Novel Antiviral Agent DTriP-22 Targets RNA-Dependent RNAPolymerase of Enterovirus 71�
Tzu-Chun Chen,1,2,5 Hwan-You Chang,5 Pei-Fen Lin,2 Jyh-Haur Chern,4 John Tsu-An Hsu,4Chu-Yi Chang,6 and Shin-Ru Shih1,2,3,4*
Research Center for Emerging Viral Infections, Chang Gung University, Taoyuan, Taiwan1; Department of Medical Biotechnology andLaboratory Science, Chang Gung University, Taoyuan, Taiwan2; Clinical Virology Laboratory, Chang Gung Memorial Hospital,Taoyuan, Taiwan3; Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Chunan,
Taiwan4; Institute of Molecular Medicine, National Tsing Hua University, Hsinchu, Taiwan5; and Department ofComputer Science and Information Engineering, Chang Gung University, Taoyuan, Taiwan6
Received 23 January 2009/Returned for modification 17 March 2009/Accepted 28 April 2009
Enterovirus 71 (EV71) has emerged as an important virulent neurotropic enterovirus in young children.DTriP-22 (4{4-[(2-bromo-phenyl)-(3-methyl-thiophen-2-yl)-methyl]-piperazin-1-yl}-1-pheny-1H-pyrazolo[3,4-d]pyrimidine) was found to be a novel and potent inhibitor of EV71. The molecular target of this compoundwas identified by analyzing DTriP-22-resistant viruses. A substitution of lysine for Arg163 in EV71 3Dpolymerase rendered the virus drug resistant. DTriP-22 exhibited the ability to inhibit viral replication byreducing viral RNA accumulation. The compound suppressed the accumulated levels of both positive- andnegative-stranded viral RNA during virus infection. An in vitro polymerase assay indicated that DTriP-22inhibited the poly(U) elongation activity, but not the VPg uridylylation activity, of EV71 polymerase. Thesefindings demonstrate that the nonnucleoside analogue DTriP-22 acts as a novel inhibitor of EV71 polymerase.DTriP-22 also exhibited a broad spectrum of antiviral activity against other picornaviruses, which highlightsits potential in the development of antiviral agents.
Enterovirus 71 (EV71), a positive-stranded RNA virus, be-longs to the genus Enterovirus in the family Picornaviridae.EV71 infection typically causes hand, foot, and mouth diseaseor herpangina, followed by severe central nervous system com-plications, including aseptic meningitis, encephalitis, poliomy-elitis-like paralysis, neurogenic cardiopulmonary failure, andeven death in some young children. Infants, following infectionby EV71 with central nervous system complications, reportedlysuffer from neurologic sequelae and delayed neurodevelop-ment (6). In 1998, a large outbreak of EV71 infection occurredin Taiwan, resulting in almost 80 fatalities and 405 severe cases(7, 21). Subsequently, many outbreaks of EV71 infection inTaiwan have been reported, with a total of 51 verified fatalcases in 2000 and 2001 (29). Severe EV71 infections continuedto occur for several years thereafter. EV71 infection also hasbeen reported to occur in many countries, such as Malaysia,Singapore, Australia, Japan, the United States, Germany, andmainland China (1–3, 5, 13, 22, 33, 34).
The development of anti-EV71 agents is important becauseEV71 is regarded as the most important neurotropic entero-virus, after poliovirus control (34). A novel series of pyridylimidazolidinones targeting VP1 protein, based on the skele-tons of WIN compounds, has been developed using computer-assisted drug design (37). The new EV71 3C protease inhibi-tors, based on rhinovirus 3C protease inhibitor AG7088,exhibit inhibitory activities in both enzymatic and cell-based
assays (25). A pharmacologically active drug library has beenemployed to identify anti-EV71 compounds (4). Ribavirin usedin combination with interferon to treat patients with hepatitisC virus infection reduces the mortality rate of EV71-infectedmice by reducing viral replication (26).
We previously discovered piperazine-containing pyrazolo[3,4-d]pyrimidine derivatives as a novel class of anti-EV71 compounds(9). DTriP-22 (4{4-[(2-bromo-phenyl)-(3-methyl-thiophen-2-yl)-methyl]-piperazin-1-yl}-1-pheny-1H-pyr-azolo[3,4-d]pyrimidine)is one such compound, containing a diacrylmethyl group at thepiperazine and a phenyl group at the pyrazolo[3,4-d]pyrimidine(9). Although DTriP-22 has a pyrazolo[3,4-d]pyrimidine struc-ture and is thus similar to pyrazolo[3,4-d]pyrimidine nucleosideanalogues, DTriP-22 differs from these analogues in that it lacksan appropriate carbocyclic ring, such as a ribose, for incorpora-tion into the growing viral RNA chains (39). Moreover, the size ofDTriP-22 differs markedly from those of the nucleoside analogues(39). In this study, we identified DTriP-22, a nonnucleoside ana-logue which targets EV71 3D polymerase, by analysis of DTriP-22-resistant viruses. Inhibition of 3D polymerase activity in vitroby DTriP-22 was also investigated.
MATERIALS AND METHODS
Cells, viruses, and chemicals. RD (rhabdomyosarcoma) cells (American TypeCulture Collection [ATCC] accession no. CCL-136) and MDCK (Madin-Darbycanine kidney) cells (ATCC accession no. CCL-34) were cultured in Dulbecco’smodified Eagle’s medium (DMEM; Gibco) supplemented with 10% fetal bovineserum (FBS). Vero (African green monkey kidney) cells (ATCC accession no.CCL-81) and HeLa cells (ATCC accession no. CCL-2) were maintained inmodified Eagle’s medium (Gibco) supplemented with 10% FBS. EV71 TW/1743/98 and TW/2231/98; coxsackieviruses A9, A10, A16, A24, B1, B2, B3, B4,B5, and B6; echovirus 9; human rhinovirus 2 (HRV-2); herpes simplex virus type1 (HSV-1); and HSV-2 were isolated from clinical specimens in the ClinicalVirology Laboratory of Chang Gung Memorial Hospital (Linkou, Taiwan).
* Corresponding author. Mailing address: Department of Medi-cal Biotechnology and Laboratory Science, Chang Gung University,259 Wen-Hua 1st Rd., Kwei-Shan, Taoyuan 333, Taiwan. Phone:886-3-2118800, ext. 5497. Fax: 886-3-2118174. E-mail: [email protected].
EV71 Tainan/4643/98 was obtained from Jen-Ren Wang, National Cheng KungUniversity (Tainan, Taiwan). EV71 BrCr, the prototype of EV71 (ATCC acces-sion no. VR 784), was obtained from the ATCC. EV71; coxsackieviruses A9,A10, A16, A24, B1, B2, B3, B4, B5, and B6; and echovirus 9 were propagated inRD cells. HRV-2 was propagated in HeLa cells. Influenza A virus (A/WSN/33)and influenza B virus (B/HK/72) were propagated in MDCK cells. HSV-1 andHSV-2 were propagated in Vero cells. DTriP-22 was synthesized at the NationalHealth Research Institutes (Taiwan) and dissolved in dimethyl sulfoxide(DMSO).
