Antiviral Research 112 (2014) 18–25

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BPR-3P0128 inhibits RNA-dependent RNA polymerase elongation and VPg uridylylation activities of Enterovirus 71 Arul Balaji Velu a,b, Guang-Wu Chen b,d, Po-Ting Hsieh a,b, Jim-Tong Horng b,e, John Tsu-An Hsu f, Hsing-Pang Hsieh f, Tzu-Chun Chen b, Kuo-Feng Weng b, Shin-Ru Shih a,b,c,g,⇑ a

Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan Research Center for Emerging Viral Infections, College of Medicine, Chang Gung University, Taoyuan, Taiwan Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, Taoyuan, Taiwan d Department of Computer Science and Information Engineering, College of Medicine, Chang Gung University, Taoyuan, Taiwan e Graduate Institute of Biomedical Sciences & Department of Biochemistry, College of Medicine, Chang Gung University, Taoyuan, Taiwan f Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Chunan, Taiwan g Clinical Virology Laboratory, Chang Gung Memorial Hospital, Taiwan b c

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Article history: Received 2 May 2014 Revised 29 September 2014 Accepted 7 October 2014 Available online 18 October 2014 Keywords: EV71-RNA-dependent RNA polymerase (RdRp) 3D polymerase inhibitor VPg uridylylation inhibitor

a b s t r a c t Enterovirus 71 (EV71) infections can cause hand, foot, and mouth disease with severe neurological complications. Because no clinical drug is available for treating EV71 infections, developing an efficient antiviral medication against EV71 infection is crucial. This study indicated that 6-bromo-2-[1-(2, 5-dimethylphenyl)-5-methyl-1H-pyrazol-4-yl] quinoline-4-carboxylic acid (BPR-3P0128) exhibits excellent antiviral activity against EV71 (EC50 = 0.0029 lM). BPR-3P0128 inhibits viral replication during the early post infection stage, targets EV71 RNA-dependent RNA polymerase and VPg uridylylation, and also reduces viral RNA accumulation levels and inhibits viral replication of EV71. Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction Enterovirus 71 (EV71) is a species of the genus Enterovirus, belonging to the Picornaviridae family. EV71 infection causes hand, foot, and mouth disease (HFMD) and herpangina. Although the symptoms of both diseases are generally relatively mild, EV71 infection can result in some cases of severe neurological complications and death (Chang et al., 1999; Ho et al., 1999). During 1998, a large EV71 outbreak occurred in Taiwan, resulting in nearly 80 deaths and 405 cases involving severe infection (AbuBakar et al., 2000). In 2000 and 2001, 51 EV71-related fatalities were reported (Bible et al., 2007), and in 2007, 38 HFMD cases were reported in India (Sarma et al., 2009). Previous studies (Lin et al., 2003; Zhao et al., 2008; Bible et al., 2008; Yang et al., 2009) have reported that during 2009, EV71 outbreaks occurred in China (115,000 cases), Hong Kong (50 fatalities and 773 cases involving severe infection), Singapore (5472 cases involving severe infection), and Taiwan ⇑ Corresponding author at: Research Center for Emerging Viral Infections, Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, 259 Wen-Hwa 1st Road, Kwei-Shan Tao-Yuan 333, Taiwan. Tel.: +886 32118800x5497. E-mail address: [email protected] (S.-R. Shih). http://dx.doi.org/10.1016/j.antiviral.2014.10.003 0166-3542/Ó 2014 Elsevier B.V. All rights reserved.

(9 cases of HFDM with severe complications). In 2010, the largest known outbreak of EV71 occurred in China, involving 1.7 million cases, 27,000 patients with severe neurological complications, and 905 fatalities (Zeng et al., 2012). Because no drug is available for treating EV71 infection, developing an efficient anti-EV71 agent is crucial (Chen et al., 2008). EV71 is a nonenveloped virus containing a single positivestranded genomic RNA of approximately 7.4 kb. The genome translates into a single polyprotein that is divided into the 3 regions P1, P2, and P3. The P1 region encodes viral capsids VP1, VP2, VP3, and VP4, and it contributes substantially to pathogenesis. VP1 plays a vital role in adsorption and uncoating processes. The P2 and P3 regions encode proteins involved in genome replication and processing (Li et al., 2007; Shih et al., 2004; Xu et al., 2010). The EV71 virion comprises several components that are potential targets for antiviral drug development, including viral capsid proteins, proteases, and proteins associated with viral RNA replication—2B, 2C, 2BC, 3A, 3B (VPg), 3AB, 3CD, and 3D (Konig and Rosenwirth, 1988; Rodriguez and Carrasco, 1993; Dragovich et al., 1998; Deszcz et al., 2004; Lee et al., 2008; Chen et al., 2008; Stone et al., 2008; Arita et al., 2010). The 3D polymerase protein functions as an RNA-dependent RNA polymerase (RdRp) that is essential for viral RNA synthesis (Gohara

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et al., 1999; Thompson and Peersen, 2004). All picornaviruses have a VPg protein that is covalently linked to the 50 -ends of theirgenomes (Takegami et al., 1993; Paul et al., 2003). EV71 genome replication involves using the plus strand as a template for minus-strand synthesis, which is subsequently used as a template for producing excess plus strands. Previous studies have asserted that the initiation of both plus- and minus-stranded RNA synthesis is primed by a uridylylated type of VPg protein, denoted as VPg-pUpU (Gamarnik and Andino, 1998; Paul et al., 2000; Paul, 2002). 6-bromo-2-[1-(2,5-dimethylphenyl)-5-methyl-1H-pyrazol 4-yl] quinoline-4-carboxylic acid (BPR-3P0128), a derivative of 2-(4 bromophenyl)-6-chloroquinoline-4-carboxylic acid (CSV0C019002), was screened from 200,000 compounds. Moreover, BPR-3P0128 exhibits antiviral activity against several RNA viruses (Hsu et al., 2011). In this study, we investigated the antiviral mechanism of BPR-3P0128. We observed that BPR-3P0128 inhibits EV71 viral RdRp activity involving the 3D protein, as well as uridylylation activity of the VPg protein. Both the viral RNA replication and yield were reduced.

