Identification and Mechanism of Action of a Novel Small-Molecule Inhibitor of Arenavirus Multiplication Nhi Ngo, Beatrice Cubitt, Masaharu Iwasaki, Juan C. de la Torre Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California, USA

ABSTRACT

Several arenaviruses cause hemorrhagic fever disease in humans and represent important public health problems in the regions where these viruses are endemic. In addition, evidence indicates that the worldwide-distributed prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is an important neglected human pathogen. There are no licensed arenavirus vaccines and current antiarenavirus therapy is limited to the use of ribavirin that is only partially effective. Therefore, there is an unmet need for novel antiarenaviral therapeutics. Here, we report the generation of a novel recombinant LCM virus and its use to develop a cell-based high-throughput screen to rapidly identify inhibitors of LCMV multiplication. We used this novel assay to screen a library of 30,400 small molecules and identified compound F3406 (chemical name: N-[3,5-bis(fluoranyl)phenyl]-2-[5,7-bis(oxidanylidene)-6-propyl-2-pyrrolidin-1-yl-[1,3]thiazolo[4,5-d]pyrimidin-4-yl]ethanamide), which exhibited strong anti-LCMV activity in the absence of cell toxicity. Mechanism-of-action studies revealed that F3406 inhibited LCMV cell entry by specifically interfering with the pH-dependent fusion in the endosome compartment that is mediated by LCMV glycoprotein GP2 and required to release the virus ribonucleoprotein into the cell cytoplasm to initiate transcription and replication of the virus genome. We identified residue M437 within the transmembrane domain of GP2 as critical for virus susceptibility to F3406. IMPORTANCE

Hemorrhagic fever arenaviruses (HFA) are important human pathogens that cause high morbidity and mortality in areas where these viruses are endemic. In addition, evidence indicates that the worldwide-distributed prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen of clinical significance. Concerns posed by arenavirus infections are aggravated by the lack of U.S. Food and Drug Administration-licensed arenavirus vaccines and current antiarenaviral therapy being limited to the off-label use of ribavirin that is only partially effective. Here we describe a novel recombinant LCMV and its use to develop a cell-based assay suitable for HTS to rapidly identify inhibitors arenavirus multiplication. The concepts and experimental strategies we describe in this work provide the bases for the rapid identification and characterization of novel antiHFA therapeutics.

A

renaviruses are enveloped viruses with a bisegmented negative-strand RNA genome. Each genome segment, large (L; ⬃7.3 kb) and small (S; ⬃3.5 kb) uses an ambisense coding strategy to direct synthesis of two polypeptides in opposite direction, separated by a noncoding intergenic region (IGR) (1, 2). The S segment encodes for the viral nucleoprotein (NP) and the glycoprotein precursor (GPC), which is co- and posttranslationally processed by the signal peptidase and SKI-1/S1P cellular proteases, respectively, to produce the mature surface virion glycoproteins GP1 and GP2 and the stable signal peptide (SSP) that together form the GP1/GP2/SSP complex present at the surface of mature virions and that mediate receptor recognition and cell entry (1, 3). The L segment encodes the viral RNA-dependent RNA polymerase (L polymerase) and the matrix Z protein. Arenaviruses cause chronic infections of rodents with worldwide distribution, where human infections occur through mucosal exposure to aerosols or by direct contact of abraded skin with infectious material. Several arenaviruses cause hemorrhagic fever (HF) disease in humans and pose important public health problems in regions where these viruses are endemic (1, 4–8). Thus, Lassa virus (LASV) infects several hundred thousand people yearly in West Africa, resulting in a high number of Lassa fever cases that are associated with high morbidity and mortality. Likewise, Junin virus (JUNV) causes Argentine HF, a severe illness endemic to Pampas in Argentina, which is characterized by hem-

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orrhagic and neurological manifestations and a fatality rate of 15 to 30% (8). Moreover, mounting evidence indicates that the worldwide-distributed prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen of clinical significance (9–12). In addition, several arenaviruses, including LASV, JUNV, and LCMV, pose a credible biodefense threat (13). Concerns about arenavirus infections of humans are exacerbated due to the lack of U.S. Food and Drug Administrationlicensed vaccines and current antiarenaviral therapy being limited to an off-label use of ribavirin (Rib) that is only partially effective (14–16). Therefore, there is an unmet need to identify novel compounds that could be developed into antiviral drugs to combat human-pathogenic arenaviruses. This task would be facilitated by the development of high-throughput screening (HTS) assays that

