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JOURNAL OF VIROLOGY July 2011 p 6381 6389 Vol 85 No 13 0022 538X 11 12 00 doi 10 1128 JVI 00078 11 Copyright 2011 American Society for Microbiology All Rights Reserved Blocking eIF4E eIF4G Interaction as a Strategy To Impair Coronavirus Replication H17188 Regina Cencic 1 Marc Desforges 2 David R Hall 3 Dima Kozakov 3 Yuhong Du 4 Jaeki Min 4 Raymond Dingledine 4 Haian Fu 4 Sandor Vajda 3 Pierre J Talbot 2 and Jerry Pelletier 1 5 Department of Biochemistry 1 and Goodman Cancer Center 5 McIntyre Medical Sciences Building McGill University Montreal Quebec Canada H3G 1Y6 Laboratory of Neuroimmunovirology INRS Institut Armand Frappier University of Quebec Laval Quebec Canada H7V 1B7 2 Department of Biomedical Engineering Boston University Boston Massachusetts 02215 3 and Emory Chemical Biology Discovery Center Emory University Atlanta Georgia 30322 4 Received 12 January 2011 Accepted 8 April 2011 Coronaviruses are a family of enveloped single stranded positive sense RNA viruses causing respiratory enteric and neurologic diseases in mammals and fowl Human coronaviruses are recognized to cause up to a third of common colds and are suspected to be involved in enteric and neurologic diseases Coronavirus replication involves the generation of nested subgenomic mRNAs sgmRNAs with a common capped 5H11541 leader sequence The translation of most of the sgmRNAs is thought to be cap dependent and displays a requirement for eukaryotic initiation factor 4F eIF4F a heterotrimeric complex needed for the recruitment of 40S ribosomes We recently reported on an ultrahigh throughput screen to discover compounds that inhibit eIF4F activity by blocking the interaction of two of its subunits R Cencic et al Proc Natl Acad Sci U S A 108 1046 1051 2011 Herein we describe a molecule from this screen that prevents the interaction between eIF4E the cap binding protein and eIF4G a large scaffolding protein inhibiting cap dependent translation This inhibitor significantly decreased human coronavirus 229E HCoV 229E replication reducing the per centage of infected cells and intra and extracellular infectious virus titers Our results support the strategy of targeting the eIF4F complex to block coronavirus infection Coronaviruses are ubiquitous enveloped single stranded positive sense RNA viruses that contain a 27 to 32 kb genome and cause a variety of respiratory enteric and neurologic diseases in mammals and fowl 12 Although they have been known since the mid 1960s human coronaviruses HCoV 23 gained notoriety following the 2002 2003 outbreak of one of their family members SARS CoV severe acute respiratory syndrome coronavirus By virtue of an RNA dependent RNA polymerase a series of 3H11032 coterminal nested subgenomic mR NAs sgmRNAs are generated that contain a common 5H11032 leader sequence but the mechanism by which they recruit ribosomes is not known 11 In eukaryotes cellular translation is thought to occur by one of two mechanisms a cap dependent mechanism involving eu karyotic initiation factor 4F eIF4F and a cap independent mechanism in which ribosomes are recruited internally to an internal ribosome entry site IRES via specific mRNA struc tural motifs present upstream of the initiation codon eIF4F consists of eIF4E the cap binding protein eIF4A an RNA helicase and eIF4G a large scaffolding protein There are two isoforms of eIF4G eIF4GI and eIF4GII that are 46 iden tical All structural features of eIF4GI are present on eIF4GII eIF4E is the least abundant initiation factor and it is thought that this renders cap recognition the rate limiting step in ini tiation The availability of eIF4E to assemble into the eIF4F complex is regulated by the mTOR pathway due to sequestra tion by binding to a negative regulator 4E BP there are three 4E BPs in a cell with the best characterized one being 4E BP1 Upon activation by upstream signals mTOR phosphor ylates 4E BP1 which causes the eIF4E 4E BP1 binary com plex to dissociate Since