Antiviral assay of DTriP-22 activity against EV71. RD cells (6 � 105 cells/well)were seeded in a six-well plates and incubated for 24 h. Cells were washed andthen infected with EV71 TW/2231/98 at a multiplicity of infection (MOI) of0.001, 0.1, or 1 PFU/cell. After 1 h of absorption at room temperature, theinfected cells were washed twice, covered with medium containing 2% FBS andvarious concentrations of DTriP-22 (0, 0.005, 0.1, 0.2, 1, 2, and 2.5 �M), andfurther incubated at 35°C. At 16 h postinfection (p.i.), the supernatant and debriswere collected together and the total virus yield was quantified by a plaque assay.
Plaque assay. RD cells (6 � 105 cells/well) were seeded in a six-well plate andincubated for 24 h. Cells were washed and infected with or without virus with a10-fold series dilution. After absorption at room temperature for 1 h, the in-fected cells were washed twice and covered with medium containing 2% FBS and0.3% agarose gel. The infected cells were further incubated at 35°C for 4 days.The plates were fixed with 0.5% formaldehyde and then stained with 0.1% crystalviolet. The plaques were then counted, and the viral titers were presented asnumbers of PFU/milliliter.
Neutralization test. The neutralization assay measured the ability of a testcompound to inhibit the cytopathic effects induced by viruses, as describedpreviously (37). Briefly, 96-well tissue culture plates were seeded with 3 � 104
cells/well in DMEM with 10% FBS. After 24 h of incubation at 37°C, RD cellswere infected with the virus at an MOI of 0.005 PFU/cell. After adsorption, theinfected cells were covered with medium containing 2% FBS and 0.5% DMSOor DTriP-22 at various concentrations (twofold dilutions from 25 �M). Theinfected cells were further incubated at 35°C for 64 h. The plates were fixed with0.5% formaldehyde and then stained with 0.1% crystal violet. The density of thewell at 570 nm was measured. Each experiment was performed in triplicate andrepeated at least two times. The 50% effective concentrations (EC50s) werecalculated according to the formula EC50 � [(Y � B)/(A � B)] � (H � L) � L],where Y represents half of the mean optical density at 570 nm (OD570) of the cellcontrol without the compound, B represents the mean OD570 of wells with thecompound dilution nearest to and below Y, A represents the mean OD570 ofwells with the compound dilution nearest to and above Y, and L and H are thecompound concentrations at B and A, respectively.
Cytotoxicity assay. Cell viability was evaluated using the MTS {tetrazoliumcompound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium, inner salt]}/PMS (phenazine methosulfate) method(Cell Titer96 AQueous cell proliferation kit; Promega) in accordance with themanufacturer’s instructions. RD cells (6.5 � 103 cells/well) were seeded into eachwell of a 96-well microtiter plate. Following incubation for 24 h, the medium wasreplaced with fresh medium with 2% FBS and a twofold serial dilution of the testcompound (each dilution in triplicate). The cells grew at 35°C for 64 h andreached 90% confluence. The culture medium was then replaced with 100 �lphenol red-free medium containing MTS (Promega) and PMS (Sigma). Theplate was incubated at 35°C for 1 to 4 h, and the absorbance at 490 nm was thenrecorded using a microplate reader. The survival rate of the cells that had beentreated with the compound was determined using the following formula: percentcell viability � (OD490 of treated cells � OD490 of blank)/(OD490 of controlcells � OD490 of blank). The 50% cytotoxic concentration (CC50) was deter-mined as the concentration of the compound at which cell viability was reducedto 50%. Data were analyzed using GraphPad Prism 4.0 software.
Generation of DTriP-22-resistant viruses. RD cells (6 � 105 cells/well) wereseeded in a six-well plate and incubated for 24 h. The cells were infected withEV71 TW/2231/98 at an MOI of 0.001 PFU/cell. After virus absorption for 1 h,the cells were washed twice and incubated for 3 days in 3 ml of DMEM-2% FBScontaining 0.2 �M DTriP-22. The clear supernatant was collected and termedpassage 1. The passage 1 virus was used to infect a new cell monolayer, which wasfurther incubated in the presence of a compound. The procedure was repeatedfor 14 passages. The susceptibility to DTriP-22 of passage 14-resistant variantswas confirmed using a neutralization test. Ten isolates of DTriP-22-resistantviruses from passage 14 virus were plaque purified. The drug susceptibilities ofthese 10 isolates were further verified using a neutralization test.
Generation of EV71 mutants. A pEV71 plasmid containing the full-lengthgenome of EV71 TW/2231/98 was used (37). The mutations in pEV71 weregenerated using a QuikChange site-directed mutagenesis kit (Stratagene). The
T256A mutation in the 2C protein and the R163K and S264L (and R163K-S264L) mutations in the 3D protein were introduced using mutagenic primerpairs comprising the sequences 5�-GATTCCTATAAGGCAGAGCTGGGCAG-3� and 5�-CTGCCCAGCTCTGCCTTATAGGAATC-3�, 5�-GTTAAAGATGAACTTAAAGCCATCGACAAGATC-3� and 5�-GATCTTGTCGATGGCTTTAAGTTCATCTTTAAC-3�, and 5�-CCGAAGACGCAGTGTTACTCATAGAAGGGATC-3� and 5�-GATCCCTTCTATGAGTAACACTGCGTCTTCGG-3�, respectively (mutated nucleotides are underlined). The mutant clones wereverified by sequencing and further used to generate virus, as described below.The plasmid was linearized with EcoRI and MluI and then transcribed using aMEGAscript T7 kit (Ambion) in accordance with the manufacturer’s instruc-tions. Vero cells in six-well plates were transfected with the transcribed RNA byusing Lipofectamine 2000 reagent (Invitrogen) in accordance with the manufac-turer’s instructions and then incubated at 35°C for 72 h. The mutant viruses fromthe culture supernatant were further plaque purified and confirmed by se-quencing.
Analysis of viral RNA accumulation. RD cells (6 � 105 cells/well in a six-wellplate) were infected with EV71 at an MOI of 1 PFU/cell. After 1 h of absorptionat room temperature, the cells were washed twice and supplemented with me-dium containing 2 �M DTriP-22. The cells were further incubated at 35°C for theindicated periods. The intracellular RNA was then extracted using an RNeasy kit(Qiagen). The viral RNA was further detected using quantitative real-time re-verse transcriptase PCR (RT-PCR) and slot blot analysis.