Received 22 June 2015 Accepted 17 August 2015 Accepted manuscript posted online 19 August 2015 Citation Ngo N, Cubitt B, Iwasaki M, de la Torre JC. 2015. Identification and mechanism of action of a novel small-molecule inhibitor of arenavirus multiplication. J Virol 89:10924 –10933. doi:10.1128/JVI.01587-15. Editor: S. Perlman Address correspondence to Juan C. de la Torre, [email protected]. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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could rapidly and accurately assess the antiarenaviral activity of compounds present in chemical libraries. Most arenaviruses exhibit a noncytolytic multiplication strategy that prevents the use of virus-induced cytopathic effect-based assays as the foundation for the development of HTS to identify antiviral drug candidates. This obstacle could be overcome by the use of recombinant arenaviruses expressing reporter genes whose expression could provide a rapid and sensitive means to monitor virus multiplication in infected cells. We have developed reversegenetics systems for LCMV (17), as well as for LASV (18) and JUNV (19). These advances in arenavirus molecular genetics have opened new avenues for the development of screening strategies to rapidly identify inhibitors of arenavirus multiplication. Furthermore, we have developed cell-based assays to examine individually each step of the arenavirus life cycle, which facilitates target identification and elucidation of mechanism of action of newly identified inhibitors of arenavirus multiplication (20–26). We document here the generation of a novel recombinant LCMV (rLCMV/GFP-P2A-NP) that expresses green fluorescent protein (GFP) from a bicistronic NP mRNA via the use of the small 2A peptide sequence (27) and its usage to establish a cell-based assay amenable to HTS for the identification of compounds with antiLCMV activity. Using this assay, we screened a library comprised of more than 30,000 compounds and identified a compound, F34062010 (F3406, chemical name: N-[3,5-bis(fluoranyl)phenyl]-2-[5,7bis(oxidanylidene)-6-propyl-2-pyrrolidin-1-yl-[1,3]thiazolo[4,5d]pyrimidin-4-yl]ethanamide), with strong anti-LCMV activity in the absence of cell toxicity as reflected by a high (⬎100) therapeutic index (TI; TI ⫽ CC50/IC50, where CC50 is the compound concentration that causes 50% reduction in cell viability, and IC50 is the 50% inhibitory concentration) in different cell lines tested. Cell-based functional assays indicated that F3406 exerts its antiviral activity by interfering with the GP2-mediated pH-dependent fusion event within the cellular endosomes that releases the virus ribonucleoprotein complex (vRNP) into the cell cytoplasm to complete the virus cell entry process. Genetic selection of viral variants resistant to F3406 and the incorporation of identified mutations into a recombinant LCMV, generated via reverse genetics, identified the methionine (M) residue at position 437 within the transmembrane domain of GP2 as critical for virus susceptibility to F3406. MATERIALS AND METHODS Cells. Vero, A549, and 293T cells were grown in Dulbecco modified Eagle medium (DMEM; Invitrogen, catalog no. 10313-021) containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 ␮g of streptomycin/ml, and 100 U of penicillin/ml. BHK-21 cells were maintained in DMEM with 10% FBS, 2 mM L-glutamine, 100 ␮g of streptomycin/ml, 100 U of penicillin/ml, and 5% tryptose phosphate broth solution (Sigma, catalog no. T8159; 100 ml). Viruses. Recombinant wild-type LCMV (rLCMV/WT) and rLCM viruses expressing vesicular stomatitis virus G (rLCMV/VSV-G), or LASV GPC (rLCMV/LASV-GPC), instead of LCMV GPC, as well as the recombinant WT VSV (VSV/WT) and rVSV expressing GPC of LCMV instead of VSV G (rVSV/LCMV-GPC), has been previously described (17, 21, 23, 24, 28). To generate rLCMV GFP-P2A-NP, we replace the NP open reading frame (ORF) in plasmid moPol-I/Sag (17, 21) by the ORF GFPP2A-NP that contained the ORF of GFP tagged to the N terminus of NP, separated by the 2A peptide sequence derived from porcine teschovirus (PVT1: P2A) (27). Rescue of rLCMV/GFP-P2A-NP was done as described previously (17). To generate rLCMV/GPC(M437I), a PCR fragment

