Abstract Rhino- and enteroviruses are important human pathogens, against which no antivirals are available. The best-studied inhibitors are “capsid binders” that fit in a hydrophobic pocket of the viral capsid. Employing a new class of entero-/rhinovirus inhibitors and by means of cryo–electron microscopy (EM), followed by resistance selection and reverse genetics, we discovered a hitherto unknown druggable pocket that is formed by viral proteins VP1 and VP3 and that is conserved across entero-/rhinovirus species. We propose that these inhibitors stabilize a key region of the virion, thereby preventing the conformational expansion needed for viral RNA release. A medicinal chemistry effort resulted in the identification of analogues targeting this pocket with broad-spectrum activity against Coxsackieviruses B (CVBs) and compounds with activity against enteroviruses (EV) of groups C and D, and even rhinoviruses (RV). Our findings provide novel insights in the biology of the entry of entero-/rhinoviruses and open new avenues for the design of broad-spectrum antivirals against these pathogens.

Citation: Abdelnabi R, Geraets JA, Ma Y, Mirabelli C, Flatt JW, Domanska A, et al. (2019) A novel druggable interprotomer pocket in the capsid of rhino- and enteroviruses. PLoS Biol 17(6): e3000281. https://doi.org/10.1371/journal.pbio.3000281 Academic Editor: Chaitan Khosla, Stanford University, UNITED STATES Received: November 12, 2018; Accepted: May 8, 2019; Published: June 11, 2019 Copyright: © 2019 Abdelnabi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the paper and its Supporting Information files. Atomic coordinates have been deposited in the Worldwide Protein Data Bank with accession code 6GZV. Density maps have been deposited in the Electron Microscopy Data Bank with accession code 0103. Raw unaligned micrographs and picked particle coordinates have been deposited in the Electron Microscopy Public Image Archive (EMPIAR) with accession code 10199. Funding: This work was funded by the BELVIR project from BELSPO (IUAP), People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme FP7/2007-2013/ under REA grant agreement number 612308 AIROPico (to SJB and JN), a postdoctoral mandate Internal Fund from KU Leuven (PDM/17/178 to RA), a Fellowship from China Scholarship Council (CSC) (grant number 201406040056 to YM), Academy of Finland (275199 to SJB), and Sigrid Juselius Foundation (to SJB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Abbreviations: A-particle, altered particle; CAR, Coxsackievirus and adenovirus receptor; COPD, chronic obstructive pulmonary disease; CPE, cytopathic effect; CVA, Coxsackievirus A; CVB, Coxsackievirus B; cVDPV, circulating vaccine-derived poliovirus; DAF, decay-accelerating factor; E-1, echovirus 1; E-7, echovirus 7; EC 50 , 50% effective concentration; EM, electron microscopy; EV, enterovirus; EVA, enterovirus A; EVB, enterovirus B; EVC, enterovirus C; EVD, enterovirus D; EVD68, enterovirus D68; EVA71, enterovirus A71; FBS, fetal bovine serum; hCAR, human Coxsackie virus and adenovirus receptor; MDFF, molecular dynamics flexible fitting; MOI, multiplicity of infection; MTS/PMS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyll-2-(4-sulfophenyl)-2-H-tetrazolium, inner salt)/phenazine methosulfate; PDB, Protein Data Bank; PISA, Proteins, Interfaces, Structures, and Assemblies; PV, poliovirus; PV1, poliovirus type I; qRT-PCR, quantitative reverse transcription PCR; RMSD, root mean square deviation; RV, rhinovirus; RVA, rhinovirus A; RVB, rhinovirus B; SAR, structure-activity relationship; SMILES, simplified molecular-input line-entry system; TCID 50 , 50% tissue culture infective dose; UCSF, University California San Francisco

Introduction Rhino- and enteroviruses (family Picornaviridae) are important human pathogens. Rhinoviruses are responsible for the common cold and are the most important trigger of exacerbations of asthma and chronic obstructive pulmonary disease (COPD) [1]. Each year, millions of people are affected by enteroviruses; there are 29 Coxsackieviruses, 28 echoviruses, and 5 other enteroviruses that cause disease (such as hand, foot and mouth disease, myocarditis, pancreatitis, aseptic meningitis, and encephalitis) in man [2]. In recent years, massive outbreaks of the enterovirus 71 (EVA71) occurred in Asia that left a large number of children with life-threatening encephalitis [3]. In 2014 and 2016, widespread outbreaks in the United States of the enterovirus 68 (EVD68) resulted in severe respiratory illness in children that was in many cases associated with acute flaccid myelitis, a condition now documented across the world in 14 countries [4]. Poliovirus (PV) is yet another important enterovirus. An enormous effort by the Global Polio Eradication Initiative (whereby 2.5 billion children have been vaccinated, requiring a $14 billion investment) has been very successful in massively reducing the number of cases [5]. However, the polio endgame appears to be much more challenging than anticipated; it is now well accepted that antivirals will be needed for a successful eradication, in particular to control outbreaks of circulating vaccine-derived polioviruses (cVDPV) and to