Background
Enterovirus 71 (EV71) is a non-enveloped, positive-sense, single stranded RNA virus belonging to the family Picornaviridae, and a major pathogen causing hand, foot, and mouth disease in children under 5 years of age. EV71 was first isolated in California, USA [
1]. Subsequently, EV71-associated hand-foot- mouse disease (HFMD) outbreaks occurred around the world [
2‐
7]. Clinical manifestations of EV71 infection range from mild HFMD to severe encephalitis, pulmonary edema, and even death [
8,
9]. In recent years, EV71 was reported to cause several large-scale outbreaks of severe complications in children involving the central nervous system, resulting in fatalities [
10,
11]. EV71 caused 126 deaths in 2008 in China [
12,
13] and the largest outbreak in China occurred in 2010, with an estimated 1.7 million infections, of which 27,000 caused severe neurological complications, resulting in 905 fatalities [
14]. Fatal cases were also reported in Asia as recently as 2012 [
13].
No approved direct-acting antiviral drug is available for EV71 infection to date [
15]. Thus, the development of effective anti-EV71 agent is quite urgent and possesses important significance. At present, the prevention of EV71 epidemics mainly depends upon public surveillance. Ribavirin, type I interferon, and pleconaril are the common agents used to treat EV71 infections [
16,
17]. In addition, some compounds, such as rupintrivir [
18,
19], showed activity against EV71 in both cell lines and animal models; however, clinical applications are not yet available, therefore more efforts are needed to develop drugs to conquer EV71 infections.
The RNA genome of EV71 encodes a large precursor polypeptide that is processed by viral protease to generate viral structural (VP1, VP2, VP3, and VP4) and nonstructural (2A, 2B, 2C, 3A, 3B, 3C, and 3D) proteins [
20]. Proteases play essential roles in the enteroviral life cycle. The virus polyprotein is co- and post-translationally processed by the viral proteases 2Apro, 3Cpro, and 3CDpro, the latter being the precursor of 3Cpro and the RNA-dependent RNA polymerase, 3Dpol. The first cleavage reaction in all enteroviral polyproteins is catalyzed by the 2Apro. These key viral proteases are the most prominent targets for consideration for development of antiviral agents [
9,
21].
Quercetin, one of the most widely distributed flavonoids in plants, has been studied for many years because of its anti-inflammatory and anti-cancer properties [
22], and has also been demonstrated to inhibit various viral infections. It has been reported to inhibit HIV reverse transcriptase and other retroviruses, such as herpes simplex virus type 1 (HSV-1), poliovirus type 1, respiratory syncytial virus (RSV) [
23], and to significantly decrease the production of infectious particles by hepatitis C virus (HCV) and affect virion integrity [
24]. Quercetin has also been reported to exhibit antiviral activity against HBV, influenza A virus (IAV) H1N1, and DENV-2 in vitro [
25‐
27]. However, previous reports on the anti-EV71 activity of quercetin described only basic evaluation of its effects [
28‐
30] and investigation of the anti-EV71 mechanism has not been reported to date.
Here, we studied the inhibitory activity of quercetin against EV71 and explored its anti-EV71 modes of action. We observed that quercetin showed powerful therapeutic effects, exhibited a degree of preventive activity against EV71, and also inhibited viral adsorption. Quercetin potently inhibited EV71 3Cpro activity, blocking EV71 replication. These findings are of potential relevance to the development of clinical therapeutics against EV71 infections.
Methods
Cells, viruses, and compounds
Human rhabdomyosarcoma cells (RD) and African green monkey kidney cells (Vero) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS) at 37 °C under a 5% CO
2 atmosphere. EV71 (strain SK-EV006) with GFP (EV71-GFP) was kindly supplied by Prof. Bo Zhang from Wuhan Institute of Virology. Live EV71 strain wuhan/3018/2010virus was kindly provided by Prof. YingZhu (State Key Laboratory of Virology, College of Life Sciences, Wuhan University, China) and propagated in the RD cell line. Virus titer was determined by the standard TCID
50 method [
31]. Quercetin (C15H10O5; CAS no. 117–39-5; molecular weight [MW] 302.24) was purchased from Calbiochem (Germany) and prepared in DMSO. Ribavirin, used as a positive control, was purchased from Sigma Chemical Co. Stock solutions of drugs were prepared in DMSO (final concentration 0.1%). A fluorogenic peptide Dabcyl-RTATVQGPSLDFE-Edans, corresponding to the EV71 polyprotein autoprocessing site between 3B–3C, was purchased from Shanghai Kai Jing Biotechnology Co. Ltd.