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containing the GPC (M437I) ORF was cloned into Pol-I-Sag plasmid and rLCMV/GPC(M437I) rescued via reverse genetics as described previously (17). Plasmids. PolIMG-CAT, pCAGGS-NP, and pCAGGS-L (22, 29), as well as pCAGGS-LCMV-Z-FLAG (30), have been described previously. Chemical library. The compound library screened consisted of approximately 34,000 small molecules and was purchased from Life Chemicals. The majority of the small molecules contained in the library adhered to Lipinski’s rules (molecular weight ⬍ 500, H-bond donors ⱖ 5, H-bond acceptors ⱖ 10, and logP ⬍ 5) and contain a low proportion of known toxicophores and unwanted functionalities. All compounds have been tested by liquid chromatography-mass spectrometry and/or nuclear magnetic resonance and guaranteed to be at least 90% pure. Compounds were provided as 10 mM stocks in dimethyl sulfoxide (DMSO). For the secondary assay validation, compounds were reordered from the original vendor as powder stocks to confirm that activity of stored DMSO stocks matched the activity when fresh compound stocks were tested. Virus titration. LCMV titers were determined by using an immunofocus assay (IFA) as described previously (31). Briefly, 10-fold serial virus dilutions were used to infect Vero cells in a 96-well plate (3 ⫻ 104 cells/ well) seeded 19 h prior infection. At 20 h postinfection (p.i.), the cells were fixed with 4% paraformaldehyde (PFA). The cells were permeabilized (0.3% Triton X-100 in phosphate-buffered saline [PBS] containing 3% bovine serum albumin [BSA]) and reacted first with a rat monoclonal antibody to NP (VL-4), followed by an Alexa Fluor 568-labeled anti-rat secondary antibody. VSV titers were determined by plaque assay. Growth kinetics. Virus was added to cells at the indicated multiplicity of infection (MOI) in a final volume of 250 ␮l/M24-well. After 90 min of adsorption, the virus inoculum was removed, the cells were washed twice with DMEM–2% FBS, and fresh complete medium was added. At the indicated hours postinfection, tissue culture supernatants (TCS) were collected, and virus titers were determined by using an IFA. Cytotoxicity assay. The compound effect on cell viability was assessed using CellTiter 96 AQueous One Solution reagent (Promega, catalog no. G3580). This method determines the number of viable cells based on the level of formazan product converted from MTS [3-(4,5-dimethylthazol2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolim] by NADPH or NADH generated in living cells. Cells were seeded at 1.8 ⫻ 104 in a 96-well plate and cultured overnight. Cells were treated with the indicated compound concentrations for 48 h before the addition of CellTiter 96 AQueous One Solution reagent. The assay was performed according to the manufacturer’s recommendations, and the absorbance was obtained using an enzyme-linked immunosorbent assay (ELISA) reader (Spectra-Max Plus384; Molecular Devices). Mean values obtained with vehicle (DMSO)-treated cells were set to 100%. LCMV MG assay. An LCMV minigenome (MG) assay was performed as described previously (22, 26). BHK-21 cells were seeded in a 24-well plate to give a confluence of 80% the next day. After 2 h of compound pretreatment, cells were transfected for 5 h with the MG-encoding plasmid (polIMG-CAT, 0.25 ␮g/well), together with pCAGGS expression plasmids for the trans-acting factors NP (60 ng/well) and L (0.3 ␮g/well), using Lipofectamine 2000 reagent (Invitrogen). After 5 h, the transfection medium was replaced with fresh medium containing the compound, and the concentrations are indicated. After 72 h of incubation, cell lysates were prepared to determine the levels of chloramphenicol acetyltransferase (CAT) protein by ELISA. Budding assay. Budding assay was done as described previously (25). 293T cells seeded in a 12-well plate were transfected with 0.2 ␮g of pCAGGS-LCMV-ZFLAG using Lipofectamine 2000. After 5 h, the transfection medium was replaced with fresh medium, and transfected cells were incubated at 37°C for 20 h, prior to the addition of the compound at the indicated concentrations. After 48 h of treatment, virus-like particle (VLP)-containing tissue culture supernatants (TCS) and cells were collected. After clarification from cell debris by centrifugation (1,500 ⫻ g at 4°C for 5 min), VLPs were collected by ultracentrifugation (100,000 ⫻ g at