Quantitative RT-PCR was performed with a TaqMan RT-PCR kit (AppliedBiosystems), using an ABI Prism 7000 apparatus. The oligonucleotide primersand the TaqMan probe for detecting EV71 RNA, designed by W. A. Verstrepen,were as follows: sense, 5�-CCCTGAATGCGGCTAATC-3�; antisense, 5�-ATTGTCACCATAAGCAGCCA-3�; and probe, FAM (6-carboxyfluorescein)-AACCGACTACTTTGGGTGTCCGTGTTTC-TAMRA (6-carboxytetramethylrho-damine) (46). 18S rRNA probe and primers obtained from TaqMan (AppliedBiosystems) were used as internal controls. Each sample was assayed in tripli-cate, and the experiment was performed three times independently. The ob-tained data were analyzed using ABI Prism 7000 sequence detection systemsoftware. The yield of EV71 RNA was normalized to that of 18S rRNA.
Slot blot analysis for detecting viral RNA was performed as described previ-ously (27). Briefly, denaturing RNA was loaded onto a nylon membrane in theslot blot manifold. The membrane was then cross-linked. Digoxigenin-labeledRNA probes, specific for the genome or antigenome of EV71, were producedusing a DIG Northern starter kit (Roche). The hybridization and detectingprocedures were performed according to the manufacturer’s instructions.
Dicistronic expression assay. pRHF-EV71-5� UTR, containing the EV71 5�untranslated region (UTR) between the Renilla and firefly luciferase genes, wasused to evaluate the internal ribosome entry site (IRES)-dependent translationof EV71, as described elsewhere (28). Briefly, RD cells (2.5 � 105 cells/well in a12-well plate) were grown to 90% confluence and transfected with the plasmid inthe presence of DTriP-22. After 2 days, cell extracts were assayed for Renilla andfirefly luciferase activity with a Lumat LB9507 bioluminometer, using a dual-luciferase reporter assay kit (Promega) in accordance with the manufacturer’sinstructions.
Expression and purification of EV71 3D polymerase. To construct pET26b-Ub-EV71-3D, the EV71 3D region of pEV71 was subcloned into pET26b-Ub-3D-GSSG-6H and used to replace the poliovirus 3D region of pET26b-Ub-3D-GSSG-6H, which encodes ubiquitin (Ub)-poliovirus 3D (a gift from C. E.Cameron) (16). pCG1 (from C. E. Cameron) encodes an Ub-specific carboxy-terminal protease (Ubp1). Expression of Ub-3D fusion protein in the presence ofUbp1 has glycine at the amino terminus of polymerase, not methionine. PlasmidspET26b-Ub-EV71-3D and pCG1 were cotransformed into BL21(DE3). Expres-sion of EV71 3D was induced by adding 50 �M isopropyl-�-D-thiogalactopyr-anoside at 25°C for 4 h. The protein expressed from lysed cells was suspended inbuffer A (50 mM Tris, pH 8.0, 20% glycerol, 1 mM dithiothreitol, 0.1% NP-40,and 60 �M ZnCl2) and loaded onto a HisTrap column (GE Healthcare Bio-sciences), which was then washed with buffer A containing 30, 50, 70, or 90 mMimidazole; the protein was then eluted with buffer A containing 500 mM imida-zole. The eluted product was dialyzed against buffer B (50 mM HEPES, pH 7.5,500 mM NaCl, 20% glycerol, 1 mM dithiothreitol, 0.1% NP-40, and 60 �MZnCl2) and stored at �70°C.
Polymerase elongation assay. The elongation assay was performed with 1 �Mpolymerase in 50 mM HEPES, pH 7.5, 10 mM 2-mercaptoethanol, 5 mM MgCl2,60 �M ZnCl2, 5 �M UTP, 0.4 �Ci/�l [�-32P]UTP, 1.8 �M dT15-0.15 �Mpoly(rA)300 (primer/template), and DTriP-22. The reaction mixtures were incu-bated for 10 min at 30°C and the reactions terminated by adding EDTA to givea concentration of 83 mM. The quenched reactants were spotted onto DE81filter paper discs (Whatman) and air dried. The discs were washed with 5%
dibasic sodium phosphate and rinsed in absolute ethanol. The radioactivity of thesample was quantified using scintillation fluid.
In vitro uridylylation assay. VPg uridylylation was assayed with a reactionmixture containing 50 mM HEPES, pH 7.5, 2.5 mM manganese(II) acetate, 8%glycerol, 0.5 �g poly(rA)300 RNA, 2 �g EV71-synthesized VPg peptide, 1 �Mpolymerase, 0.04 �M [�-32P]UTP, 10 �M unlabeled UTP, and DTriP-22. Thereaction mixture was incubated at 33°C for 60 min. The sample was analyzed byTricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis with 13.5%polyacrylamide. The radioactivity of the sample was exposed to X-ray film.
Data analysis. Data were plotted and statistical significance was determinedusing the GraphPad Prism 4 program. The curves in the figures were plottedusing nonlinear regression analysis. The related elongation activity of 3D poly-merase is shown in the bar graph. Significance of differences among groups wasassessed by one-way analysis of variance followed by Tukey’s post hoc multiple-comparison test.
RESULTS
Antiviral activity of DTriP-22 against EV71. The chemicalstructure of DTriP-22, shown in Fig. 1A, contains pyrazolo[3,4-d]pyrimidine. According to a previous investigation of struc-ture-activity relationship, the phenyl group at the pyrazolo[3,4-d]pyrimidine and the hydrophobic diarylmethyl group at thepiperazine in a series of pyrazolo[3,4-d]pyrimidine-containingcompounds significantly influence antienterovirus activity (9).DTriP-22 exhibited better anti-EV71 activity than the otherderivatives (9). Therefore, it was used to select drug-resistantviruses. The anti-EV71 activity of DTriP-22 was evaluated withhigh and low viral titers. RD cells were infected with EV71 atan MOI of 0.001, 0.1, or 1 PFU/cell in the presence of DTriP-22. The total viral yields at 16 h p.i. were then detected using
a plaque assay. The result, shown in Fig. 1B, demonstrates thatDTriP-22 has the ability to inhibit EV71 at both high and lowMOI. DTriP-22 at 0.1 �M reduced the viral yield by 95% whenthe cells were infected with an MOI of 0.001 PFU/cell. TheEC50s were 0.023 and 0.16 �M for 0.001 MOI and 1 MOI(PFU/cell), respectively. The cytotoxicity of the compound wasevaluated using an MTS-based assay. The CC50 of the com-pound was larger than 100 �M. The selective index (ratio ofCC50 to EC50) of DTriP-22 for EV71 exceeds 625 for an MOIof 1 PFU/cell.