Antiviral activity and cytotoxicity
The anti-EV71 activity of quercetin was evaluated by phenotype screening. Briefly, RD cells were seeded in 96-well microtiter plates, after an overnight culture at 37 °C in 5% CO
2, infected with 100 TCID
50 of EV71-GFP virus (EV71 labeled with a stable eGFP reporter) [
32] for 1.5 h, then incubated in a quercetin dilution series (3–100 μM) for 48 h, and the expression level of GFP monitored using an epifluorescence microscope.
The antiviral activity of quercetin against EV71 was further determined by a CPE reduction method. RD and Vero cells were seeded onto a 96-well culture plate. Next day, medium was removed and the cells were infected with 100 TCID50 of EV71 in the presence of serial dilutions of quercetin for 1.5 h at 37 °C, inocula were aspirated and the cells were further incubated with equal concentrations of quercetin at 37 °C in 5% CO2 for 2 days until appropriate CPE was achieved. Controls included uninfected cells and cells infected with virus in the absence of quercetin. The morphology of the cells to observation the effect of quercetin on EV71-induced CPE was observed under microscope, and images were recorded. The MTT assay was used to determine cell viability. The concentration required for the tested compound to reduce the CPE by 50% (EC50) was determined. The cytotoxicity of quercetin was also evaluated via the MTT-method after exposing uninfected cells to various concentrations of quercetin for 48 h at 37 °C. The results were expressed as CC50, defined as the concentration causing a 50% reduction or inhibition of cell viability. SIs were calculated as the ratio of CC50: EC50.
Progeny virus titration
The titers of the virus stocks were determined by a TCID
50 assay. Serially diluted viruses from 10
− 1 to 10
− 8 in DMEM were inoculated to RD cells in 96-well plates, and the cells were incubated for 2 days at 37 °C. TCID
50 were calculated by counting the wells with cytopathic effect (CPE) in infected RD cells using the Reed–Muench method [
31] .
Modes of action assay
To explore the mechanisms underline the effects of quercetin on EV71 infection, three action modes (prevention action mode, mixture action mode, and treatment action mode) were applied. In the prevention action mode, RD cells were pre-incubated with various concentrations of quercetin for 2 h 37 °C, washed with PBS, and then infected with EV71 (100 TCID50). In the mixture action mode, an equal volume of EV71 suspension and quercetin, in DMEM were mixed and added to RD cells for 2 h at 37 °C, then inocula were aspirated and the cells were incubated with medium. In the treatment action mode, RD cells were first infected with EV71 (100 TCID50) for 2 h, the supernatants were removed and the infected cells were then treated with quercetin. The antiviral activities of quercetin were detected at 48 h post-infection using MTT assays.
Time-of-addition assay
RD cells were infected with 100 TCID50 of EV71 and then 50 μM quercetin for different periods of time: -2 h to 12 h, 0–12 h, 2 h–12 h, 4 h–12 h, 6 h–12 h, and 8 h–12 h. At 12 h post infection, culture supernatants and cell lysates were collected following freeze-thaw cycles and then subjected to virus titration by TCID50 assay.
RNA extraction and quantitative reverse transcriptase PCR
Total intracellular RNA was extracted using Trizol reagent (TaKaRa). Viral RNA samples were detected using an RT-PCR instrument (LightCycler 96, Roche) in accordance with the manufacturer’s instructions. A quantitative RT-PCR assay was performed using a PrimeScript RT reagent kit and SYBR Premix Ex Taq II (TaKaRa) following the manufacture’s protocol. Primer sequences were: EV71 VP1 forward, 5′-GCAGCCCAAAAGAACTTCAC-3′ and reverse, 5′-ATTTCAGCAG CTTGGAGTGC-3′; and β-actin, forward, 5′-GGCGGGACCACCATGTACCCT-3′ and reverse 5′-AGGGGCCGGACTCGTCATACT-3′. EV71 and β-actin transcript levels were determined using the ΔΔCT method.