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4°C for 30 min through a 20% sucrose cushion) and resuspended in PBS. Cells were collected in lysis buffer (1% NP-40, 50 mM Tris-HCl [pH 8.0], 62.5 mM EDTA, 0.4% sodium deoxycholate). Both cell lysates and VLPs were analyzed by Western blotting. Western blotting. Cell lysates and VLP samples were mixed with 4⫻ sodium dodecyl sulfate (SDS) loading buffer (50 mM Tris [pH 6.8], 100 mM dithiothreitol, 2% SDS, 0.1% bromophenol blue, 10% glycerol) and boiled for 5 min. Clarified protein samples were fractionated by SDSpolyacrylamide gel electrophoresis (PAGE) using 4 to 20% gradient polyacrylamide gels and electroblotted onto polyvinylidene difluoride membranes (Immobilon-P transfer membranes; Millipore). To detect FLAG or actin, membranes were incubated with rabbit polyclonal antibody to FLAG (Cayman) or actin (Santa Cruz), followed by incubation with secondary horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG) antibody (Pierce). SuperSignal West Dura chemiluminescent substrate (Thermo Scientific) was used to elicit chemiluminescent signals that were visualized using ImageQuant LAS 4000 (GE Healthcare Life Science). Detection of cell syncytium formation. 293T cells were seeded in a 24-well plate (1.25 ⫻ 105 cells/well) and 18 h later transfected, using Lipofectamine 2000, with pCAGGS expression plasmids for LCMV or LASV GPC or with empty pCAGGS as a control. After 5 h of transfection, the cells were washed, and DMEM containing 10% FBS was added. After 20 h, the cells were treated with compound F3406 (10 ␮M) or vehicle (DMSO) for 3 h, followed by incubation for 15 min with acidified (pH 5.0) DMEM. After treatment with acidified medium, the cells were washed and placed in DMEM containing 10% FBS. Then, 5 h later, the cells were fixed with 4% PFA-PBS, and syncytium formation was visualized by light microcopy.

RESULTS

Generation and characterization of rLCMV/GFP-P2A-NP virus. To rescue rLCMV/GFP-P2A-GFP, we replaced the NP ORF in plasmid pol-I-S (17, 21) by the GFP-P2A-NP sequence that contained the ORF of GFP tagged to the N terminus of NP, separated by the PTV1 2A peptide sequence (P2A) (27). The P2A sequence allowed for production of both GFP and NP proteins from the same bicistronic mRNA transcribed from the NP locus of the S genome segment (Fig. 1A). The rLCMV/GFP-P2A-NP was rescued according to described methods (17). To characterize the newly generated rLCMV/GFP-P2A-NP, we first compared its GFP expression to that of our previously described tri-segmented r3LCMV/GFP that expresses GFP from both the GPC and NP loci (21). GFP expression levels were higher in rLCMV/GFP-P2A-NPinfected cells (Fig. 1B). A comparison of growth kinetics between rLCMV/WT and rLCMV/GFP-P2A-NP showed that in all three cell lines (BHK-21, A549, and Vero) tested, rLCMV/GFP-P2A-NP exhibited slower growth early in the infection, but reached similar peak titers as rLCMV/WT (Fig. 1C). As predicted, Rib inhibited in a dose-dependent manner multiplication of rLCMV/GFP-P2ANP, as determined by the expression of GFP in infected cells (Fig. 1D). Use of rLCMV/GFP-P2A-NP to develop a cell-based assay suitable for HTS to identify inhibitors of LCMV multiplication. We first determined the cell density and MOI conditions that resulted in optimal discrimination between virus-and mock-infected control cells using levels of GFP expression as surrogate of virus multiplication. Both interferon (IFN)-competent (A549) and -deficient (Vero) cell lines were used. We seeded cells at various densities (ranging from 1 ⫻ 104 to 4 ⫻ 104 cells/well) on black-wall, clear-bottom 96-well plates in a total volume of 80 ␮l of DMEM–10% FBS. After 10 min at room temperature, we trans-