Identification of mutations that confer resistance to DTriP-22. To understand the antiviral mechanism of DTriP-22, themolecular target that can render the virus resistant to thiscompound was initially identified. EV71 TW/2231/98 at anMOI of 0.001 PFU/cell was cultivated to select resistant virusesat increasing concentrations (0.2 to 2 �M, representing anapproximately 8.7- to 87-fold increase in EC50) of DTriP-22.Virus pools in passages 5, 10, and 14 were tested to evaluatetheir drug susceptibilities by using a neutralization test. Virusesharvested from passages 10 and 14 were found to have EC50sof over 25 �M (Table 1), indicating that those viruses hadbecome resistant to the compound. Ten plaque-purified vi-ruses from passage 14 were at least 187 times more resistant toDTriP-22 than were the parental viruses (data not shown).Full-genome sequence analysis of the plaque-purified resistantviruses revealed consistent mutations at five amino acids inthese resistant viruses relative to the sequence for the parentalstrain. They are Y106C and P243S in the VP1 region, T256Ain the 2C region, and R163K and S264L in the 3D region. Toidentify the mutations that are involved in drug resistance, theamino acid mutations at these five positions in various passagesof viruses were monitored. The data revealed that the sensitiveviruses became resistant in passage 10; two mutations (R163Kand S264L) in the 3D region were observed in this passage(Table 1). However, one mutation in the 2C region (T256A)and two mutations in the VP1 region (Y106C and P243S) wereobserved in passage 5, and those mutations were also observedin passages 10 and 14. These results indicate that R163K andS264L mutations may render EV71 drug resistant but thatT256A, Y106C, and P243S mutations do not.
The above-mentioned mutations were introduced into theEV71 infectious clone to confirm that the resistance markersare located in the 3D region. The other mutation in the 2Cregion was also used, as a control. The drug susceptibilities ofthese recombinant viruses were tested by using a neutralization
TABLE 1. Drug susceptibilities and sequence changes observed forviruses harvested from different passages
Passageno. EC50 (�M)a
Amino acid(s) at indicated position
VP1 106 VP1 243 2C 256 3D 163 3D 264
0 0.74 0.03 Y P T R S5 0.25 0.02 C S A R S10 25 C S A K S/Lb
14 25 C S A K L
a Data are presented as means standard deviations of results from at leasttwo independent experiments.
b Two kinds of amino acid (serine and leucine) at position 264 in the 3D regionwere observed in passage 10 virus. A leucine residue at position 264 in the 3Dregion was predominant.
FIG. 1. (A) Structural formula of DTriP-22. (B) Anti-EV71 activityof DTriP-22. RD cells (6 � 105 cells/well) were seeded in a six-wellplate and incubated for 24 h. RD cells were infected with EV71 (MOIof 0.001, 0.1, or 1 PFU/cell, separately). After absorption, the infectedcells were treated with various concentrations of the compound (0,0.05, 0.1, 0.2, 1, 2, and 2.5 �M) for 16 h. The infected cells were lysedinto culture medium, and the total virus yield was quantified by aplaque assay. Data are presented as means standard errors of themeans (SEM) of results from three independent experiments. The50% inhibitory concentrations were calculated using nonlinear regres-sion analysis.
test (Table 2). An arginine-to-lysine amino acid substitution atresidue 163 (R163K) in the 3D polymerase reduced the drugsusceptibility of the mutant virus (EC50 25 �M). A change atposition 264 in the 3D region from serine to leucine (S264L)was a lethal mutation for EV71. This lethality can be rescuedby a compensatory mutation at the R163K residue in the 3Dregion rather than at the T256A residue in the 2C region. Boththe 3D R163K and the 3D R163K-S264L mutants had sensi-tivities to DTriP-22 at least 81 times lower than that observedfor the wild-type virus. However, the 2C T256V mutant and thewild-type virus had similar drug susceptibilities. The VP1V192M mutant virus, resistant to pyridyl imidazolidinones (anEV71 capsid binder), was also sensitive to DTriP-22 (37). Insummary, the change at residue 163 in the 3D polymerase iscrucial to drug resistance. However, DTriP-22-resistant virusesexhibited sensitivity to pyridyl imidazolidinone.
Time-of-addition experiments with EV71-infected cells. Theresults of the genetic approach indicated that 3D-involvingviral replication is likely to be the target of DTriP-22. Todetermine which stage(s) of the EV71 replication cycle is af-fected by DTriP-22, the time course of a single viral replicationcycle was first determined. The growth curve for the viralprogeny production initially rose exponentially, reaching a sta-tionary phase (approximately 8 � 107 PFU/ml) at 12 to 14 h p.i.(Fig. 2A). Therefore, DTriP-22 was added to the culture me-dium 2 h prior to the end of EV71 absorption, at the end ofEV71 absorption, or at 2-h intervals until 14 h p.i. At 16 h p.i.,the total virus yield was quantified by a plaque assay. DTriP-22inhibited progeny virus production by 79 to 97% when it wasadded before 2 h p.i. (Fig. 2B). A significant increase in virusyield (to 76%) was observed when the compound was added at4 h postadsorption. Almost no inhibition occurred when thecompound was added after 6 h postadsorption. This resultdiffers from that obtained by adding pyridyl imidazolidinone, acapsid inhibitor of EV71. Pyridyl imidazolidinone exhibited aloss of antiviral activity when it was added to EV71-infectedcells later than 1 h p.i. (37). These observations indicate thatthe molecular target of DTriP-22 differed from that of thecapsid binder. The results of the time-of-addition assay withEV71-infected cells supported the involvement of DTriP-22 inviral replication.
DTriP-22 decreased the level of accumulated EV71 RNA. Toverify that DTriP-22 inhibits viral replication, we first moni-tored viral RNA production in EV71-infected cells followingDTriP-22 treatment. Virus-infected cells (at an MOI of 1 PFU/cell) were treated with 2 �M DTriP-22 after virus absorption.Intracellular RNA was isolated at different intervals p.i. Theamounts of EV71 RNA were measured using both quantitativereal-time RT-PCR and slot blot analysis. The results of real-time RT-PCR showed that the presence of 2 �M DTriP-22reduced viral RNA production at 6 to 14 h p.i. by 63% to 87%from that obtained by DMSO treatment at each time point(Fig. 3A). The reduction in the amount of viral RNA due toDTriP-22 treatment was also observed with slot blot analysis(Fig. 3B). Treating infected cells with DTriP-22 reduced yieldsof both positive- and negative-stranded viral RNA. Also, wemonitored the effect of DTriP-22 on IRES-medicated transla-tion. EV71 IRES activity was detected when a dicistronic plas-mid with Renilla and firefly luciferase reporter genes was used.No significant decrease in IRES-driven translation was ob-served in the presence of DTriP-22 (Fig. 3C). These results
FIG. 2. (A) Single step growth curve of EV71. RD cells (6 � 105
cells/well) were seeded in a six-well plate and incubated for 24 h. RDcells were infected with EV71 at an MOI of 1 PFU/cell. After 1 h ofabsorption at room temperature, cells were covered with 2 ml mediumcontaining 2% FBS. At the indicated times, total virus yield was quan-tified by a plaque assay. (B) Time-of-addition assay. The condition ofvirus infection was the same as that described above. After 1 h ofabsorption at room temperature, the infected cells were treated with 2�M DTriP-22 at the indicated times (2 h before the end of viralabsorption and at 0, 2, 4, 6, 8, 10, 12, and 14 h postabsorption). At 16 hp.i., the culture supernatant and cell lysate were collected together.Total viral yield was measured by a plaque assay. Data are presentedas means SEM of results from three independent experiments.