Immunofluorescence microscopy
RD cells (1 × 105) were seeded in 24-well plates and incubated overnight. Cells were infected with 100 TCID50 of EV71 for 1.5 h with or without quercetin (50 μM) and the wells were fixed with 4% paraformaldehyde for 30 min at room temperature at various time points post-infection (p.i.). Plates were incubated with blocking solution (0.5% bovine serum albumin in phosphate-buffered saline (PBS)) for 1 h at room temperature and then reacted with mouse anti-enterovirus 71 monoclonal antibody diluted in blocking solution overnight at 4 °C. After plates had been washed three times with PBST (0.1% Tween-20 in PBS), they were incubated with the appropriate Alexa-Fluor-488-labeled secondary antibody and fluorescence evaluated using an OLYMPUS IX73 microscope.
Flow cytometry
To assess levels of apoptosis, RD cells in 12-well plates were untreated or infected with 100 TCID50 of EV71. After viral adsorption, the cells were incubated in the absence or presence of 50 μM quercetin for 36–48 h, until the CPE of the virus control reached 70–90%. Cells were then stained with Annexin-V-fluorescein and propidium iodideand subsequently subjected to flow cytometry analysis, according to the manufacturer’s instructions. Apoptosis levels were determined using FlowJo software (USA).
In vivo 2A protease activity assay
Plasmids encoding the 2A viral protease (pEGFP-C1-2A) or the 2A viral protease with a single point mutation (C110S) [pEGFP-C1-2Amut (C110S)] and a reporter plasmid, pCMV-FLuc, harboring firefly luciferase under the control of the CMV enhancer/promoter region, were co-transfected into RD cells seeded in 24-well plates using Lipofectamine 2000 transfection reagent (Life Technologies). Serial concentrations of quercetin were added 5 h post-transfection. After 24 h, cell lysates were collected and analyzed to determine FLuc activity using a Firefly luciferase assay system, following the manufacturer’s instructions (Multiskan®, Thermo).
In vitro activity assay of 3C protease activity
Expression and purification of EV71 3C protease
The purification of EV71 3C protease was based on a previously reported protocol [
33]. Briefly, the genomic region encoding EV71 3C protease was amplified and cloned into the pET-28a vector using the NcoI and HindIII restriction sites. Target proteins were expressed in
Escherichia coli BL21 (DE3) after induction using isopropyl β-D-1-thiogalactopyranoside, and purified by affinity chromatography using a Ni-NTA column (Qiagen, Germany). The purified proteins were concentrated to 1 mg/mL in 20 mM Tris-HCl (pH 7.0), 500 mM NaCl, 2 mM DTT buffer for storage.
In vitro protease activity assay
EV71 3C
pro is a specific protease that recognizes peptide substrates containing a Q-G junction [
34]. Thus, the substrate Dabcyl-RTATVQGPSLDFE- Edans was synthesized with fluorescence and quenching groups attached. Activity assays were performed using 1 μM 3C protease in 50 mM Tris-HCl, 200 mM NaCl, 2 mM DTT, and different concentrations of quercetin. Rutin was used as a positive control [
35]. Reactions were incubated at room temperature for 6 h in a final volume of 100 μL. Subsequently, fluorogenic peptide substrate was added to a final concentration of 20 μM, then relative fluorescence was determined using an excitation wavelength of 340 nm and monitoring the emission at 500 nm every 30 s. All experiments were conducted in triplicate. Initial velocities of proteolysis were plotted as the function of quercetin concentrations by fitting the following equation:
A(
I) =
A(0) × {1-([
I])/([
I] + IC
50)}, where
A(
I) is the difference between the enzyme activity with quercetin concentration [
I] and
A(0), which is the enzyme activity without inhibitor.