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ferred the cells to 37°C and 5% CO2. After 20 h, the cells were infected with rLCMV/GFP-P2A-NP at different MOIs (0.25, 0.5, 1.0, and 2.0) by adding 20 ␮l of the required amount of virus in DMEM–2% FBS. At 40 h p.i., the cells were fixed with 4% PFAPBS for 25 min at room temperature, washed once with PBS, and stained with DAPI (4=,6=-diamidino-2-phenylindole; 1 ␮g/ml in buffer [0.3% Triton X-100 in PBS containing 3% BSA]) for 15 min at room temperature. After DAPI staining, we washed the cells once with PBS and then left them in PBS. We determined GFP expression using the Advanced Hybrid microplate reader Synergy H4. For Vero cells, optimal conditions (S/B ⫽ 16 and Z= ⫽ 0.68, when comparing untreated and Rib-treated infected cells) corresponded to a seeding cell density of 3 ⫻ 104 cells/well and an MOI of 0.25. For A549, optimal conditions (S/B ⫽ 4 and Z= ⫽ 0.61, when comparing untreated and Rib-treated infected cells) corresponded to a seeding cell density of 2 ⫻ 104 cells/well and an MOI of 1. Library screen. To assess the performance of our newly developed cell-based assay, we conducted a pilot screen using a Life Chemical library containing 30,400 compounds to identify inhibitors of rLCMV/GFP-P2A-NP multiplication, as determined by reduced levels of GFP expression in compound treated compared to vehicle treated and rLCMV/GFP-P2A-NP-infected cells (Fig. 2A). We use Rib (100 ␮M), a validated inhibitor of LCMV multiplication, as positive control. The primary screen was conducted at one single compound concentration (10 ␮M) and primary hits were selected based on low toxicity (cell biomass ⬎ 60%, as determined by DAPI staining) and strong (⬎50%) inhibition of GFP expression compared to vehicle-treated and infected cells (Fig. 2B). Primary hits were rescreened using the same criteria, and reconfirmed hits were selected and retested using fresh solutions prepared using reordered compounds. Freshly made compound stocks were tested using the same screening process as in the primary screen, but hits were selected using more stringent criteria: biomass ⬎ 90% and GFP inhibition ⬎ 90% compared to vehicletreated cells. This resulted in the identification of two hits, F34062010 (F3406) and F2927-0885 (F2927) (Fig. 2C), which also exhibited anti-LCMV activity in the IFN-deficient Vero cells (Fig. 2D), indicating that their anti-LCMV activity was unrelated to induction of the IFN response. We selected compound F3406 for further studies aimed at investigating mechanisms of action. Dose-dependent effect of F3406 on LCMV multiplication. We compared the dose-dependent effect of F3406 and Rib on rLCMV/GFP-P2A-NP propagation and production of infectious progeny in A549, Vero, and BHK-21 cells. We pretreated cells 2 h prior to infection with rLCMV/GFP-P2A-NP with F3406 or Rib and maintained the compounds throughout the infection time. F3406 exhibited stronger inhibitory effect than Rib on both the propagation (Fig. 3A) and the production of infectious progeny (Fig. 3B) of LCMV in all of the cell lines tested. In A549, BHK-21, and Vero cells, F3406 had IC50s (␮M) of 0.4, 0.7, and 0.9, respectively (Fig. 3C), whereas at concentrations below 50 ␮M F3406 did not have significant effects on the viability of any of the cell lines tested (Fig. 3D), which resulted in an TI values of ⬎250 (A549 cells), 106 (BHK-21 cells), and ⬎111 (Vero cells). Steps of the LCMV life cycle affected by F3406. To gain insights about the mechanisms by which F3406 exerted its antiLCMV activity, we examined the effect of F3406 on distinct steps of the LCMV life cycle. We first examined whether F3406 directly affected viral RNA synthesis. For this, we used an

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G S G A T N F S L L K Q A G D V E E N P G P GGA TCC GGA GCC ACG AAC TTC TCT CTG TTA AAG CAA GCA GGA GAC GTG GAA GAA AAC CCC CGT CCT P2A