TABLE 2. Plaque formation and drug susceptibilities forrecombinant EV71 viruses
a Data are presented as means standard deviations of results from at leasttwo independent experiments.
b NT, not tested.c The VP1 V192M mutant is a pyridyl imidazolinone-resistant virus.d Pyridyl imidazolinone is an EV71 inhibitor targeting the VP1 capsid protein.
demonstrated that DTriP-22 inhibits EV71 RNA accumulationduring virus infection but that it does not reduce IRES-driventranslation.
DTriP-22 inhibited the poly(U) polymerase activity of re-combinant EV71 3D protein. To investigate the effect ofDTriP-22 on in vitro EV71 3D polymerase activity, EV71 3Dpolymerase activity was evaluated by detecting the amount ofradiolabeled UMP incorporated into poly(U) RNA in thepresence of poly(A) templates and oligo(dT) primers. Therecombinant EV71 3D polymerase in the presence of 5%DMSO exhibited an activity of 110 pmol UMP incorporated/min/�g. A dose-dependent decrease in polymerase activity wasobserved in the presence of DTriP-22 (P � 0.001; one-wayanalysis of variance) (Fig. 4A). As a negative control, pyridylimidazolidinone, a capsid inhibitor of EV71, did not inhibit 3Dpolymerase activity. The experiments were repeated severaltimes, and the results consistently demonstrated that the EV713D polymerase activity was reduced in the presence of DTriP-22. The activity of the mutant EV71 3D polymerase with aresistance marker (R163K) treated with DTriP-22 was as-sessed. The mutant polymerase exhibited 28 to 29% moreactivity than wild-type polymerase in the presence of DTriP-22at 250 and 500 �M (P � 0.01 and P � 0.05, respectively;one-way analysis of variance followed by Tukey’s post hoc test)(Fig. 4A).
The 3D polymerase of enterovirus has been observed tohave another function, VPg uridylylation, in virus-infected cells(41). Therefore, an in vitro uridylylation assay was performedto detect the ability of EV71 polymerase to generate VPg-pU(pU) in the presence of DTriP-22. Recombinant 3D poly-merase uridylylated VPg in the presence of poly(A) RNAtemplates (Fig. 4B, lane 3). As a control, the uridylylationactivity of 3D polymerase was inhibited in the presence of 0.1M NaCl (Fig. 4B, lane 7). DTriP-22 could not inhibit theVPg-pU(pU) synthesis at the indicated concentrations (Fig.4B, upper panel, lanes 4 to 6). However, a capsid inhibitor ofEV71, pyridyl imidazolidinone, also did not inhibit the EV713D polymerase activity in uridylylating VPg. These results in-dicate that DTriP-22 may inhibit EV71 3D polymerase activityassociated with chain elongation but not that associated withVPg uridylylation.
DTriP-22 exhibited broad-spectrum activity against otherRNA viruses. Several viral polymerase inhibitors, such as riba-virin, exhibit broad-spectrum activity against various viruses(10, 23, 38). We next examined the activities of DTriP-22against other viruses by using a neutralization test. DTriP-22inhibited the cytopathic effects induced by all three genotypes
FIG. 3. Effect of DTriP-22 on level of accumulated viral RNA.(A) RD cells (6 � 105 cells/well) were seeded in a six-well plate andincubated for 24 h. RD cells were infected with EV71 at an MOI of 1PFU/cell. After absorption, the virus-infected cells were treated withor without 2 �M DTriP-22. The intracellular viral RNA was isolated at2, 4, 6, 8, 10, 12, and 14 h p.i. The amount of viral RNA was determinedby real-time RT-PCR. The amount of viral RNA at 12 h p.i. in theabsence of the compound was set to 100%. The relative amount ofviral RNA isolated at each time point is presented as a percentage onthe vertical axis. Data are presented as means SEM. (B) The con-dition of the treatment of the infected cells with DTriP-22 was asdescribed above. Cytoplasmic RNA was extracted at the indicated
times. The amounts of positive- and negative-strand viral RNA weredetected by slot blot analysis. For the loading control, the total RNAin each test was measured using a �-actin probe. The data are from onerepresentative experiment of three. (C) Effect of DTriP-22 on IRESactivity of EV71. RD cells were transfected with pRHF-EV71-5�UTRor pRHF-EV71-5�UTR-AS (antisense EV71 5� UTR) in the presenceof DTriP-22 or DMSO. After 48 h posttransfection, the Renilla lucif-erase (RLuc) and firefly luciferase (FLuc) activities were analyzed.The FLuc/RLuc activity of pRHF-EV71-5�UTR without DTriP-22-treatment is set to 100%, and the other relative FLuc/RLuc activitiesare presented. Data are presented as means SEM.
of EV71. These three genotypes were those associated withprototype BrCr (genotype A), TW/1743/98 (genotype B), andTainan/4643/98 (genotype C), with EC50s from 0.13 to 0.44 �M(Table 3). DTriP-22 also had strong antiviral activity againstother enteroviruses, including coxsackieviruses A and B andechovirus 9 (EC50s of 0.07 to 1.22 �M). Additionally, thiscompound exhibited antiviral activity against HRV-2 (EC50 �1.69 �M) and influenza viruses A and B, whereas the prototypecompound (compound 1) in this series of compounds did not(9). However, DTriP-22 failed to inhibit two DNA viruses,HSV-1 and HSV-2 (EC50s of 25 �M). The CC50 values ofDTriP-22 for Vero, MDCK, and HeLa cells were all greaterthan 100 �M, according to MTS assays. The results show thatDTriP-22 has a broader spectrum than the prototype pyr-azolo[3,4-d]pyrimidine-containing compound (compound 1).
DTriP-22, targeting the EV71 3D polymerase, exhibits a broaderspectrum of activity against RNA viruses, especially picornavi-ruses. However, the antiviral mechanisms of DTriP-22 activityagainst the aforementioned non-EV71 viruses, especially the en-veloped viruses, need to be further studied.
DISCUSSION
In this study, we demonstrated that R163K mutations inEV71 polymerase render the virus resistant to DTriP-22. In thepolymerase elongation assay, DTriP-22 affected the R163Kmutant polymerase activity significantly less than it affected thewild-type polymerase activity. The sequence alignment of theEV71 polymerase region with other enteroviruses shows thatthe Arg-163 residue is highly conserved within the Enterovirusgenus (present in 99.3% of 136 isolates). The Arg-163 residueis also found in HRV 3D polymerase, which may explain theactivity of DTriP-22 against HRV 2. The crystal structure ofthe poliovirus 3D polymerase revealed that the Arg-163 resi-due is located in the ring finger domain of the right-handstructure, which contains conserved basic residues (Arg-163,Lys-167, and Arg-174) that interact with the incoming nucleo-side triphosphate for chain elongation (43). Therefore,DTriP-22 may interfere with 3D activity by obstructing thenucleoside triphosphate entry cavity of 3D polymerase but notby incorporation into the growing RNA chains.