RNA elongation assay
The detailed protocol for the expression of EV71 3D polymerase and the polymerase elongation assay has been previously reported [
36]. RNA elongation assays were performed using 0.1 μM 3D polymerase in 50 mM HEPES, 10 mM 2-mercaptoethanol, 5 mM MgCl
2, 60 μM ZnCl
2, 5 μM UTP, 0.4 μCi/μL [α-
32P]UTP, 1.8 μM dT
15-primer, 0.15 μM poly (rA) 300 and various concentrations of quercetin. Reactions were incubated for 5–10 min at 30 °C, and then quenched by adding 2 × gel loading buffer (90% formamide, 0.5% EDTA, 0.1% xylene cyanol and 0.1% bromophenol). After separating the reactions on 6 M urea polyacrylamide denaturing gels, RNA products were measured using a Cyclone Plus storage phosphor system (PerkinElmer).
Molecular docking
The X-ray structure of EV71 3C protease (PDB no. 3SJO) was prepared using a Protein Preparation Wizard and Maestro 9.3 (Schrödinger, USA). The model of EV71 3C protease structure (EV71/wuhan/3018/2010) was used as a template to predicate whether quercetin affects 3C protease. The study of docking of EV71 3C protease with quercetin was performed using AUTODOCK 4.2.6, with atomic affinity potential energy calculated using AUTOGRID supporting software.
Statistical analysis
The data shown are the means ± standard deviations (SD) of three independent experiments. The levels of significance for the differences between the test and control groups were analyzed using Student t-test, and the differences were considered significant if P values were < 0.05.
Discussion
HFMD is a common pediatric illness primarily caused by infection with EV71 [
39,
40]. No effective or specific therapeutic measures against HFMD are available. Hence, an effective antiviral therapy against EV71 infection is urgently needed. Flavonoids are associated with multiple biological and pharmacological activities. Some flavonoids and isoflavonoids have also been reported to exhibit anti-EV71 activities [
23,
26]; however, the majority of compounds were only evaluated as anti-EV71 agents and research into the anti-EV71 mechanism of flavonoids and isoflavonoids has rarely been reported. In this study, we tested the anti-EV71 activity of the natural flavonoid, quercetin, and explored its mode of action against EV71 for the first time.
In this study, a two-step detection system, consisting of an enhanced GFP reporter infection assay and a cell viability-based assay, was developed to assess the antiviral activity of quercetin against EV71. A major limitation of these assays is their inability to discriminate between drug toxicity and viral-induced CPE. Moreover, reporter virus-based assays are only suitable for determining the antiviral activity of non-toxic compounds, since toxic compounds decrease the fluorescence or luminescence signal. Thus, the EV71 antiviral assay used in this study was conducted by combining these two approaches. Moreover, EC50 values of quercetin against EV71 were also obtained by other approaches, including CPE microscopy and classic virus yield reduction assay (data not shown), in addition to the two assays described above. GFP reporter infection assays, using a full-length infectious clone, allowed screening for inhibitors of any step(s) of the replication cycle of EV71. CPE detection, MTT and virus yield assays reflected changes in cellular phenotypes, metabolism, and viral multiplication, respectively. The similarity of the EC50s obtained using these different approaches suggested that quercetin alone did not inhibit cell growth and could effectively inhibit viral infection.
Furthermore, we determined the CC50 values at a maximum quercetin concentration of 200 μM, because when the concentration was higher than 200 μM, a large amount of particles would adsorb on the cell surface, resulting in an inability to visualize the state of the cells, or to accurately determine cell viability using the MTT method. The value of CC50 for quercetin was > 200 μM, which was the same as the positive control, ribavirin. Quercetin exhibited dose-dependent antiviral effects against EV71, which meant quercetin could inhibit EV71 infection only when it reached a certain concentration. Actually, quercetin had an EC50 of 12.1 μM for RD cells and 8.8 μM for Vero cells against EV71 infection, with selective indexes of > 16.6 and > 22.8, respectively. An approximately ten-fold inhibitory activity against EV71 relative to ribavirin was observed. Selectivity index (SI) values ≥4 are considered suitable for antiviral agents [
41], which suggesting that quercetin is an effective anti-EV71 agent with a relatively safe profile. Previous reports also demonstrated that quercetin showed potent anti-EV71 activity without toxic effects [
29]. These results suggest that quercetin has potential for use in therapeutic application against EV71 infection.