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FIG 1 Generation and characterization of rLCMV/GFP-P2A-NP. (A) Schematic of rLCMV/GFP-P2A-NP. The rLCMV/GFP-P2A-NP was generated as described in Materials and Methods. (B) Comparison of rLCMV/GFP-P2A-NP and r3LCMV/GFP propagation in cultured cells. Vero cells were infected with either the rLCMV/GFP-P2A-NP or r3LCMV/GFP at an MOI of 0.01 or 0.1; at 36 h p.i., the cells were fixed with 4% PFA, and GFP expression was assessed by fluorescence microscopy. The panels show representative fields using the same magnifications and exposure times. (C) Comparison of rLCMV/GFP-P2A-NP and rLCMV/WT growth kinetics in cultured cells. BHK-21, A549, and Vero cells were infected with rLCMV/WT or rLCMV/GFP-P2A-NP at an MOI of 0.01 or 0.1. At the indicated times, TCS were collected, and virus titers were determined. The data represent the means ⫾ the standard deviations (SD) of two independent experiments. Each independent experiment consisted of three replicates. (D) Inhibition of rLCMV/GFP-P2A-NP by Rib. Cells were treated with Rib at the indicated concentrations and infected with rLCMV/GFP-P2A-NP (MOI ⫽ 0.01). At 36 h p.i., the cells were fixed with 4% PFA, and GFP expression was examined by fluorescence microscopy. Nuclei were visualized by DAPI staining. The limit of detection (LoD; shown as dashed lines) indicates the threshold of level of detection of infectious virus.

LCMV minigenome (MG) assay that recapitulates the biosynthetic processes of RNA replication and gene transcription directed by the virus polymerase complex (22, 26). At 10 ␮M, F3406 caused a slight decrease on MG activity compared to vehicle (DMSO) (Fig. 4A), but this effect could not account for the strong inhibition of LCMV multiplication observed in cells treated with 10 ␮M F3406. We next examined the effect of F3406 on LCMV budding, a process directed by the LCMV Z protein (1, 25, 32). For this we used a previously described assay that is based on transfection of cells with a Z-expressing plasmid and quantification of Z present in VLPs collected from the TCS of transfected cells (25). F3406 did not affect Z budding efficiency (Fig. 4B). These results indicated that F3406 was likely interfering with LCMV cell entry. To examine this, we

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performed a time of addition experiment (Fig. 4C). Addition of F3406 prior or at the time of infection resulted in strong inhibition of LCMV multiplication during the first round of infection. In contrast, addition of F4306 1 h after infection resulted only in a slight decrease in LCMV multiplication. Since LCMV cell entry is mostly completed within 60 min, these results indicated that F3406 likely affects early stages of cell entry. To assess the specificity of the effects of F3406 on LCMV entry, we examined the compound effects on cell entry of a recombinant VSV expressing either its homologous surface glycoprotein G (rVSV/WT) or the GPC from LCMV (rVSV/LCMV-GPC), as well as on a rLCMV expressing either its homologous GPC (rLCMV/WT) or G from VSV (rLCMV/VSV-G). F3406 exhibited a strong dose-dependent inhibition of rLCMV/WT and

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FIG 2 Cell-based HTS using rLCMV/GFP-P2A-NP to identify inhibitors of LCMV multiplication. (A) Screening flow chart. (B) Summary of results from the

library screening. (C) Anti-LCMV activity of the top two identified hits F2927 and F3406. The data represent the means ⫾ the SD of two independent experiments. Each independent experiment consisted of three replicates. (D) Structures of F2927 and F3406.

rVSV/LCMV-GPC, whereas cell entry of rVSV/WT and rLCMV/VSV-G were not affected by F3406 (Fig. 4D). Effect of F3406 on pH-dependent membrane fusion mediated by GP2. Arenaviruses enter cells via receptor-mediated endocytosis (2, 33–35). Once the virus is delivered to the endosome, GP2 mediates a pH-dependent fusion between viral and cellular membranes that releases the virus ribonuclear protein (vRNP) to the cell cytoplasm. To determine the effect of F3406 on LCMV GP-mediated membrane fusion, we examined whether treatment with F3406 prevented GP-mediated cell fusion following treatment with low pH. For this we transfected 293T cells with a plasmid expressing LCMV GPC and after treatment with F3406, or vehicle control, the cells were exposed to a pH of 5.0 for 15 min (Fig. 5). Cells transfected with LCMV GPC and exposed to acidified medium developed prominent syncytia that was prevented by prior treatment with F3406. Effect of F3406 on virion infectivity. We next examined whether the treatment of cell-free LCMV virions with F3406 resulted in inhibition of virus infectivity. We treated 2 ⫻ 105 focusforming units (FFU) of rLCMV/WT and VSV/WT with F3406 (2 and 10 ␮M), or vehicle control, for 30 min at 37°C and 5%CO2 in DMEM–2% FBS. Compound-treated virus samples were diluted 1,000-fold to reduce the concentration (⬍10 nM) of F3406 to levels at which it did not exhibit noticeable anti-LCMV activity. These diluted samples were used to infect (MOI ⫽ 0.01) BHK-21 in the absence of F3406. At 24 h p.i., the cells were fixed and stained for either LCMV NP or VSV N. Treatment of rLCMV/ WT, but not of VSV/WT, virions with F3406 resulted in signif-