DTriP-22 efficiently reduced the amount of EV71 RNA ac-cumulation in the cell base system. However, DTriP-22 wasless effective in inhibiting the in vitro polymerase activity ofEV71 3D (Fig. 4A). The amounts of DTrip-22 required toinhibit purified EV71 3D polymerase in vitro were more than100 times larger than those required to inhibit virus replicationin RD cells. One possibility is that DTriP-22 may accumulate
FIG. 4. Effect of DTriP-22 on in vitro EV71 3D polymerase activ-ity. (A) Poly(U) polymerase activity was measured with 1 �M poly-merase in the reaction buffer as described in Materials and Methods.The percent activity on the vertical axis represents the activity of thetested polymerase (in the presence of the compound) divided by thatof the untreated polymerase. Black and white bars represent wild-type(wt) EV71 3D polymerase. Patterned bars represent the R163K mu-tant polymerase. Data are presented as means SEM of results fromthree independent experiments. The activities of wild-type and mutant3D polymerase observed in the presence of 250 and 500 �M DTriP-22,respectively, are compared. Difference among different groups wasassessed by one-way analysis of variance followed by Tukey’s post hocmultiple-comparison test (���, P � 0.001 versus the control group; a,P � 0.05; b, P � 0.01 versus the same treatment with wild-type poly-merase). (B) Effect of DTriP-22 on VPg uridylylation in vitro.Poly(A) RNA was used as a template in the presence of DTriP-22. Thereaction conditions for the upper and lower panels are the same,except for the treatment with different compounds in lanes 4 to 6.
TABLE 3. Antiviral activities of DTriP-22 against various viruses
to higher concentrations in cells than in the extracellular en-vironment. Another explanation is that DTriP-22 needs to bemetabolized in cells to exhibit full efficacy. A previous reportdescribed a similar scenario in which the anti-influenza com-pound T-705 needed to be modified to T-705RTP (T-705-4-ribofuranosyl-5-triphosphate) by cellular kinases, subsequentlyinhibiting influenza RNA polymerase activity (14). Anotherpossibility is that DTriP-22 may not only inhibit 3D polymeraseelongation activity but also interfere with other 3D polymer-ase-involving cellular functions during viral replication in in-fected cells.
DTriP-22 inhibited EV71 3D polymerase activity associatedwith chain elongation but not that associated with VPg uri-dylylation. The possibility that the inability of DTriP-22 toinhibit VPg uridylylation is due to reactions that are not similarto the physiological conditions cannot be excluded. The VPguridylylation assay employed poly(A) as the template andMn2� as a cofactor of EV71 3D polymerase. Mn2� in the VPguridylylation reaction mixture allows for enhanced/exagger-ated poliovirus 3D polymerase activity, relative to the levels inreaction mixtures containing Mg2�, when poly(A) is used asthe template (35). However, Mn2�-based conditions allow 3Dpolymerase-catalyzed VPg uridylylation, which would not oc-cur naturally. Another consideration is that appropriate RNAtemplates (cis-acting replication elements [CREs]) in the invitro VPg uridylylation reaction are closer than a poly(A) tem-plate to the physiological conditions. The CRE structures arelocated in the 2C-encoding region of poliovirus, the capsid-encoding region of HRV-14 and cardiovirus, the 2A-encodingregion of HRV-2, the 5� noncoding region of foot-and-mouth-disease virus, and the 3D-encoding region of hepatitis A virus(15, 17, 30–32, 47). However, the CRE of EV71 has not yetbeen identified in any biochemical experiment. A stem-loopstructure was observed in the 2C region of EV71, with a typicalAAACA/G CRE motif in its top loop, with the MFOLD pro-gram (data not shown). Biochemical analysis needs to be per-formed to map the CRE of EV71. When the appropriate RNAtemplate is used under Mg2�-based conditions, DTriP-22 maybe able to inhibit the VPg uridylylation of EV71 3D polymer-ase in vitro.
The high mutation rate of enteroviruses could result in theemergence of drug-resistant viruses. The present study and ourearlier studies have shown that two EV71 inhibitors, DTriP-22and pyridyl imidazolidinone, target different molecules andmay be effective in drug combinations for delaying or prevent-ing the generation of drug-resistant viruses. The results of thetime-of-addition assay show that unlike pyridyl imidazolidi-none, DTriP-22 acted after virus absorption. Moreover,DTriP-22 efficiently inhibited the viral replication of the VP1V192M mutant, a pyridyl imidazolidinone-resistant virus (Ta-ble 2). However, pyridyl imidazolidinone exhibited great activ-ity in the inhibition of DTriP-22-resistant viruses (Table 2).DTriP-22 had a broader spectrum of antiviral activity thanpyridyl imidazolidinone against enteroviruses, which result isconsistent with the fact that 3D polymerase is a more con-served target than is that of the capsid protein VP1.
Viral polymerases have been considered to be potent targetsfor drug development. Successful clinical studies of nonnucleo-side reverse transcriptase inhibitor-based human immunodefi-ciency virus (HIV)/AIDS therapies have been reported. Nevi-
rapine and efavirenz (nonnucleoside reverse transcriptaseinhibitors) have been reported to combine with other anti-HIVagents in treatment against HIV-1 (36, 40). Several nonnucleo-side inhibitors of hepatitis C virus polymerase have also beenshown, including benzothiadiazines (12, 42, 44, 45). Althoughmany polymerase inhibitors have been shown to exhibit anti-enterovirus activity, most are nucleoside analogues (8, 11, 18–20, 24). Here, we report on a novel nonnucleoside analoguewhich targets 3D polymerase and may have great potential inthe development of a broad-spectrum antienteroviral agent.
ACKNOWLEDGMENTS
This research was supported by the National Science Council of theRepublic of China, Taiwan, under contract no. NSC-94IDP002-1.
We thank Yi-Yu Ke for helpful discussions.
REFERENCES
1. AbuBakar, S., Y. F. Chan, and S. K. Lam. 2000. Outbreaks of enterovirus 71infection. N. Engl. J. Med. 342:355–356.
2. AbuBakar, S., H. Y. Chee, M. F. Al-Kobaisi, J. Xiaoshan, K. B. Chua, andS. K. Lam. 1999. Identification of enterovirus 71 isolates from an outbreakof hand, foot and mouth disease (HFMD) with fatal cases of encephalomy-elitis in Malaysia. Virus Res. 61:1–9.
3. Alexander, J. P., Jr., L. Baden, M. A. Pallansch, and L. J. Anderson. 1994.Enterovirus 71 infections and neurologic disease—United States, 1977–1991.J. Infect. Dis. 169:905–908.
4. Arita, M., T. Wakita, and H. Shimizu. 2008. Characterization of pharmaco-logically active compounds that inhibit poliovirus and enterovirus 71 infec-tivity. J. Gen. Virol. 89:2518–2530.