eIF4G and the 4E BPs share binding sites on eIF4E the removal of 4E BP1 allows eIF4E to enter the eIF4F complex When mTOR activity is reduced e g amino acid starvation or energy depletion eIF4F levels are diminished due to the sequestration of eIF4E by 4E BPs and cap dependent translation is squelched The overex pression of 4E BP1 in cells impairs coronavirus replication suggesting that the translation of some viral sgmRNAs or mRNA encoding a cellular factor s required for viral rep lication is cap dependent 5 We recently reported on an ultrahigh throughput screen that identified several new inhibitors of eIF4E eIF4G interac tion 7 Herein we report on the characterization of an in hibitor of eIF4E eIF4G interaction called 4E2RCat We dem onstrate that this inhibitor is capable of blocking coronavirus replication as monitored by viral protein expression and the production of infectious virus MATERIALS AND METHODS Ultrahigh throughput screening for eIF4E eIF4G inhibitors High throughput screening for eIF4E eIF4G interaction inhibitors has been described in detail previously 7 8 In brief a time resolved fluorescence resonance energy transfer TR FRET based high throughput assay was miniaturized to a 1 536 well for mat and optimized It was used to screen a library of 217 341 compounds from the Molecule Library Screening Centers Network from which 4E2RCat PubChem no 2287238 was identified Corresponding author Mailing address McIntyre Medical Sci ences Building Rm 810 3655 Promenade Sir William Osler McGill University Montreal Quebec Canada H3G 1Y6 Phone 514 398 2323 Fax 514 398 7384 E mail jerry pelletier mcgill ca H17188 Published ahead of print on 20 April 2011 6381 on May 1 2015 by Univ of Massachusetts Amherst http jvi asm org Downloaded from In vitro translations In vitro transcriptions and translations of bicistronic mRNA reporters were performed as described previously 16 Firefly FF and renilla Ren luciferase Luc activities RLU were measured using a Berthold Lumat LB 9507 luminometer To visualize in vitro translated proteins reactions were performed in micrococcal nuclease treated Krebs extract in the presence of 35 S methionine Translations were analyzed on a 10 SDS polyacrylamide gel followed by staining with Coomassie blue and destaining to ensure equal loading Gels were further incubated for1hinEn 3 Hance Perkin dried and exposed to X Omat film Kodak GST pulldowns For pulldown experiments either glutathione S transferase GST eIF4GI 517 606 or bovine serum albumin BSA was coupled to Affi Gel 10 Bio Rad To this end 1 mg of protein was incubated at 2 5 mg ml with 400 H9262l of a 50 slurry of Affi Gel 10 beads in 10 ml AC buffer 20 mM HEPES pH 7 5 10 glycerol 1 mM dithiothreitol DTT 1 mM EDTA end over end for 12 h After being centrifuged for 10 min at 1 200 H11003 g the supernatant was removed and beads incubated with AC buffer supplemented with 75 mM NaCl and 80 mM ethanolamine for 1 h end over end at 4 C After an additional spin to remove the supernatant beads were blocked for 1 h with 8 ml AC buffer supplemented with 75 mM NaCl and 1 mg ml BSA Beads were washed once with 20 ml AC buffer supplemented with 1 M NaCl and three times with AC buffer supplemented with 75 mM NaCl Beads were stored as 50 slurry in AC buffer containing 75 mM NaCl For pulldown experiments involving eIF4E 20 H9262l of a 50 slurry of Affi Gel matrix coupled to either GST eIF4GI 517 606 or BSA was preincubated with 100 H9262M 4E2RCat Recombinant eIF4E also was preincubated with 4E2RCat or vehicle DMSO for 1 h end over end at room temperature Recombinant eIF4E and Affi Gel bound proteins then were mixed and incubated for another hour end over end at room temperature Beads were washed three times for 10 min with 10 volumes of binding buffer resuspended in SDS PAGE loading dye and resolved by 10 SDS PAGE Western blotting was used to detect the presence of eIF4E Santa Cruz Pulldowns with GST eIF4GII 555 658 and GST 4E BP1 were performed with 2 5 H9262g of the GST