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(phenazine methosulfate) method (Cell Titer96 AQueous cell proliferation kit; Promega, Madison, WI, USA) in accordance with the manufacturer’s instructions. RD cells (1  105 cells/well) were seeded into each well of a 96-well microtiter plate. After incubating the cells for 24 h, the medium was replaced with fresh medium with 2% FBS and a 2-fold serial dilution of the test compound (each dilution was performed in triplicate). The cells grew for 64 h at 37 °C, and 90% confluence was attained. Subsequently, the culture medium was replaced with 100 lL of phenol red-free medium containing MTS (Promega, Madison, WI, USA) and PMS (Sigma, Saint Louis, Missouri, USA). The plate was incubated for 1–4 h at 37 °C. Subsequently, the absorbance at 490 nm was recorded using a microplate reader. The survival rate of the compound-treated cells was determined using the following formula: cell viability (%) = (OD490 of treated cells  OD490 of blank)/(OD490 of control cells  OD490 of blank). The 50% cytotoxic concentration (CC50) was determined based on the compound concentration at which the cell viability was reduced to 50%. Data were analyzed using GraphPad Prism 5.0 software. 2.4. Plaque inhibition assay

2. Materials and methods 2.1. Virus and cell cultures Rhabdomyosarcoma (RD) cells (American Type Culture Collection accession No. CCL-136) were grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS). We employed EV71 Tainan/4643/98/MP4 virus to examine the antiviral spectrum of BPR-3P0128. Coxsackieviruses A6, A10, and A16 were isolated from clinical specimens in the Clinical Virology Laboratory at Chang Gung Memorial Hospital (Linkou, Taiwan). EV71 Tainan/4643/98/MP4 and coxsackieviruses A6, A10, and A16 were propagated in RD cells. BPR-3P0128, DTrip-22 (Chen et al., 2009) and Pyridyl imidazolidinone (Shia et al., 2002) were synthesized at the National Health Research Institutes (Taiwan) and dissolved in dimethyl sulfoxide (DMSO). 2.2. Cytopathic effect inhibition assay We employed a cytopathic effect inhibition assay to determine the inhibition of BPR-3P0128 virus-induced cytopathic effects. Each well of a 96-well plate was seeded with 1  105 RD cells in DMEM containing 10% FBS (E10). After 24 h incubation at 37 °C, the RD cells were infected with EV71 at an multiplicity of infection (MOI) of 0.005 PFU/cell. After 1 h of viral adsorption, the infected cells were immersed in a medium containing 2% FBS (E2) and 0.5% DMSO or various concentrations of BPR-3P0128 (2-fold dilutions from 25 lM). The infected cells were further incubated for 64 h at 37 °C. Subsequently, the cells were fixed in 10% formaldehyde and stained with 0.1% crystal violet. The formula EC50 = [(Y  B)/(A  B)]  (H  L) + L] was applied to calculate the 50% effective concentrations (EC50), where Y represents 50% of the mean quantity of control cells at an optical density of 570 nm (OD570), B is the mean OD570 of the wells with compound dilutions close to or less than Y, A denotes the mean OD570 of the wells with compound dilutions close to or higher than Y, and L and H are the respective compound concentrations of B and A (Chen et al., 2009).

RD cells (6  105 cells/well) were seeded into 6-well plates and incubated for 24 h at 37 °C. After washing the cells with 1% PBS, they were infected with EV71 Tainan/4643/98 at an MOI of 50. After 1 h of viral adsorption at 37 °C, the cells were washed twice with 1% PBS and subsequently immersed in 3 mL of a medium containing 2% FBS supplemented with 0.3% agarose and various concentrations of BPR-3P0128 (0, 0.01, 0.02, 0.03, 0.04, 0.05, and 0.06 lM). After the cells were incubated for 4d at 37 °C, they were fixed in 10% formaldehyde for 1 h at room temperature, and then stained with 0.1% crystal violet. 2.5. Antiviral assay of BPR-3P0128 on EV71 RD cells (6  105) were seeded in 6-well plates and incubated for 24 h at 37 °C. The cells were subsequently washed with 1% PBS and then infected with EV71 Tainan/4643/98/MP4 at MOIs of 1, 0.1, and 0.01 PFU/cell. After 1 h of absorption at room temperature, the infected cells were washed twice, covered with medium containing 2% FBS and various concentrations of BPR-3P0128 (0, 0.5, 1, 1.5, 2, and 2.5 lM). After being incubated for 16 h at 37 °C, the supernatant and debris were collected, and the total viral yield was quantified using a plaque assay. The 50% inhibitory concentrations were calculated by performing a nonlinear regression analysis (GraphPad Prism 5). 2.6. Plaque assay RD cells (6  105 cells/well) were seeded in a 6-well plate and incubated for 24 h. The cells were washed and infected with or without virus by performing 10-fold serial dilutions. Following viral absorption at room temperature for 1 h, the infected cells were washed twice with 1% PBS and subsequently covered with a medium containing E2 and 0.3% agarose gel. The infected cells were further incubated for 4 d at 37 °C. The plates were fixed in 10% formaldehyde and stained with 0.1% crystal violet. Finally, the plaques were counted. The viral titers are presented as the number of plaque-forming units (PFUs) per milliliter. 2.7. Time-of-addition assay