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icantly reduced numbers of infected cells (Fig. 6). These findings suggested that F3406 binds with a low dissociation rate (Koff) to the prefusion GP complex present in LCMV virions and that this interaction interfered with the GP2-mediated fusion activity. Selection of F3406-resistant LCMV variants. To gain further information about the viral gene product targeted by F3406, we conducted experiments aimed at selecting viral variants resistant to F3406. For this, we conducted serial passages (MOI ⫽ 0.1) of rLCMV/WT in BHK-21 cells in the presence of F3406 (5 ␮M). TCS were collected from each passage, and the corresponding virus titers were determined. After three passages (P3) under constant selection with F3406, we observed the emergence of an F3406-resistant viral population, called rLCMV(F3406)R (Fig. 7A). The drug-resistant phenotype of rLCMV(F3406)R was stable through passages P3 to P5 (Fig. 7A). We did not observe significant differences in growth kinetics and peak titers of between rLCMV/WT and P5-rLCMV/WT(F3406)R in BHK-21 cells infected at an MOI of 0.1 in the absence of F3406 (Fig. 7B), indicating that the selection of rLCMV/WT(F3406)R did not result in a virus with altered fitness compared to the parental rLCMV/WT. To identify the viral genetic determinants responsible for resistance to F3406, we isolated RNA from cells infected with rLCMV(F3406)R and used reverse transcription-PCR (RT-PCR) to amplify the complete GPC gene, which sequence analysis revealed a single mutation, M437I, located with the N terminus of the transmembrane domain on GP2 (36, 37) (Fig. 7C). To confirm that no additional mutations within the genome of

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FIG 3 Effect of F3406 on virus propagation and production of infectious progeny. (A) Propagation of rLCMV/GFP-P2A-NP. BHK-21, A549, and Vero cells

were treated with the indicated concentrations of either Rib or F3406 starting 2 h prior infection with rLCMV/GFP-P2A-NP (MOI ⫽ 0.1). At 30 h p.i., the cells were fixed (4% PFA), and GFP expression was assessed by fluorescence microscopy. Nuclei were visualized by DAPI staining. (B) TCS samples from panel A were collected at 0 and 30 h p.i., and virus titers were determined. Titers obtained at 0 h p.i. were in all cases less than the LoD. The data represent the means ⫾ the standard deviation (SD) of two independent experiments. Each independent experiment consisted of three replicates. (C) Determination of IC50. Cells (104 cells/96-well plate) were treated with F3406 at the indicated concentrations, or vehicle treated as control, and infected with rLCMV/WT (70 FFU/well). At 18 h p.i., the cells were fixed, and NP expression was detected by immunofluorescence using a rat monoclonal antibody (VL4) to NP. The numbers of infected cells were normalized (%), considering as 100% the values for NP⫹ cells in vehicle-treated cells. The data represent the means (normalized) ⫾ the SD of two independent experiments. Each independent experiment consisted of three replicates. (D) Cell toxicity of F3406. BHK-21, A549, and Vero cells were treated with 2, 10, 25, 50, and 100 ␮M F3406 for 48 h, and the cell toxicity was determined using the CellTiter 96 AQueous One solution reagent. The results were normalized to values obtained with vehicle (DMSO)-treated cells. The data represent the means ⫾ the SD of two independent experiments. Each independent experiment consisted of four replicates.

rLCMV(F3406)R contributed to the drug resistant phenotype, we used reverse genetics to generate a rLCMV containing the single M437I mutation within GP2 (rLCMV(M437I)) and compared it with rLCMV(F3406)R. Both rLCMV/GPC(M437I) and rLCMV(F3406)R exhibited similar resistance to F3406 as determined by their ability to grow to similar titers in the absence and presence (10 ␮M) of F3406, whereas rLCMV/WT multiplication was abrogated in the presence of 10 ␮M F3406 (Fig. 7D). Effects of F3406 on cell entry mediated by LASV GPC. LASV has a great impact in public health in West Africa. The lack of LASV vaccines and currently very limited therapeutic options to treat LASV infections, underscore the importance to identify and