stop excretion in chronic shedders (www.polioeradication.org). There are, however, no antivirals available for the treatment and/or prophylaxis of infections with entero- (including polio) and/or rhinoviruses. The best studied inhibitors are the so-called “capsid binders” (such as pleconaril, pirodavir, vapendavir, and pocapavir). These compounds fit in a hydrophobic pocket in the viral particle, thereby preventing binding of the virus to the receptor and/or uncoating [6]. Capsid binders have the disadvantage that they select rapidly for drug-resistant variants; this was also recently documented when assessing the effect of pocapavir in healthy volunteers challenged with the oral live attenuated polio vaccine virus [7]. Moreover, some capsid binders, such as pleconaril, lack activity against certain enteroviruses such as EVA71 [8]. Also, rhinoviruses of group C (infections with these viruses are associated with severe respiratory infections and childhood asthma exacerbations) are completely resistant to the capsid binders because the hydrophobic pocket is collapsed in the particle [9]. Today, the hydrophobic pocket is the only druggable surface pocket of the entero-/rhinoviruses particle. Now 32 years after its discovery, we report on a novel druggable pocket within a conserved VP1-VP3 interprotomer interface of the viral capsid. We propose that this pocket is crucial for conformational changes required for viral RNA release, and we describe a class of compounds targeting this pocket with broad-spectrum activity against entero-/rhinoviruses.

Discussion Pockets traditionally make more attractive targets for drug design and optimization than flat surfaces, as they can accommodate larger surface areas and hence contribute more residues for interaction. For the same reason, pockets such as the hydrophobic site found within VP1 of most enteroviruses play important functional roles during virus entry and replication. Until now, the hydrophobic canyon site has been the only explored surface pocket for enteroviruses. The cryo-EM structure of CVB3 in complex with compound 17 revealed that 16 residues line the interprotomer pocket and provided a plausible explanation for most of the observed resistance mutations identified by selection. To confirm the druggable site, several of the residues were mutated, and the resulting viruses had indeed reduced sensitivity to compound 17. There were several amino acid residues in the pocket that could not be mutated, highlighting the importance of this pocket in the biology of enterovirus replication. Residues in the pocket allow quaternary structural changes in the capsid as it progresses from the virion to an expanded altered particle (A-particle) during entry, primed for RNA release [2,18], a process common to many picornaviruses [2]. This expansion requires rotation and translation of VP1, relative to itself, and to VP3 and VP2, a process dependent on movements along the interprotomer interfaces, including the region containing the pocket described here. Comparison of several structures of expanded picornavirus particles revealed that in the expanded particle, the pocket has an altered shape caused by the movement of the VP3 C terminus; this includes the residues that line the pocket (Q233, Q234, N235, F236). Hence, we propose that binding of compounds in this pocket stabilizes the conformation of a key region of the virion, preventing rearrangements that allow transition to the A-particle. The resistant mutant distant to the pocket (VP1-D133G) destabilizes the particle sufficiently so that the energy barrier to the transition is lowered, compensating for the increased stability induced by the drug, and thus still allowing release of the RNA. To conclude, compound 17 and its analogues are selective inhibitors of CVB3 replication that target a novel pocket on the surface of the capsid that is important in conformational changes required for RNA release from the viral particle. Cryo-EM structural data and in vitro resistance selection together with reverse engineering revealed a set of amino acid residues in the virion that are crucial for the compound activity and allowed us to propose a unique mechanism of antiviral action. Antiviral evaluation of a set of compound 17 analogues indicates that it is possible to target EV-B, EV-C, EV-D, and even rhinovirus (A and B) species. In addition, computational analysis suggests that the pocket is partially conserved across species, including the RV-C species that are naturally resistant to pleconaril (and molecules with a similar mechanism of action) and EV-A. However, the shape and electrostatic properties of the pocket are sufficiently different in those latter species so that further study is required to design molecules that also block viruses belonging to these species (S3 and S4 Tables). Medicinal chemistry efforts are ongoing to replace the carboxylic acid moiety in the core structure of this class of compounds with more stable moieties, such as (bio)isosteres. Hence, we foresee the development of a future series of analogues with improved activity and spectrum against an important group of viruses that cause significant infectious illnesses worldwide.