2.3. Cytotoxicity assay Cell viability was evaluated using the MTS {tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt]}/PMS

A time-of-addition assay involved infecting RD cells (6  105 cells/well) with EV71 at an MOI of 1. After 1 h of absorption at room temperature, the infected cells were treated with 3 lM BPR-3P0128 at specific times (0, 1, 2, 4, 6, 8, 10, 12 and 14 h). At

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16 h postinfection, the culture supernatants and cell lysate were collected, and the viral yield was quantified using a plaque assay. 2.8. Quantitative real-time reverse-transcriptase polymerase chain reaction RD cells (6  105 cells/well) were infected with EV71 at an MOI of 1 PFU/cell. After 1 h of absorption at room temperature, the cells were washed twice and supplemented with or without medium containing 3 lM BPR-3P0128. The cells were further incubated at 37 °C for the indicated periods. Total intracellular RNA was extracted using an NucleoSpin RNA II (Macherey–Nagel, Bethlehem, PA). Total viral RNA samples were detected using a real-time reverse-transcriptase polymerase chain reaction (RT-PCR) instrument (SuperScript III Reverse Transcriptase kit, Invitrogen, and LightCycler LC480, Roche) in accordance with the manufacturer’s instructions. The following EV71 RNA primers were used: (1) sense 50 -CCC TGA ATG CGG CTA ATC C-30 ; and (2) antisense 50 -ATT GTC ACC ATA AGC AGC CA-30 . We employed b-actin sense 50 -GCT CGT CGT CGA CAA CGG CTC-30 and antisense 50 -CAA ACA TGA TCC TGG GTC ATC TTC TC-30 as the internal control (Chen et al., 2009). Each sample was assayed in triplicate. 2.9. Slot-blot Total RNA was extracted and dissolved for 30 min in 20 SSC containing formaldehyde at 60 °C. Subsequently, the reactant was loaded onto a nitrocellulose membrane in a slot-blot manifold. After washing it twice with 6 SSC, the membrane was removed, air dried and crosslinked in a stratalinker (Stratagene) to obtain an optimal crosslinking performance of 1200 J. The membrane was prehybridized for 30 min at 68 °C in a DIG Easy Hybridization buffer (Roche). Genome- or antigenome-specific digoxigenin (DIG)-labeled RNA probes were produced using a DIG Northern Starter kit (Roche). After adding the RNA probes at 100 ng mL1, the blots were incubated for 16 h at 68 °C. After hybridization, the membrane was immediately submerged in a tray containing low-stringency buffer (2 SSC containing 0.1% SDS) at room temperature for 5 min while shaking. The blot was then incubated twice (15 min each while shaking) in a high-stringency buffer (0.1 SSC containing 0.1% SDS) at 68 °C. Subsequently, the membrane was incubated using a washing buffer (Roche) for 5 min at room temperature while shaking. After the membrane had been blocked with a blocking solution (Roche) for 30 min, it was incubated with alkaline phosphatase-conjugated anti-DIG antibody solution for 30 min, and then washed twice with maleic acid buffer (0.1 M maleic acid, 0.15 M NaCl, 0.3% Tween 20, pH 7.5; Roche). Subsequently, the sample was equilibrated for 5 min in 20 mL of detection buffer (0.1 M Tris/HCl [pH 9.5], 0.1 M NaCl; Roche). Finally, a chemiluminescent substrate (CDP-Star; Roche) was added and the membrane was exposed to Kodak film.

(3  10 min in 5% dibasic sodium phosphate) and subsequently rinsed with absolute ethanol. Sample radioactivity was quantified in 1.5 mL scintillation fluid. The related elongation activity of 3D polymerase is shown in bar graph. Significance was assessed by one-way analysis of variance followed by Turkey’s post hoc multiple comparison test. 2.11. In vitro uridylylation assay VPg uridylylation was assayed with a reaction mixture containing HEPES (50 mM, pH 7.5), manganese(II) acetate (2.5 mM), glycerol (8%), poly(rA)300 RNA (0.5 lg), EV71-synthesized VPg peptide (2 lg), polymerase (1 lM), [a-32P]UTP (0.04 lM), unlabeled UTP (10 lM), and BPR-3P0128. The reaction mixture was incubated for 60 min at 34 °C. The sample was analyzed using Tricine sodium dodecyl sulfate–polyacrylamide gel electrophoresis with 13.5% polyacrylamide. The sample radioactivity was exposed to X-ray film (Chen et al., 2009). 2.12. Generation of BPR-3P0128-resistant viruses RD cells (6  105 cells/well) were seeded in a 6-well plate and incubated for 24 h. The cells were infected with EV71 Tainan/ 4643/98/MP4 at an MOI of 0.2 PFU/cell. After virus absorption for 1 h, the cells were washed twice and incubated for 3d in 3 mL of DMEM–2% FBS containing 0.01 lM BPR-3P0128. The supernatants from the 6-well plates were pooled and centrifuged at 4000 rpm for 10 min at 4 °C. Subsequently, the debris was freeze–thawed 3 times and centrifuged again. Finally, the clear supernatant was denoted as Passage 1. The Passage 1 virus was used to infect a new cell monolayer, which was further incubated in the presence of a compound. Passages 2–5, 6–8, and 9–11 were incubated with 0.01, 0.02, and 0.03 lM BPR-3P0128. After Passage 11 was completed, a plaque assay was performed. 3. Results 3.1. Efficiency of BPR-3P0128 against EV71 BPR-3P0128 was an analog synthesized based on CSV0C019002. Fig. 1 presents the chemical structure of BPR-3P0128. We employed a cytopathic effect inhibition assay to determine the EC50 value of BPR-3P0128 against EV71. The formula EC50 = [(Y  B)/(A  B)]  (H  L) + L] was applied to calculate the 50% effective concentrations (EC50). Based on cytopathic effect inhibition assay results, BPR-3P0128 is an inhibitor against EV71 Tainan/4643/98/MP4 (EC50 = 0.0029 lM). We evaluated the cytotoxicity of the compound by using an MTS-based assay in RD cell. The CC50 of the compound was higher than 20 lM. We analyzed the efficiency of BPR-3P0128 against EV71 (MOI of 50 PFU/well) by using plaque reduction assays. Various