fusion proteins and 0 25 H9262g eIF4E Proteins were individually incubated for 1 h at room temperature in the presence of 25 H9262M 4E2RCat in binding buffer 20 mM Tris pH 7 5 100 mM KCl 10 glycerol and 0 1 NP 40 After incubation eIF4E was added to the GST tagged partners and incubated for another hour followed by addition to glutathione beads for an other hour at room temperature Beads were washed three times with 10 volumes of binding buffer and eluted for 1 h using reduced glutathione 10 mM The eluents were separated on 10 SDS PAGE followed by transfer to a poly vinylidene difluoride PVDF membrane Millipore and Western blot analysis Primary anti GST and anti eIF4E antibodies were from Santa Cruz Secondary antibodies were from Jackson Immuno Research In vivo metabolic labeling In vivo 35 S methionine labeling was performed by seeding 60 000 cells well in 24 well plates 24 h prior to treatment Cells were treated for 4 or 24 h in the presence of increasing concentrations of 4E2RCat For the last hour medium was replaced by methionine free Dulbecco s modified essential medium DMEM supplemented with 10 dialyzed serum and for the last 15 min cells were labeled with 35 S methionine 150 to 225 H9262Ci ml Cells were washed with PBS and lysed in RIPA buffer 20 mM Tris 7 5 100 mM NaCl 1 mM EDTA 1 mM EGTA 0 1 NP 40 0 5 sodium deoxycholate 0 1 SDS 20 mM glycerophosphate 10 mM NaF 1 mM phenylmethylsulfonyl fluoride PMSF 4 H9262g ml aprotinin 2 H9262g ml leupeptin 2 H9262g ml pepstatin Samples were precipitated with trichloroacetic acid TCA and radioactivity was determined by scintillation counting Protein concentrations in each sample were measured using the Bio Rad D C protein assay Bio Rad Laboratories and used to stan dardize the counts obtained after TCA precipitation eIF4F pulldown experiments For pulldown experiments a ribosome salt wash RSW was incubated in the presence of 1 dimethylsulfoxide DMSO or 25 H9262M 4E2RCat for1hat30 C Following incubation 50 H9262l of 50 m 7 GTP Sepharose beads GE Healthcare was added to the reaction mixtures After end over end rotation for2hat4 C beads were washed three times with 500 H9262l of LCB 20 mM HEPES pH 7 5 100 mM KCl 0 2 mM EDTA and once with 50 H9262l of LCB containing 1 mM GTP Proteins were eluted with 50 H9262lofLCB containing 1 mM m 7 GTP for 10 min on ice Eluents were separated by 10 SDS PAGE and transferred to a PVDF membrane Millipore and Western blot analysis was performed Primary antibodies used were anti eIF4E Santa Cruz anti eIF4GI Bethyl and anti eIF4A 9 Secondary antibodies were from Jackson Immuno Research Modeling of 4E2RCat to eIF4E Computational solvent mapping 4 of the three dimensional structures of eIF4E has been performed previously 7 and was used to identify the binding site for 4E2RCat The available X ray structures of human eIF4E were downloaded from the Protein Data Bank Mapping was performed after removing all other molecules except for m 7 GTP since 4E2RCat does not compete with 5H11032 mRNA cap structures for binding to eIF4E The atomic coordinates of 4E2RCat were obtained from PubChem but the molecule was considered flexible in docking MGLtools version 1 5 4 was used to prepare the ligand and receptor for docking and the graphical front end for setting up and running the AutoDock docking software AutoDock Vina 1 1 0 with standard settings was used to perform the docking 24 The most likely binding pose for 4E2RCat was selected using an algorithm that scores bound poses based on the degree of overlap between the compound and the atom densities calculated from the solvent mapping results and the most likely binding pose for 4E2RCat was also the lowest energy pose Virus and cell lines Strain 229E of human coronavirus HCoV 229E origi nally was obtained from the American Type Culture Collection ATCC and was grown in the human L132 cell line ATCC CCL5 which was expanded in alpha minimum essential medium H9251 MEM supplemented with 10 vol vol fetal bovine serum FBS L132 cells were