Material and methods Cells and viruses African green monkey kidney (Vero A, ATCC CCL-81) cells, Buffalo green monkey (BGM, ECACC 90092601) cells, HeLa Rh cells (kindly provided by Dr. K. Andries [Janssen Infectious Diseases, Beerse, Belgium]), and human rhabdosarcoma (RD, ECACC 85111502) cells were maintained in minimal essential medium (MEM Rega-3, Gibco, Belgium) supplemented with 10% fetal bovine serum (FBS, Gibco, Belgium), 2 mM l-glutamine (Gibco, Belgium), and 1% sodium bicarbonate (Gibco, Belgium). Antiviral assays medium was supplemented with 2% FBS instead of 10%. All cell cultures were maintained at 37°C in an atmosphere of 5% CO 2 except for rhinovirus 14, which was incubated at 35°C. Coxsackievirus B3 strain Nancy (CVB3, derived from plasmid P53CB3/T7), Coxsackievirus B1 (Conn-5), Coxsackievirus B2 (CVB2, OH), Coxsackievirus B5 (CVB5, Dekking), Coxsackievirus B6 (CVB6, stam P2183), enterovirus 71 (EVA71, BrCr), and enterovirus 68 (EVD68, CU70) were kind gifts from Prof. F. van Kuppeveld (Universiteit Utrecht, Utrecht, the Netherlands). Coxsackievirus B4 (CVB4, Edwards) was kindly provided by Dr. J.W. Yoon (Julia McFarlane Diabetes Research Centre, Canada). The Sabin vaccine strain of PV1 was kindly provided by Dr. A.J. Macadam (Centre for Infectious Diseases, University of Edinburgh, Edinburgh, United Kingdom). Human rhinovirus type 14 (RVB14) and echovirus 11 (ECHO11) was kindly provided by Dr. K. Andries (Janssen Infectious Diseases, Beerse, Belgium). Compounds Compound 17 and its inactive analogue compound 15 (where the carboxyl group has been replaced with a methyl group) were synthesized as described previously [19]. Favipiravir (T-705) was purchased as a custom synthesis product from BOC Sciences (NY). Rupintrivir was purchased from Axon Medchem (the Netherlands). Pleconaril was kindly provided by V. Makarov (RAS Institute of Biochemistry, Russia). All compounds were dissolved in analytical grade DMSO (10 mg/mL). Cytopathic effect reduction assay Cells were seeded (at a density of 2.5 × 104 cells/well for Vero A, BGM cells, and RD cells and 1.8 × 104 cells/well for HeLa Rh cells) in 96-well tissue culture plates (BD Falcon) and were allowed to adhere overnight. The next day, cells were treated with serial dilutions of the compound and infected with the selected virus strain at a multiplicity of infection (MOI) of 0.01. On days 2–3 postinfection, the antiviral effect of the compound was quantified using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyll-2-(4-sulfophenyl)-2-H-tetrazolium, inner salt)/phenazine methosulfate (MTS/PMS) assay, as described by the manufacturer Promega (the Netherlands). In addition, the cells were also checked microscopically for minor signs of virus-induced cytopathic effects (CPE) or compound-induced toxicity (changes in cell and/or monolayer morphology). The EC 50 , which is the concentration of compound that is required to inhibit virus-induced cell death by 50%, was determined using logarithmic interpolation. Virus yield assay Vero A cells were seeded at a density of 5 × 104 cells/well in 96-well tissue culture plates. The next day, cells were treated with serial dilutions of the compound and then infected with CVB3 (MOI, 0.01). After a 2-h incubation, the cells were washed three times with assay medium to remove non-adsorbed virus. The cells were treated with the same serial dilutions of the compound as for the CPE reduction assay and incubated for 2 days at 37°C. At the end of the incubation period, the viral RNA was isolated from the culture supernatants in different wells using NucleoSpin 96 virus kit (Macherey Nagel, Germany) and was quantified by real-time quantitative reverse transcription PCR (qRT-PCR) (see below). In addition, the number of infectious virions in the collected supernatants was determined