2.10. RdRp (3D polymerase) elongation assay The expression of EV71 3D polymerase and a polymerase elongation assay were performed as detailed in previous research (Gohara et al., 1999; Thompson and Peersen, 2004). RNA polymerase assays were performed using 3D polymerase (1 lg/lL) in HEPES (50 mM, pH 7.5), 2-mercaptoethanol (10 mM), MgCl2 (5 mM), ZnCl2 (60 lM), UTP (10 mM), [a-P32] UTP (2 lCi/lL), dT15 (40 pmol/lL), polyA RNA (2 lg/lL) (primer/template), and various concentrations of BPR-3P0128. The reactants were incubated for 10 min at 30 °C and terminated by adding 0.5 M ethylenediaminetetraacetic acid. A 10 mL sample of the quenched reactant was spotted onto DE81 filter paper discs (Whatman) and dried at room temperature. The discs were washed

Fig. 1. Structure of BPR-3P0128: BPR-3P0128-6-bromo-2-[1-(2,5-dimethylphenyl)5-methyl-1H-pyrazol-4-yl] quinoline-4-carboxylic acid.

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concentrations of BPR-3P0128 (0, 0.01, 0.02, 0.03, 0.04, 0.05, and 0.06 lM) covered the infected monolayer in medium for a 4 d incubation. We observed that the plaque numbers diminished in a dose-dependent manner (Fig. 2a). These results confirmed that BPR-3P0128 exhibits excellent antiviral efficiency against EV71. Anti-EV71 activity was evaluated at MOIs of 1, 0.1, and 0.01 PFU/cell after incubation for 16 hpi in the presence of BPR3P0128 (0, 0.5, 1.0, 1.5, 2.0, and 2.5 lM). Total virus yields were determined using a plaque assay, and the EC50 values were calculated using a nonlinear regression analysis (Graph Pad Prism5). The results presented in Fig. 2b indicated that BPR-3P0128 inhibited EV71 at both low and high MOI. BPR-3P0128 at 0.5 lM reduced viral yield by 95% for an MOI equal to 1, and reduced the yield by 100% for infections with MOIs of 0.1 and 0.01 PFU/cell. The EC50 values were 0.397, 0.076, and 0.028 lM, for MOIs of 1, 0.1, and 0.01, respectively.

16 h. Supernatants were collected, and the virus yield for a single viral replication cycle was quantified using a plaque assay. The yields were compared to that of the virus control. The viral yield at 0, 1, 2, and 4 h inhibited 100% of virus in cells exposed to the inhibitor following viral absorption compared with the control. Subsequently, the yield gradually increased in conjunction with the incubation time from 6 to 8 h, and 50% of the virus yield was attained at 10 h (Fig. 3). No inhibition was observed at 12 h. The antiviral effects of BPR-3P0128 significantly inhibited the virus at 0, 1, 2 and 4 h. The time-of-addition assay results indicated that BPR-3P0128 exerts antiviral effects during the early stages of the EV71 replication cycle. Subsequently, genetic and biochemical approaches were used to probe the BPR-3P0128 antiviral mechanism against the EV71 virus.

3.2. BPR-3P0128 inhibited EV71 replication at an early stage of infection

We treated infected cells (MOI of 1) with and without 3 lM of BPR-3P0128 to examine the effect of BPR-3P0128 on the synthesis of EV71 RNA. Total intracellular and extracellular RNA was isolated at various hpi times (Fig. 4a). The viral RNA accumulation level was quantified using a quantitative real-time RT-PCR. The real-time RTPCR results indicated that the presence of 3 lM BPR-3P0128 reduced the viral RNA production by 97–99% at 6–12 hpi in samples that were compared with treated DMSO at each time point (Fig. 4a). The reduction in viral RNA resulting from BPR-3P0128 treatment was also observed during the slot-blot analysis. BPR3P0128 reduced the viral yield of both plus- and minus-stranded viral RNAs (Fig. 4b). These results indicated that BPR-3P0128 inhibits EV71 RNA accumulation during viral replication.

A time-of-addition assay was performed to determine which EV71 replication stages BPR-3P0128 inhibits. RD cells were infected at an MOI of 1, and a 3 lM concentration of BPR-3P0128 was added at various times following viral absorption (0, 1, 2, 4, 6, 8, 10, 12, and 14 h). Subsequently, the cells were incubated for

3.3. BPR-3P0128 reduced viral RNA accumulation of EV71

3.4. BPR-3P0128 inhibited EV71 3D RdRp activity To examine the effect of BPR-3P0128 on in vitro EV71 RdRp activity, we measured the amount of radiolabeled Uridine triphosphate (UTP) incorporated into poly (U) RNA in the presence of poly (A) templates and oligo (dT) primers, and applied the rate of UTP incorporation of EV71 3D RdRp in the presence of 5% DMSO as the control. RdRp polymerase activity decreased in a dose-dependent manner in the presence of BPR-3P0128 and DTrip-22 (Fig. 5a) (Chen et al., 2009). BPR-3P0128 concentrations of 100–500 lM markedly reduced the RdRp in a dose-dependent manner. The negative control pyridyl imidazolidinone was a capsid