infected in triplicate at a multiplicity of infec tion MOI of 0 1 and incubated for2hat33 C in the absence of any compound After two washes with phosphate buffered saline PBS cells were incubated in H9251 MEM supplemented with 1 vol vol FBS and containing DMSO negative control or compounds at the indicated concentrations at 33 C for up to 48 h For poliovirus infections 3 H11003 10 5 HeLa cells were seeded per well in a six well plate the day before infection For virus adsorption cells were washed with PBS and the Mahoney strain of poliovirus type 1 was added at 2 PFU cell in 200 H9262l serum free DMEM Cells were incubated at room temperature for 30 min with gentle rocking followed by the removal of the medium and two washes with PBS At this point fresh DMEM containing 10 heat inactivated FBS containing either vehicle 1 DMSO or 50 H9262M 4E2RCat was added to the cells and cells were incubated for4hat37 C For the last 30 min 35 S methionine 150 H9262Ci ml was added to the wells For harvesting cells were washed with PBS and lysed in PLB PBS 0 1 SDS 0 5 sodium deoxycholate 1 Triton X 100 1 mM PMSF Samples were analyzed on a 10 SDS polyacrylamide gel followed by being stained with Coomassie blue and being destained to ensure equal loading Gels were further incubated for1hinEn 3 Hance Perkin dried and exposed to X Omat film Kodak Quantitation of infectious virus titers by IPA The immunoperoxidase assay IPA was performed on L132 cells as previously described 13 Briefly the primary antibody used was monoclonal antibody MAb 5 11H 6 directed against the S protein of HCoV 229E The secondary antibody was horseradish peroxi dase conjugated goat anti mouse immunoglobulin KPL Immune complexes were detected by incubation with 0 025 wt vol 3 3H11032 diaminobenzidine tetra hydrochloride Bio Rad and 0 01 hydrogen peroxide in PBS and infectious virus titers were calculated by the Karber method as previously described 13 Determination of cell viability To determine cell viability in the presence of 4E2RCat 200 000 L132 cells well were seeded in six well plates 24 h before treatment The next day cells were treated with 12 5 H9262M 4E2RCat for the indicated times after which the cells were processed for annexin V propidium iodide staining To this end cell medium was collected Cells were washed with 1 ml PBS which was collected as well and trypsinized in 200 H9262l 0 05 trypsin EDTA Cells were pooled with previously collected supernatants and spun for 2 min at 2 000 rpm and 4 C The cell pellet was washed with 2 ml cold PBS followed by another spin After the second spin the cell pellet was resuspended in 100 H9262l annexin V binding buffer 10 mM HEPES pH 7 4 140 mM NaCl 2 5 mM CaCl 2 and propidium iodide added to a final concentration of 5 H9262g ml After the addition of 5 H9262l annexin V fluorescein isothiocyanate FITC BD Biosciences samples were incubated for 15 min in the dark at room tempera ture and diluted with 400 H9262l annexin V binding buffer Fluorescence activated cell sorter FACS analyses were performed using a FACScan instrument from BD Biosciences and CELLQUEST software Immunofluorescence assay IFA and semiquantitative analyses to determine percent S protein positive cells L132 9 H11003 10 4 cells were seeded on coverslips and grown overnight in H9251 MEM supplemented with 10 vol vol FBS and infected the next day At specific time points following treatment cells were fixed with 4 paraformaldehyde PFA in PBS for 30 min at room temperature Fixed cells were permeabilized by incubation with cold methanol for 5 min followed by a wash in PBS and the detection of viral antigens The primary antibody was mouse MAb 5 11H 6 and the secondary antibody was Alexa Fluor 488 mouse specific goat antibody Invitrogen After three washes with PBS fixed cells were incubated for 5 min with 4H11032 6H11032 diamidino 2 phenylindole DAPI Sigma Al drich at 1 H9262g ml to stain the DNA in the nucleus To determine the percent viral S protein positive cells 10 fields of L132 