by an end-point titration assay, as described by Reed and Muench (TCID 50 /mL) [20]. qRT-PCR For each RT-PCR reaction, 25 μL of a PCR reagent mixture was prepared to contain 12.5 μL One-Step qRT-PCR mix (Eurogentec, Seraing, Belgium), a 900 nM concentration of each primer, a 200 nM concentration of the specific TaqMan probe, RNase-free water, and 5 μL RNA. The nucleotide sequences of the used primers and probe were as follows: forward primer (5′-ACG AAT CCC AGT GTG TTT TGG-3′), reverse primer (5′-TGC TCA AAA ACG GTA TGG ACA T-3′), and CVB3 probe (5′-FAM-CGA GGG AAA CGC CCC GCC-TAMRA-3′). The RT-PCR reaction was done using the ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Branchburg, NJ) according to the following protocol: 30 min at 48°C and 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. The RNA copy number in each sample was determined by a standard curve generated using serial dilutions of CVB3 standard cDNA that were included in the run [21]. Time-of-drug-addition assay Vero-A cells were seeded in 96-well tissue culture plates at a density of 5 × 104 cells/well in assay medium and incubated overnight. Cells were treated with the appropriate concentration of the compound before or after the infection with CVB3 VP1_L92I variant (pleconaril sensitive, MOI = 1) at the following time points (−2 h, zero time, 2 h, 4 h, and 6 h). Following 24 h of incubation, the intracellular viral RNA from treated and untreated cells was extracted using Cells-to-cDNA lysis buffer (Life Technologies, the Netherlands) and was quantified by quantitative RT-PCR, as described before. Thermostability assay CVB3 WT (5 × 105 TCID 50 /mL) was incubated with 20 μM compound 17, 20 μM compound 15 (an inactive analogue) [22], or an equal volume of assay medium) at six different temperatures ranging from 37–52°C for 2 min, followed by rapid cooling to 4°C. Subsequently, the infectious virus load of the different samples was quantified by end-point titration. Large-scale virus purification For large-scale virus purification for structural study, CVB3 Nancy strain was grown on Vero A cells, maintained in MEM (Eagle, without L-glutamine, Sigma M2279) supplemented with sodium bicarbonate (1%), Glutamax (2 mM, Gibco, 35050–038), antibiotic/antimycotic solution (1%, Sigma, A-5955), and 10% FBS. For proliferation, 30× T175 flasks with confluent cell layers (approximately 90%) were inoculated with CVB3 Nancy strain at an MOI of 0.02 in serum-free medium. After CPE was observed over the entire cell monolayer, the contents of the flask were collected, freeze-thawed three times, and the lysate cleared by centrifugation (3,000g, 277 K, 15 min). The supernatant was filtered and concentrated by ultrafiltration in Centricon 100-kDa filter units (Millipore). Ultracentrifugation was then performed on a CsCl step gradient, for which the top density was 1.25 g/cm3 and the bottom 1.48 g/cm3 CsCl (32,000g, Beckmann SW41Ti, 277 K, 18 h), in buffer (0.01 M HEPES, pH 7.5, 0.1 M MgCl 2, ) with additional 0.5% CHAPS detergent [23]. The virus band was collected, and the CsCl and CHAPS removed in a buffer exchange by ultrafiltration in Centricon 100-kDa filters (Millipore). Ultracentrifugation, band collection, and buffer exchange steps were performed twice. Cryo-EM Purified CVB3 was mixed with compound 17 at a 1:2,500 molar ratio of capsid to inhibitor and incubated for 1 h at 37°C prior to cryo sample preparation on glow-discharged ultrathin carbon on lacey carbon grids (#01824, Ted Pella, US). Micrographs were collected at the Diamond Light Source, UK, via iNEXT access using a FEI Titan Krios electron microscope equipped with a Gatan energy filter (post-GIF) and a Gatan K2 Summit direct electron detector. The K2 detector was operated in counting mode, recording movies at a nominal magnification of 