Fig. 2. (a) BPR-3P0128 inhibits EV71: RD cells were infected with EV71 (MOI of 50 PFU/well) and BPR-3P0128 in agarose-containing medium. BPR-3P0128 reduced the EV71 (Tainan/4643/98/MP4) plaque numbers in a dose-dependent manner. The experiments were performed in triplicate, and a single representative result is presented. (b) Antiviral assay of BPR-3P0128 activity against EV71: RD cells were infected with EV71 (MOI of 1, 0.1 and 0.01 PFU/cell, separately) and treated with various concentrations of BPR-3P0128 at 16 hpi. BPR-3P0128 inhibited EV71 at both low and high MOIs. The 50% inhibitory concentrations were obtained by performing a nonlinear regression analysis (GraphPad Prism 5). The results of 3 independent experiments are presented as the mean ± the standard error of the mean (SEM).

Fig. 3. Time-of-addition assay: RD cells were infected with EV71 (MOI of 1) and treated with BPR-3P0128 at 3 lM at various times. At 16 h post infection the culture supernatant and cell lysate were collected. Total virus yield was measured using a plaque assay. The results of 3 independent experiments are presented as the mean ± the standard error of the mean (SEM). VC – Virus control. The statistical significance was assessed by the Student’s-t-test (⁄P 6 0.05 and ⁄⁄P 6 0.01 treated versus the control).

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Fig. 4. (a) Effect of BPR-3P0128 on the level of accumulated viral RNA: RD cells were infected with EV71 (MOI of 1) and treated with or without 3 lM BPR-3P0128 at various hpi times. Total viral RNA was extracted and determined by applying a quantitative RT-PCR technique. The EV71 viral RNA accumulation level was reduced by BPR-3P0128. The amount of viral RNA at 12 hpi in the absence of the compound was set to 100%. The relative amount of viral RNA isolated at each time point is presented as a percentage on the vertical axis. The results of 3 independent experiments are presented as the mean ± the standard error of the mean (SEM). The statistical significance was assessed by the Student’s-t-test (⁄⁄⁄P 6 0.001 treated versus the control group). (b) Slot-blot analysis: the condition of the treatment of the infected cells with BPR-3P0128 is detailed in (a). Cytoplasmic RNA was extracted at the indicated times. The amounts of plus- and minus-stranded viral RNA were detected using a slot-blot analysis. Regarding the loading control, the total RNA in each test was measured using a b-actin probe.

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(a)

(b) BPR-3P0128 (µM)

VPg pU (pU) 1

2

3

4

5

6

7

8

9

10

Fig. 5. (a) EV71 3D viral polymerase activity: Poly (U) polymerase activity was measured with 1 lM polymerase in a reaction buffer. The percent of activity shown on the vertical axis represents the activity of tested polymerase (in the presence of BPR-3P0128) divided by that of the untreated polymerase. BPR-3P0128 and DTrip-22 inhibited the activity of 3D RdRp activity in a dose-dependent manner. Pyridyl imidazolidinone did not inhibit the RdRp activity. The results of 3 independent experiments are presented as the mean ± the standard error of the mean (SEM). The difference among the various groups was assessed by performing a one-way analysis of variance and post hoc Tukey’s multiple comparison test (⁄⁄⁄P 6 0.001 and ⁄⁄P 6 0.01 versus the control group). (b) Effect of BPR-3P0128 on VPg uridylylation in vitro: Poly (A) RNA was used as a template in the presence of BPR-3P0128 and DTrip-22. The reaction conditions for the upper and lower panels are identical. BPR-3P0128 reduced the VPg uridylylation in a dose-dependent manner, and DTrip-22 did not reduce the VPg uridylylation.

inhibitor of EV71, and it did not inhibit 3D polymerase activity (Shih et al., 2004). The experiments were performed in triplicate, and the results consistently indicated that the EV71 3D polymerase activity was reduced in the presence of BPR-3P0128, which confirmed that BPR-3P0128 inhibits EV71 3D RdRp activity. 3.5. BPR-3P0128 inhibited the 3D polymerase of VPg uridylylation An in vitro assay was performed to detect the ability of EV71 polymerase to generate VPg-pU (pU) in the presence of BPR3P0128. The 3D polymerase was uridylylated VPg in the presence of poly (A) RNA templates (Fig. 5b, upper panel Lane 4). The uridylylation activity of the 3D polymerase was inhibited in the presence of 0.1 M NaCl (Fig. 5b, Lane 10) when it was used as a control. BPR-3P0128 inhibited the VPg-pU(pU) synthesis in a dose-dependent manner at concentrations of 100–500 lM. However, the DTrip-22 was not inhibited by uridylylating VPg (Fig. 5b, lower panel) (Chen et al., 2009). These results indicated

that BPR-3P0128 can inhibit the EV71 3D polymerase activity associated with chain elongation and VPg uridylylation. 3.6. BPR-3P0128-resistant viruses The cells were infected with EV71 Tainan/4643/98/MP4 at an MOI of 0.2 PFU/cell. After virus absorption for 1 h, the cells were washed twice and incubated for 3 d in 3 mL of DMEM with 2% FBS containing 0.01 lM BPR-3P0128. The supernatants were pooled and the clear supernatant was denoted as Passage 1. The Passage 1 virus was used to infect a new cell monolayer, which was further incubated in the presence of a compound. Passages 2–5, 6–8, and 9– 11 were incubated with 0.01, 0.02, and 0.03 lM BPR-3P0128. We performed plaque assay for the virus from Passages 6 to 11. The control (untreated) passages demonstrated fully detectable and stable viruses. This observation implied that no escape mutant viruses were isolated and the virus was driven to undetectable levels in Passages 8–11.

