38]. Consistently, we found here that a novel entry pathway might be engaged by EV71 as well. Initially, we suspected this pathway was clathrin independent but still required dynamin. However, further investigation using dynamin inhibitors and siRNA certainly ruled out the involvement of dynamin. We are attempted to speculate that this novel endocytic pathway might mimic the one utilized by the
Enterovirus B species which has been associated with micropinocytosis [
36], due to their similarities with
Enterovirus A. However, the exact mechanism of this new route, the receptor usages, the endocytic compartment for uncoating etc. are still not clear yet and certainly should be pursued next.
Acknowledgments
We thank Dr. Wenhui Li from NIBS for kindly providing the plasmids of EV71 subgenomic replicon and EV71 capsid.