130,000× (sampling 1.06 Å/pixel), using a total electron dose of approximately 47 e/Å2 separated into 20-dose fractions. Example micrographs are shown in S1A Fig. Image processing For each movie collected, all 20-dose fractions were aligned and compensated for drift using MotionCor2 [24]. The defocus level and other CTF parameters of each micrograph were determined using GCTF [25]. Micrographs were discarded if the power spectrum indicated noticeable astigmatism, crystalline ice, or uncorrected drift. For each of the remaining 2,121 movies, 71,034 particles were automatically picked on aligned averaged images using Relion 2.0.4 [26]. Two-dimensional classification was used to select 10,182 particles for further processing. The starting model was generated by Fourier filtering a CVB3 capsid atomic model (PDB ID: 1COV) [14] to a resolution of 40 Å in UCSF Chimera [27]. Three-dimensional classification in Relion [28] was used to remove empty, broken, and overlapping particles. All 20 frames were used for classification, refinement, and calculation of the final density map in Relion 2.1 beta from 4,891 particles. The final refinement step combining two independent data sets gave a resolution of 4.0 Å, as assessed by 0.143 criterion Fourier shell correlation [29]. A B-factor of −170 Å2 was automatically calculated in Relion and applied. The reconstruction central section is shown in S1B Fig, and resolution estimates are shown in S2 Fig. Atomic model building and refinement An initial atomic model for the Nancy strain CVB3-inhibitor complex was generated using I-TASSER and UCSF Chimera [27,30]. I-TASSER threaded the Nancy CVB3 sequence onto the crystal structure of the CVB3 coat protein (PDB ID: 1COV [14]). UCSF Chimera built compound 17 atom by atom from the simplified molecular-input line-entry system (SMILES) line notation that described the structure of the chemical species. Docking of atomic coordinates was done manually using UCSF Chimera and the fit was further optimized using the “Fit in Map” command. Inspection and further refinement of the viral atomic coordinates was done using Coot, and this served as input for molecular dynamics flexible fitting (MDFF) [31]. The MDFF program was used together with NAMD and VMD to further enhance the fit of the model into the cryo-EM density [32–34]. A scale factor of 1 was employed to weight the contribution of the cryo-EM map to the overall potential function used in MDFF. Simulations included 20,000 steps of minimization and 100,000 steps of molecular dynamics under implicit solvent conditions with secondary structure restraints in place. Model statistics are shown in S2 Table. Interface analysis was done with the PDBePISA www server [35] and with Coot [31] (S3 Table). RMSD analysis of the pocket conservation was calculated in UCSF Chimera (S4 Table). Isolation of compound 17–resistant CVB3 variants and reverse genetics A multistep clonal selection protocol was performed to obtain compound 17–resistant CVB3 variants [13]. Whole-genome sequencing was performed to detect any mutations in the genome of the putative compound-resistant variants (3130 Genetic Analyzer automatic sequencer, Applied Biosystems). Desired mutations were introduced into the CVB3 strain Nancy infectious cDNA clone kindly provided by Prof. F. van Kuppeveld (University of Utrecht, Utrecht, the Netherlands) using the QuikChange mutagenesis kit (Agilent, CA) according to the manufacturer’s instructions. The presence of the desired mutation in the produced construct was confirmed by sequencing. Full-length infectious viral RNA was then transcribed from each plasmid using the RiboMAX Large Scale RNA Production System-T7 (Promega, the Netherlands) and transfected into Vero A cells with a TransIT-mRNA Transfection Kit (Mirus) to produce virus stocks of the