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3.7. Antiviral activity of BPR-3P0128 against other RNA viruses We examined the antiviral activity of BPR-3P0128 against other RNA viruses by performing a Cytopathic effect inhibition assay. BPR-3P0128 exhibited antiviral activity against Coxsackieviruses A6, A10, A16 (EC50, 0.60, 0.73, and 0.48 lM), and B3 (EC50, 0.062 lM). This compound exhibited antiviral activity against human rhinovirus 2, and influenza types A and B. However, BPR3P018 did not inhibit the DNA of HSV2 or the adenovirus (EC50, >20 lM) (Hsu et al., 2011). BPR-3P0128 exhibits broad spectrum antiviral activity against RNA viruses.

4. Discussion BPR-3P0128 was initially designed and developed to inhibit the influenza type A virus. BPR-3P0128 exhibits antiviral activity against RNA viruses EV71, Coxsackieviruses A6, A10, A16, and B3, influenza types A and B, and HRV2 (Hsu et al., 2011). The results of this study indicated that BPR-3P0128 is a potent inhibitor against EV71. Our biochemical and genetic approaches indicated that BPR-3P0128 inhibited EV71 RNA-dependent RNA polymerase 3D proteins and VPg uridylylation, and reduces viral replication. The plaque inhibition and antiviral assay results indicated that BPR-3P0128 reduced EV71 (Tainan/4643/98/MP4) plaque numbers in a dose-dependent manner (Fig. 2a), and inhibited low and high MOI titers of the virus (Fig. 2b). These results confirmed that BPR3P0128 is a potent inhibitor of EV71. The time-of-addition assay results indicated that BPR-3P0128 inhibition began following viral absorption by the target cells, and that inhibition occurred during the early stages of viral replication (Fig. 3). BPR-3P0128 efficiently reduced the amount of EV71 RNA accumulation (Fig. 4a), as well as the plus and minus RNA strands (Fig. 4b) in the cell-based assay during viral replication. BPR-3P0128 effectively inhibited EV71 RdRp (3Dpol) activity when a chain elongation assay was performed (Fig. 5a). The amount of BPR-3P0128 required inhibiting pure EV71 3D polymerase was 100-times more than that used in the in vivo study against infected RD cells. This result correlates with the report of BPR3P0128 inhibited influenza A virus polymerase activity (Hsu et al., 2011). This report showed that Hsu et al. used a higher concentration (EC50 = 0.125 lM), which inhibited polymerase activity; this activity was 50 times higher than that observed in virus propagation in cell culture (68.4 nM). As a pervious reports of other antiviral compound expressed, that DTrip-22 and aurintricarboxylic acid are EV71 3D polymerase inhibitors (Chen et al., 2009; Hung et al., 2010) and T-705RTP is an influenza A virus RdRp inhibitor ( Furuta et al., 2013). All these results indicate that a large amount of compound is required to inhibit RdRp activity for in vitro assays. In addition, another report indicated that 5D9 inhibited FMDV (foot-and-mouth disease virus) 3D polymerase at a 100 lM concentration. However, the concentration was 3 times higher than that used in the plaque reduction assay (25 lM) (Rai et al., 2013). These results indicated that the polymerase activity inhibitory effect of antiviral compound required a higher concentration in the polymerase activity assay than in the cytopathic inhibition assay. Previous studies have reported that the 3D polymerase of EV71 exhibits a VPg uridylylation function in virus-infected cells. The purified EV71 RNA polymerase catalyzed the uridylylation of VPg on a poly (A) template to direct the transfer of 1–2 uridylate residues to VPg, thereby forming VPgpU and VPgpUpU. The 3D polymerase can catalyze the inter molecular and intra molecular uridylyation of 3D polymerase molecules (Paul et al., 2003; Richards et al., 2006).

The VPg uridylylation assay employed poly (A) as the template and Mn2+ as a cofactor, and the EV71 3D polymerase activity was enhanced relative to the level of Mg2+ in the reaction mixture (Paul et al., 2003). However, an Mn2+ condition allows the 3D polymerase to catalyze the VPg uridylylation. BPR-3P0128 inhibited the VPg-pU(pU) synthesis in a dose-dependent manner (Fig. 5b). Either the in vitro assay VPg failed to bind with the active sites of the 3D polymerase, or the 3D polymerase cannot catalyze the formation of a phosphodiester linkage between a uridylated residue and the hydroxyl group on Tyr3 of VPg uridylylation synthesis. The in vitro RdRp elongation assay results further confirmed that BPR-3P0128 inhibited 3D polymerase. Another possibility is that BPR-3P0128 may interfere with and inhibit other 3D polymerase activities involving associated proteins (3CD) during viral replication in infected cells. The results of this study indicated that BPR3P0128 exhibits excellent properties for inhibiting VPg uridylylation. We have suggested that VPg is a potent molecular target for drug development against EV71. We did not find a resistant EV71 virus during more than 10 passages of viral propagation with BPR-3P0128. We also examined whether DTrip-22 resistant EV71 (3D R163K) indentified before (Chen et al., 2009) can be resistant to BPR-3P0128. However; the 3D R163K mutant virus is not resistant to BPR-3P0128 treatment (EC50 value = 0.45 lM). This result indicated that 3D R163 K mutant is not the relevant target site for BPR-3P0128. However, BPR-3P0128 is able inhibited both 3D polymerases activity and VPg uridylylation (Fig. 5a and b), but DTrip-22 inhibited only the polymerase activity. This observation suggested BPR-3P0128 inhibited EV71 by multiple target sites and that’s reason why we failed to get a resistant virus.