An internal ribosomal entry site (IRES) required for viral protein translation is a potential EV71 drug target. As previously reported, several flavonoids, including kaempferol and hesperetin, exhibit antiviral activity against EV71 through inhibition of the IRES; however, quercetin does not inhibit IRES-driven translation [
29]. Another dietary flavonoid, apigenin, suppresses EV71 replication through a novel mechanism by targeting trans-acting factors [
28]. The results of this study demonstrated that quercetin was able to effectively inhibit EV71 replication by inhibition of the activity of the virus-encoded 3C
pro, and the results verified that flavonoids with similar structures exert different mechanisms against EV71. Further study of the affinity of quercetin for 3C
pro is warranted to ascertain whether the compound affects other viral or cellular functions crucial for EV71 replication.
In this study, recombinant 3Cpro and fluorogenic substrate were used to evaluate the effect of quercetin on protease activity. The amounts of quercetin required to inhibit purified EV71 3Cpro activity in vitro were higher than those required to block EV71 replication, this is because the higher concentrations of 3Cpro present in vitro required more quercetin for inhibition.
Viral 3C
pro is a multifunctional protein involved in binding viral RNA and RNA replication, in addition to a number of other biological processes. It has also been reported that 3C
pro can impair host RNA processing and polyadenylation by cleavage of the cellular CstF-64 protein, leading to enhanced viral RNA replication [
7]. The 3C protein can also trigger apoptosis through the caspase pathway in neuronal cells [
42]. In this study, treatment of infected cells with quercetin reduced yields of viral RNA and inhibited EV71-induced apoptosis. These results led us to conclude that the inhibition of viral RNA synthesis and EV71-induced apoptosis in RD cells by quercetin was the result of its inhibition of viral 3C
pro activity.
The 3C-like protease (3CL
pro) of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is vital for SARS-CoV replication, and quercetin displays strong inhibition of 3CLpro [
43]. Quercetin has also been identified as a potent inhibitor of reverse transcriptases from rauscher murine leukemia virus (RLV) and human immunodeficiency virus (HIV) [
44]. In this study, quercetin significantly inhibited the activity of 3Cpro of EV71. Further, we predicted the interaction between EV71 3C
pro and quercetin and calculated the binding energy using molecular docking. Docking calculations between 3C
pro and other flavonoids have been reported previously, and the accurate prediction of biomolecular conformation and binding energies of protein-ligand complexes exhibited similarities, including common binding sites Leu4, Leu8, Ser111, Met112, Phe113, and Pro115 [
45,
46]. The amino acid sites predicted to be involved in 3Cpro-quercetin complexes included Met0, Arg4, Pro5, Ser111, Met112, Phe113, Leu4, Thr101, Pro115 (rank 1 and rank 2 in Table 2). Although these amino acid sites were explored for important roles in the interaction between EV71 3C
pro and other flavonoids (or its phosphate ester), they have not been identified as involved in biological catalysis to date. Analysis of the 3C
pro-quercetin interaction revealed that quercetin interacted with the 3C
pro catalytic residues, His40, Glu71 and Cys147 (rank 7 and rank 8 in Table 2), which is different to other flavonoids, this binding may decrease EV71 3C
pro activity. Moreover, for the initial manner of binding, predicted by the docking results (rank 0 in Table 2), quercetin was predicted to interact with numerous residues in 3C
pro, including Tyr138, Phe140, Ala144, His161, Ile162, Gly163, Gly164, Gln168, Gly169, and Phe170. The molecular binding model of the 3C
pro-quercetin complex revealed that quercetin may stably insert into the substrate-binding pocket of EV71 3C
pro via numerous hydrogen bond interactions. Similar binding mechanisms have been reported for the interactions between flavonoids and 3CLpro in SARS-CoV [
43], but not EV71. Therefore, these modes of interaction between different flavonoids and various viruses could be used as a reference for the design of 3C
pro inhibitors.
EV71 infection is often accompanied by the apoptosis of host cells, and it can be a mechanism for viral spread to neighboring cells, which may promote culminating a lytic infection and cause viremia and severe central nervous system complications. Here, quercetin was found to protect host cells from apoptosis induced by viral infections, suggesting that quercetin may play an important role in EV71-induced critically severe and fatal cases.