reverse-engineered mutants. Growth kinetics The in vitro growth kinetics of CVB3 WT and VP1 variants were determined in Vero A cells (96-well tissue culture plates, 5 × 104 cells/well). At time zero, cells were infected with the respective virus variants (MOI = 1). After 2 h of infection, the cells were washed three times with 2% medium to remove non-adsorbed virus, and 200 μL medium were added to each well. At 8 and 24 h postinfection, the culture supernatants were collected to quantify the number of infectious virus particles at each time point by end-point titration. Plaque phenotyping Vero A cells were seeded in 6-well plates (BD Falcon 6-Well Cell Culture Plate) at a density of 1 × 106 cells/well. The next day, cells were infected with 1 mL of 10-fold serial dilutions of the respective virus variants in assay medium. At 2 h postinfection, the cell monolayers were washed three times with PBS, after which 3 mL of a freshly prepared 1:1 mixture of 1% low-melting-point agarose (Invitrogen, Belgium) and 2× MEM medium (Gibco, Belgium) was added. After 3 d of incubation at 37°C, cells were fixed with 3.7% formaldehyde followed by removal of the agarose overlay and staining with 2% Giemsa staining solution to visualize the virus-induced plaques. Expression of a flag-tagged CAR The pcDNA3.1-hCAR plasmid was kindly provided by Prof. F. van Kuppeveld (Universiteit Utrecht, Utrecht, the Netherlands). A flag tag was added to the C terminus of an hCAR sequence by fusion PCR using the following set of primers: forward, GAAAAGCTTCCACCATGGCGCTCCTGCTGTGC; reverse, GGA TCC CTA CTT GTC GTC ATC GTC TTT GTA GTC TAC TAT AGA CCC ATC CTT GCT. The primers were designed to add HindIII and BamHI restriction sites at the N and C termini of the hCAR sequence, respectively. Following PCR amplification, the hCAR-flag fragment was digested with HindIII and BamHI restriction enzymes and cloned back into pcDNA3.1 vector (that was digested with the same enzymes). The pcDNA3.1-hCAR-flag plasmid was then transfected into HEK293T cells in 6-well plates (7 × 105 cells/well) using Mirus TransIT-LT1 Transfection Reagent according to the manufacturer’s protocol. The expression of flag-tagged hCAR protein on day 2 post-transfection was checked by immunostaining using ANTI-FLAG antibody (Sigma-Aldrich) rabbit IgG as a 1ry antibody and Alexa Fluor 488 goat anti-rabbit (Thermo Fisher Scientific, Belgium) as a secondary antibody. Fluorescence was detected using the FLoid Cell Imaging Station (Thermo Fisher Scientific, Belgium). Testing the effect of compound 17 on the binding of CVB3 to the CAR The lysate from HEK293T cells transfected with a flag-tagged pcDNA3.1-hCAR plasmid was incubated with ANTI-FLAG M2 Magnetic Beads (Sigma-Aldrich) at 4°C overnight. After washing with PBS, the CAR-coated beads were incubated with CVB3 or mixtures of the virus with different concentrations of compound 17 for 2 h at 37°C. At the end of incubation, the beads were washed three times with PBS followed by elution of CAR-bound virus using RLT lysis buffer (Qiagen, Germany). The viral RNA in each sample was then extracted using an RNeasy kit (Qiagen, Germany) and quantified by real-time quantitative RT-PCR as described before [21].

Acknowledgments We are grateful to Pasi Laurinmäki and Benita Löflund, University of Helsinki, for excellent technical assistance. We acknowledge CSC–IT Center for Science, Finland, for computational resources. Microscopy was carried out with the support of the Biocenter Finland National Cryo-EM Unit and Instruct-FI. We acknowledge Diamond Light Source, UK, for access and support of the cryo-EM facilities at the UK National Electron Bio-Imaging Centre (eBIC), funded by the Wellcome Trust, MRC, and BBSRC, with funding from the Horizon 2020 programme of the European Union, n°653706 iNEXT (proposal n°1973).