5. Conclusion BPR-3P0128 inhibits multiple molecular targeting sites in EV71, such as RdRp 3D protein and VPg uridylylation synthesis and inhibits viral RNA replication. Our results indicated that BPR3P0128 exhibited broad-spectrum antiviral activity against Enterovirus 71, and that it is potential candidate for developing potent multi target antienteroviral compounds.

Funding This study was supported by grants from National Science Council Taiwan (102-2325-B-182-015) and from Chang Gung Memorial Hospital, Taiwan (CMRPD1A0672).

Transparency declarations None to declare. References AbuBakar, S., Chan, Y.F., Lam, S.K., 2000. Outbreaks of enterovirus 71 infection. N. Engl. J. Med. 342, 355–356. Arita, M., Takebe, Y., Wakita, T., Shimizu, H., 2010. A bifunctional anti-enterovirus compound that 1 inhibits replication and early stage 2 of enterovirus 71 infection. J. Gen. Virol. 91, 2734–2744. Bible, J.M., Pantelidis, P., Chan, P.K., Tong, C.Y., 2007. Genetic evolution of enterovirus 71 epidemiological and pathological implications. Rev. Med. Virol. 17, 371–379. Bible, J.M., Iturriza-Gomara, M., Megson, B., Brown, D., Pantelidis, P., Earl, P., Bendig, J., Tong, C.Y., 2008. Molecular epidemiology of human enterovirus 71 in the United Kingdom from 1998 to 2006. J. Clin. Microbiol. 46, 3192–3200. Chang, L.Y., Lin, T.Y., Hsu, K.H., Huang, Y.C., Lin, K.L., Hsueh, C., Shih, S.R., Ning, H.C., Hwang, M.S., Wang, H.S., Lee, C.Y., 1999. Clinical features and risk factors of pulmonary oedema after enterovirus-71-related hand, foot, and mouth disease. Lancet 354, 1682–1686.

A.B. Velu et al. / Antiviral Research 112 (2014) 18–25 Chen, T.C., Weng, K.F., Chang, S.C., Lin, J.Y., Huang, P.N., Shih, S.R., 2008. Development of antiviral agents for enteroviruses. J. Antimicrob. Chemother. 62 (6), 1169–1173. Chen, T.C., Chang, H.Y., Lin, P.F., Chern, J.H., Hsu, J.T., Chang, C.Y., Shih, S.R., 2009. Novel antiviral agent DTriP-22 targets RNA-dependent RNA polymerase of enterovirus 71. Antimicrob. Agents Chemother., 2740–2747. Deszcz, L., Seipelt, J., Vassilieva, E., Roetzer, A., Kuechler, E., 2004. Antiviral activity of caspase inhibitors effect on picornaviral 2A proteinase. FEBS Lett. 560, 51–55. Rai, Devendra K., Schafer, Elizabeth A., Singh, Kamalendra, McIntosh, Mark A., Sarafianos, Stefan G., Rieder, Elizabeth, 2013. Repeated exposure to 5D9, an inhibitor of 3D polymerase, effectively limits the replication of foot-and-mouth disease virus in host cells. Antiviral Res. 98, 380–385. Dragovich, P.S., Webber, S.E., Babine, R.E., Fuhrman, S.A., Patick, A.K., Matthews, D.A., Lee, C.A., Reich, S.H., Prins, T.J., Marakovits, J.T., Littlefield, E.S., Zhou, R., Tikhe, J., Ford, C.E., Wallace, M.B., Meador 3rd, J.W., Ferre, R.A., Brown, E.L., Binford, S.L., Harr, J.E., DeLisle, D.M., Worland, S.T., 1998. Structure-based design, synthesis and biological evaluation of irreversible human rhinovirus 3C protease inhibitors. 1. Michael acceptor structure–activity studies. J. Med. Chem. 41, 2806–2818. Gohara, D.W., Ha, C.S., Kumar, S., Ghosh, B., Arnold, J.J., Wisniewski, T.J., Cameron, C.E., 1999. Production of ‘‘authentic’’ poliovirus RNA-dependent RNA polymerase (3D(pol)) by ubiquitin-protease-mediated cleavage in Escherichia coli. Protein Expr. Purif. 17, 128–138. Gamarnik, A.V., Andino, R., 1998. Switch from translation to RNA replication in a positive-stranded RNA virus. Genes Dev. 12, 2293–2304. Ho, M., Chen, E.R., Hsu, K.H., Twu, S.J., Chen, K.T., Tsai, S.F., Wang, J.R., Shih, S.R., 1999. An epidemic of enterovirus 71 infection in Taiwan. N. Engl. J. Med. 341, 929–935. Hsu, J.T., Yeh, J.Y., Lin, T.J., Li, M.L., Wu, M.S., Hsieh, C.F., Chou, Y.C., Tang, W.F., Lau, K.S., Hung, H.C., Fang, M.Y., Ko, S., Hsieh, H.P., Horng, J.T., 2011. Identification of BPR3P0128 as an inhibitor of cap-snatching activities of influenza virus. Antimicrob. Agents Chemother., 647–657. Hung, H.C., Chen, T.C., Fang, M.Y., Yen, K.J., Shih, S.R., Hsu, J.T., Tseng, C.P., 2010. Inhibition of enterovirus 71 replication and the viral 3D polymerase by aurintricarboxylic acid. J. Antimicrob. Chemother. 65, 676–683. Konig, H., Rosenwirth, B., 1988. Purification and partial characterization of poliovirus protease 2A by means of a functional assay. J. Virol. 62, 1243–1250. Lin, T.Y., Twu, S.J., Ho, M.S., Chang, L.Y., Lee, C.Y., 2003. Enterovirus 71 outbreaks, Taiwan occurrence and recognition. Emerg. Infect. Dis. 9 (3), 291–293. Li, C., Wang, H., Shih, S.R., Chen, T.C., Li, M.L., 2007. The efficacy of viral capsid inhibitors in human enterovirus infection and associated diseases. Curr. Med. Chem. 14 (8), 847–856. Lee, J.C., Shih, S.R., Chang, T.Y., Tseng, H.Y., Shih, Y.F., Yen, K.J., Chen, W.C., Shie, J.J., Fang, J.M., Liang, P.H., Chao, Y.S., Hsu, J.T., 2008. A mammalian cell-based reverse two-hybrid system for functional analysis of 3C viral protease of human enterovirus 71. Anal. Biochem. 375, 115–123. Paul, A.V., Peters, J., Mugavero, J., Yin, J., van Boom, J.H., Wimmer, E., 2003. Biochemical and genetic studies of the VPg uridylylation reaction catalyzed by the RNA polymerase of poliovirus. J. Virol. 7, 891–904.