cells containing a total of 150 to 250 cells H11003200 magnification with a Nikon Eclipse E800 microscope were counted for each compound tested in three independent experiments Green cells were scored as HCoV 229E S protein positive out of the total amount of cells DAPI stained in blue for each condition 6382 CENCIC ET AL J VIROL on May 1 2015 by Univ of Massachusetts Amherst http jvi asm org Downloaded from RESULTS Characterization of 4E2RCat an inhibitor of eIF4E eIF4G interaction Compound 4E2RCat was identified from an HTS campaign undertaken to identify inhibitors of eIF4E eIF4G interaction Fig 1A 7 The titration of 4E2RCat in a TR FRET based assay monitoring the interaction between eIF4E and eIF4GI revealed a 50 inhibitory concentration IC 50 of 13 5 H9262M Fig 1A The activity of 4E2RCat was tested on the translation of FF hepatitis C virus HCV Ren mRNA a bi FIG 1 Inhibition of cap dependent translation by 4E2RCat A Schematic diagram illustrating the structure of 4E2RCat An 8 point dose response curve of 4E2RCat in a TR FRET assay is provided to the right B Inhibition of translation by 4E2RCat Schematic representation of FF HCV Ren bicistronic construct used for in vitro translation studies top In vitro translations were performed in Krebs extracts programmed with FF HCV Ren in the presence of 35 S methionine and a representative autoradiograph of the products after fractionation on 10 SDS PAGE is provided bottom left Translations contained vehicle 1 DMSO lane 1 500 H9262Mm 7 GDP lane 2 500 H9262M GDP lane 3 50 H9262M anisomycin lane 4 the indicated concentrations of 4E2RCat lanes 5 to 10 or no RNA lane 11 FF and Ren RLU values relative to DMSO controls from two independent experiments are provided with the standard errors of the means SEM indicated bottom right C Schematic representation of FF EMCV Ren bicistronic construct used for in vitro translation studies top RLU values relative to those of the DMSO control from two independent in vitro translations performed in Krebs extract programmed with FF EMCV Ren mRNA are provided with the SEM indicated bottom VOL 85 2011 INHIBITING eIF4F BLOCKS CORONAVIRUS REPLICATION 6383 on May 1 2015 by Univ of Massachusetts Amherst http jvi asm org Downloaded from cistronic mRNA construct that contains a cap dependent fire fly FF cistron and a viral IRES dependent renilla HCV Ren cistron that is not dependent on eIF4F for its initiation Fig 1B The specificity of the bicistronic mRNA was assessed using m 7 GDP as a specific inhibitor and GDP as a nonspecific inhibitor of cap dependent in vitro translation As expected m 7 GDP inhibited the translation of FF but not HCV Ren Fig 1B compare lane 2 to lane 1 whereas GDP had no effect on the translation of either of the two cistrons Fig 1B compare lane 3 to lane 1 The elongation inhibitor anisomycin equally inhibited the translation of the cap dependent FF and IRES dependent Ren cistron compare lane 4 to lane 1 4E2RCat inhibited cap dependent translation in a dose dependent man ner Fig 1B compare lanes 5 to 10 to lane 1 Translation from the HCV IRES was not affected by 4E2RCat except at 100 H9262M suggesting some off target effects at this very high con centration Fig 1B We note that an increase in HCV driven translation is observed upon the inhibition of cap dependent translation at 25 H9262M and likely reflects the increased availabil ity of free ribosomes an effect previously reported with other eIF4F inhibitors 2 3 7 Another IRES the encephalomyo carditis virus EMCV IRES which requires eIF4A and eIF4G but not eIF4E for initiation also was tested and we observed that 4E2RCat inhibited cap dependent FF translation but not EMCV IRES driven Ren translation Fig 1C 4E2RCat blocks interaction of eIF4E with its binding part ners We next investigated the ability of 4E2RCat to inhibit the interaction of eIF4E with its binding partners Fig 2 To this end we performed pulldown assays with eIF4E and either eIF4GI eIF4GII or 4E BP1 Fig 2A We noted that 4E2RCat inhibi