5. Chan, K. P., K. T. Goh, C. Y. Chong, E. S. Teo, G. Lau, and A. E. Ling. 2003.Epidemic hand, foot and mouth disease caused by human enterovirus 71,Singapore. Emerg. Infect. Dis. 9:78–85.
6. Chang, L. Y., L. M. Huang, S. S. Gau, Y. Y. Wu, S. H. Hsia, T. Y. Fan, K. L.Lin, Y. C. Huang, C. Y. Lu, and T. Y. Lin. 2007. Neurodevelopment andcognition in children after enterovirus 71 infection. N. Engl. J. Med. 356:1226–1234.
7. Chang, L. Y., T. Y. Lin, K. H. Hsu, Y. C. Huang, K. L. Lin, C. Hsueh, S. R.Shih, H. C. Ning, M. S. Hwang, H. S. Wang, and C. Y. Lee. 1999. Clinicalfeatures and risk factors of pulmonary oedema after enterovirus-71-relatedhand, foot, and mouth disease. Lancet 354:1682–1686.
8. Chen, T. C., K. F. Weng, S. C. Chang, J. Y. Lin, P. N. Huang, and S. R. Shih.2008. Development of antiviral agents for enteroviruses. J. Antimicrob. Che-mother. 62:1169–1173.
9. Chern, J. H., K. S. Shia, T. A. Hsu, C. L. Tai, C. C. Lee, Y. C. Lee, C. S.Chang, S. N. Tseng, and S. R. Shih. 2004. Design, synthesis, and structure-activity relationships of pyrazolo[3,4-d]pyrimidines: a novel class of potententerovirus inhibitors. Bioorg. Med. Chem. Lett. 14:2519–2525.
10. De Clercq, E. 1993. Antiviral agents: characteristic activity spectrumdepending on the molecular target with which they interact. Adv. VirusRes. 42:1–55.
11. De Palma, A. M., G. Purstinger, E. Wimmer, A. K. Patick, K. Andries, B.Rombaut, E. De Clercq, and J. Neyts. 2008. Potential use of antiviral agentsin polio eradication. Emerg. Infect. Dis. 14:545–551.
12. Di Marco, S., C. Volpari, L. Tomei, S. Altamura, S. Harper, F. Narjes, U.Koch, M. Rowley, R. De Francesco, G. Migliaccio, and A. Carfi. 2005.Interdomain communication in hepatitis C virus polymerase abolished bysmall molecule inhibitors bound to a novel allosteric site. J. Biol. Chem.280:29765–29770.
13. Fujimoto, T., M. Chikahira, S. Yoshida, H. Ebira, A. Hasegawa, A. Totsuka,and O. Nishio. 2002. Outbreak of central nervous system disease associatedwith hand, foot, and mouth disease in Japan during the summer of 2000:detection and molecular epidemiology of enterovirus 71. Microbiol. Immu-nol. 46:621–627.
14. Furuta, Y., K. Takahashi, M. Kuno-Maekawa, H. Sangawa, S. Uehara, K.Kozaki, N. Nomura, H. Egawa, and K. Shiraki. 2005. Mechanism of actionof T-705 against influenza virus. Antimicrob. Agents Chemother. 49:981–986.
15. Gerber, K., E. Wimmer, and A. V. Paul. 2001. Biochemical and geneticstudies of the initiation of human rhinovirus 2 RNA replication: identifica-tion of a cis-replicating element in the coding sequence of 2Apro. J. Virol.75:10979–10990.
16. Gohara, D. W., C. S. Ha, S. Kumar, B. Ghosh, J. J. Arnold, T. J. Wisniewski,and C. E. Cameron. 1999. Production of “authentic” poliovirus RNA-de-pendent RNA polymerase (3D(pol)) by ubiquitin-protease-mediated cleav-age in Escherichia coli. Protein Expr. Purif. 17:128–138.
17. Goodfellow, I., Y. Chaudhry, A. Richardson, J. Meredith, J. W. Almond, W.
Barclay, and D. J. Evans. 2000. Identification of a cis-acting replicationelement within the poliovirus coding region. J. Virol. 74:4590–4600.
18. Goris, N., A. De Palma, J. F. Toussaint, I. Musch, J. Neyts, and K. De Clercq.2007. 2�-C-methylcytidine as a potent and selective inhibitor of the replica-tion of foot-and-mouth disease virus. Antivir. Res. 73:161–168.
19. Graci, J. D., K. Too, E. D. Smidansky, J. P. Edathil, E. W. Barr, D. A. Harki,J. E. Galarraga, J. M. Bollinger, Jr., B. R. Peterson, D. Loakes, D. M. Brown,and C. E. Cameron. 2008. Lethal mutagenesis of picornaviruses with N-6-modified purine nucleoside analogues. Antimicrob. Agents Chemother. 52:971–979.
20. Harki, D. A., J. D. Graci, J. E. Galarraga, W. J. Chain, C. E. Cameron, andB. R. Peterson. 2006. Synthesis and antiviral activity of 5-substituted cytidineanalogues: identification of a potent inhibitor of viral RNA-dependent RNApolymerases. J. Med. Chem. 49:6166–6169.
21. Ho, M., E. R. Chen, K. H. Hsu, S. J. Twu, K. T. Chen, S. F. Tsai, J. R. Wang,S. R. Shih, et al. 1999. An epidemic of enterovirus 71 infection in Taiwan.N. Engl. J. Med. 341:929–935.
22. Kehle, J., B. Roth, C. Metzger, A. Pfitzner, and G. Enders. 2003. Molecularcharacterization of an Enterovirus 71 causing neurological disease in Ger-many. J. Neurovirol. 9:126–128.
23. Kirsi, J. J., J. A. North, P. A. McKernan, B. K. Murray, P. G. Canonico, J. W.Huggins, P. C. Srivastava, and R. K. Robins. 1983. Broad-spectrum antiviralactivity of 2-beta-D-ribofuranosylselenazole-4-carboxamide, a new antiviralagent. Antimicrob. Agents Chemother. 24:353–361.
24. Kishimoto, C., C. S. Crumpacker, and W. H. Abelmann. 1988. Ribavirintreatment of murine coxsackievirus B3 myocarditis with analyses of lympho-cyte subsets. J. Am. Coll. Cardiol. 12:1334–1341.
25. Kuo, C. J., J. J. Shie, J. M. Fang, G. R. Yen, J. T. Hsu, H. G. Liu, S. N. Tseng,S. C. Chang, C. Y. Lee, S. R. Shih, and P. H. Liang. 2008. Design, synthesis,and evaluation of 3C protease inhibitors as anti-enterovirus 71 agents.Bioorg. Med. Chem. 16:7388–7398.
26. Li, Z. H., C. M. Li, P. Ling, F. H. Shen, S. H. Chen, C. C. Liu, C. K. Yu, andS. H. Chen. 2008. Ribavirin reduces mortality in enterovirus 71-infected miceby decreasing viral replication. J. Infect. Dis. 197:854–857.