25

Paul, A.V., Rieder, E., Kim, D.W., van Boom, J.H., Wimmer, E., 2000. Identification of an RNA hairpin in poliovirus RNA that serves as the primary template in the in vitro uridylylation of VPg. J. Virol. 74, 10359–10370. Paul, A.V., 2002. In: Semler, B.L., Wimmer, E. (Eds.), Molecular Biology of Picornaviruses, first ed. American Society for Microbiology Press, Washington, DC, pp. 227–246. Richards, O.C., Spagnolo, J.F., Lyle, J.M., Vleck, S.E., Kuchta, R.D., Kirkegaard, K., 2006. Intramolecular and intermolecular uridylylation by poliovirus RNA-dependent rna polymerase. J. Virol., 7405–7415. Rodriguez, P.L., Carrasco, L., 1993. Poliovirus protein 2C has ATPase and GTPase activities. J. Biol. Chem. 268, 8105–8110. Sarma, N., Sarkar, A., Mukherjee, A., Ghosh, A., Dhar, S., Malakar, R., 2009. Epidemic of hand, foot and mouth disease in West Bengal, India in August 2007: a multicentric study. Indian J. Dermatol. 54 (1), 26–30. Shia, K.S., Li, W.T., Chang, C.M., Hsu, M.C., Chern, J.H., Leong, M.K., Tseng, S.N., Lee, C.C., Lee, Y.C., Chen, S.J., Peng, K.C., Tseng, H.Y., Chang, Y.L., Tai, C.L., Shih, S.R., 2002. Design, synthesis, and structureactivity relationship of pyridyl imidazolidinones: a novel class of potent and selective human enterovirus 71 inhibitors. J. Med. Chem. 45, 1644–1655. Shih, S.R., Tsai, M.C., Tseng, S.N., Won, K.F., Shia, K.S., Li, W.T., Chern, J.H., Chen, G.W., Lee, C.C., Lee, Y.C., Peng, K.C., Chao, Y.S., 2004. Mutation in enterovirus 71 capsid proteinVP1 confers resistance to the inhibitory effects of pyridyl imidazolidinone. Antimicrob. Agents Chemother. 48, 3523–3529. Stone, J.K., Rijnbrand, R., Stein, D.A., Ma, Y., Yang, Y., Iversen, P.L., Andino, R., 2008. A morpholino oligomer targeting highly conserved internal ribosome entry site sequence is able to inhibit multiple species of picornavirus. Antimicrob. Agents Chemother. 52, 1970–1981. Thompson, A.A., Peersen, O.B., 2004. Structural basis for proteolysis-dependent activation of the poliovirus RNA-dependent RNA polymerase. EMBO J. 23, 3462– 3471. Takegami, T., Kuhn, R.J., Anderson, C.W., Wimmer, E., 1993. Membrane-dependent uridylylation of the genome-linked protein VPg of poliovirus. Proc. Natl. Acad. Sci. U.S.A. 80, 7447–7451. Xu, J., Qian, Y., Wang, S., Serrano, J.M., Li, W., Huang, Z., Lu, S., 2010. EV71: an emerging infectious disease vaccine target in the far East? Vaccine 28, 3516– 3521. Yang, F., Ren, L., Xiong, Z., Li, J., Xiao, Y., Zhao, R., He, Y., Bu, G., Zhou, S., Wang, J., Qi, J., 2009. Enterovirus 71 outbreak in the People’s Republic of China in 2008. J. Clin. Microbiol. 47, 2351–2352. Furuta, Yousuke, Gowen, Brian B., Takahashi, Kazumi, Shiraki, Kimiyasu, Smee, Donald F., Barnard, Dale L., 2013. Favipiravir (T-705), a novel viral RNA polymerase inhibitor. Antiviral Res. 100, 446–454. Zhao, H., Zhang, Y., Zhang, Y., 2008. Etiology and clinical Characteristics of the 2007 outbreak of hand-foot-mouth disease in children in Beijing. J. Clin. Pediatr. 26 (6), 467–469. Zeng, M., E. Khatib, NF, Tu, S., Xu, S., Zhu, Q., Mo, X., Pu, D., Wang, X., Altmeyer, R., 2012. Seroepidemiology of enterovirus 71 infection prior to the 2011 season in children in Shanghai. J. Clin. Virol. 53, 285–289.

BPR-3P0128 inhibits RNA-dependent RNA polymerase elongation and VPg uridylylation activities of Enterovirus 71.

Enterovirus 71 (EV71) infections can cause hand, foot, and mouth disease with severe neurological complications. Because no clinical drug is available...
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