27. Lin, J. Y., M. L. Li, P. N. Huang, K. Y. Chien, J. T. Horng, and S. R. Shih.2008. Heterogeneous nuclear ribonuclear protein K interacts with theenterovirus 71 5� untranslated region and participates in virus replication.J. Gen. Virol. 89:2540–2549.
28. Lin, J. Y., M. L. Li, and S. R. Shih. 2009. Far upstream element bindingprotein 2 interacts with enterovirus 71 internal ribosomal entry site andnegatively regulates viral translation. Nucleic Acids Res. 37:47–59.
29. Lin, T. Y., S. J. Twu, M. S. Ho, L. Y. Chang, and C. Y. Lee. 2003. Enterovirus71 outbreaks, Taiwan: occurrence and recognition. Emerg. Infect. Dis.9:291–293.
30. Lobert, P. E., N. Escriou, J. Ruelle, and T. Michiels. 1999. A coding RNAsequence acts as a replication signal in cardioviruses. Proc. Natl. Acad. Sci.USA 96:11560–11565.
31. Mason, P. W., S. V. Bezborodova, and T. M. Henry. 2002. Identification andcharacterization of a cis-acting replication element (cre) adjacent to theinternal ribosome entry site of foot-and-mouth disease virus. J. Virol. 76:9686–9694.
32. McKnight, K. L., and S. M. Lemon. 1998. The rhinovirus type 14 genomecontains an internally located RNA structure that is required for viral rep-lication. RNA 4:1569–1584.
33. McMinn, P., I. Stratov, L. Nagarajan, and S. Davis. 2001. Neurologicalmanifestations of enterovirus 71 infection in children during an outbreak ofhand, foot, and mouth disease in Western Australia. Clin. Infect. Dis. 32:236–242.
34. McMinn, P. C. 2002. An overview of the evolution of enterovirus 71 and itsclinical and public health significance. FEMS Microbiol. Rev. 26:91–107.
35. Paul, A. V., J. Peters, J. Mugavero, J. Yin, J. H. van Boom, and E. Wimmer.2003. Biochemical and genetic studies of the VPg uridylylation reactioncatalyzed by the RNA polymerase of poliovirus. J. Virol. 77:891–904.
36. Raboud, J. M., S. Rae, S. Vella, P. R. Harrigan, R. Bucciardini, V. Fragola,D. Ricciardulli, and J. S. Montaner, et al. 1999. Meta-analysis of two ran-domized controlled trials comparing combined zidovudine and didanosinetherapy with combined zidovudine, didanosine, and nevirapine therapy inpatients with HIV. J. Acquir. Immune Defic. Syndr. 22:260–266.
37. Shih, S. R., M. C. Tsai, S. N. Tseng, K. F. Won, K. S. Shia, W. T. Li, J. H.Chern, G. W. Chen, C. C. Lee, Y. C. Lee, K. C. Peng, and Y. S. Chao. 2004.Mutation in enterovirus 71 capsid protein VP1 confers resistance to theinhibitory effects of pyridyl imidazolidinone. Antimicrob. Agents Che-mother. 48:3523–3529.
38. Sidwell, R. W., J. H. Huffman, G. P. Khare, L. B. Allen, J. T. Witkowski, andR. K. Robins. 1972. Broad-spectrum antiviral activity of Virazole: 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide. Science 177:705–706.
39. Smee, D. F., P. A. McKernan, L. D. Nord, R. C. Willis, C. R. Petrie, T. M.Riley, G. R. Revankar, R. K. Robins, and R. A. Smith. 1987. Novel pyr-azolo[3,4-d]pyrimidine nucleoside analog with broad-spectrum antiviral ac-tivity. Antimicrob. Agents Chemother. 31:1535–1541.
40. Staszewski, S., J. Morales-Ramirez, K. T. Tashima, A. Rachlis, D. Skiest, J.Stanford, R. Stryker, P. Johnson, D. F. Labriola, D. Farina, D. J. Manion,N. M. Ruiz, et al. 1999. Efavirenz plus zidovudine and lamivudine, efavirenzplus indinavir, and indinavir plus zidovudine and lamivudine in the treatmentof HIV-1 infection in adults. N. Engl. J. Med. 341:1865–1873.
41. Takegami, T., R. J. Kuhn, C. W. Anderson, and E. Wimmer. 1983. Mem-brane-dependent uridylylation of the genome-linked protein VPg of polio-virus. Proc. Natl. Acad. Sci. USA 80:7447–7451.
42. Tedesco, R., A. N. Shaw, R. Bambal, D. Chai, N. O. Concha, M. G. Darcy, D.Dhanak, D. M. Fitch, A. Gates, W. G. Gerhardt, D. L. Halegoua, C. Han,G. A. Hofmann, V. K. Johnston, A. C. Kaura, N. Liu, R. M. Keenan, J.Lin-Goerke, R. T. Sarisky, K. J. Wiggall, M. N. Zimmerman, and K. J. Duffy.2006. 3-(1,1-dioxo-2H-(1,2,4)-benzothiadiazin-3-yl)-4-hydroxy-2(1H)-quino-linones, potent inhibitors of hepatitis C virus RNA-dependent RNA poly-merase. J. Med. Chem. 49:971–983.
43. Thompson, A. A., and O. B. Peersen. 2004. Structural basis for proteolysis-dependent activation of the poliovirus RNA-dependent RNA polymerase.EMBO J. 23:3462–3471.
44. Tomei, L., S. Altamura, L. Bartholomew, A. Biroccio, A. Ceccacci, L. Pacini,F. Narjes, N. Gennari, M. Bisbocci, I. Incitti, L. Orsatti, S. Harper, I.Stansfield, M. Rowley, R. De Francesco, and G. Migliaccio. 2003. Mecha-nism of action and antiviral activity of benzimidazole-based allosteric inhib-itors of the hepatitis C virus RNA-dependent RNA polymerase. J. Virol.77:13225–13231.
45. Tomei, L., S. Altamura, L. Bartholomew, M. Bisbocci, C. Bailey, M. Bosser-man, A. Cellucci, E. Forte, I. Incitti, L. Orsatti, U. Koch, R. De Francesco,D. B. Olsen, S. S. Carroll, and G. Migliaccio. 2004. Characterization of theinhibition of hepatitis C virus RNA replication by nonnucleosides. J. Virol.78:938–946.
46. Verstrepen, W. A., S. Kuhn, M. M. Kockx, M. E. Van De Vyvere, and A. H.Mertens. 2001. Rapid detection of enterovirus RNA in cerebrospinal fluidspecimens with a novel single-tube real-time reverse transcription-PCR as-say. J. Clin. Microbiol. 39:4093–4096.
47. Yang, Y., M. Yi, D. J. Evans, P. Simmonds, and S. M. Lemon. 2008. Iden-tification of a conserved RNA replication element (cre) within the 3Dpol-coding sequence of hepatoviruses. J. Virol. 82:10118–10128.