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characterize compounds that could be developed into antiviral drugs against LASV. Hence, our interest to examine whether F3406 was also able to inhibit cell entry mediated by LASV GPC. Inspection of the LASV GPC sequence revealed a valine (V) residue at the GP2 position corresponding to LCMV M437. Because the structural similarities between I and V residues, we reasoned that, unfortunately, F3406 would not have an effect on LASV GPC. To test this, we examined the effect of F3406 on cell entry of an rLCMV where LASV GPC substituted for LCMV GPC (rLCMV/ LASV-GPC) (28). BHK-21 cells were treated with vehicle (DMSO) or F3406 (2 ␮M or 10 ␮M) starting 2 h before infection (MOI ⫽ 0.1) and maintained throughout the infection. At 24 h p.i., we collected TCS and determined virus titers. F3406 did not affect rLCMV/

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were transfected with pCAGGS-GPC, or empty pCAGGS as a control, and treated 20 h later with compound F3406 (10 ␮M) or vehicle (DMSO) for 3 h, followed by incubation for 15 min with acidified (pH 5.0) DMEM. After treatment with acidified medium, the cells were washed and placed in DMEM containing 10% FBS. After 5 h, the cells were fixed with 4% PFA-PBS, and syncytium formation visualized by using light microcopy. The panels correspond to representative fields of results observed in three independent experiments.

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FIG 4 Effect of F3406 on different steps of the LCMV life cycle. (A) Effect of F3406 on the LCMV minigenome rescue assay. BHK-21 cells were pretreated with F3406 for 2 h prior to transfection with the MG rescue plasmids. After 5 h, the transfection medium was replaced with DMEM–2% FBS medium containing the appropriate concentration of compound. At 72 h posttransfection, cell lysates were prepared, and the levels of CAT protein were determined by ELISA. The data represent the means (normalized) ⫾ the SD of two independent experiments. Each independent experiment consisted of two replicates, and each CAT value determination by ELISA was performed in triplicate. (B) Effect of F3406 on Z budding. 293T cells were transfected with pC-ZFLAG and treated 24 h later with the indicated compound concentrations. At 24 h after treatment, VLPs were collected from TCS. (i) Both VLP and cell lysate samples were analyzed by Western blotting with antibodies to FLAG and actin. (ii) Western blot signals were quantified using ImageQuant, and the expression levels of actin were used to normalize the amount of protein loaded for each cell lysate. Budding efficiency was determined as Z (VLP)/Z (VLP⫹ cell lysate). The data represent the means (normalized) ⫾ the SD of two independent experiments. (C) Effect of F3406 on cell entry. BHK-21 cells were treated with the indicated F3406 concentrations starting at 2 h prior infection, at the start of infection, or 1 h after infection with LCMV/WT (MOI ⫽ 0.1). At 16 h p.i., the cells were fixed and stained with an antibody to LCMV NP. (D) F3406 affects specifically cell entry mediated by GPC of LCMV. BHK-21 cells were pretreated 2 h with vehicle (DMSO) or F3406 prior to infection with either rVSV/WT (MOI ⫽ 0.01), rVSV/LCMV-GPC (MOI ⫽ 0.01), rLCMV/WT (MOI ⫽ 0.1), or rLCMV/VSV-G (MOI ⫽ 0.01). After 90 min of adsorption, the cells were washed once, and medium containing the indicated concentration of F3406 was added. At 24 h p.i., TCS were collected, and virus titers were determined by plaque assay (VSV) or immunofocus assay (LCMV). The data represent the means ⫾ the SD of two independent experiments. Each independent experiment consisted of triplicate replicates.

LASV GPC multiplication (Fig. 8A). Consistent with this result, F3406 did not affect infectivity of rLCMV/LASVGPC virions (Fig. 8B). Likewise, treatment with F3406 of cells transfected with LASV GPC did not prevent cell fusion after the exposure of transfected cells to acidified medium (Fig. 8C).

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In this study we have documented the generation of a novel recombinant LCMV, rLCMV/GFP-P2A-NP, which facilitated rapid identification of inhibitors of LCMV infection in the context of a cell-based HTS format. The rLCMV/GFP-P2A-NP expressed GFP and the virus NP from the same bicistronic mRNA, and therefore GFP expression levels served as an accurate surrogate of levels of virus multiplication in infected cells (Fig. 1A). The robust GFP expression in rLCMV/GFP-P2A-NP-infected cells provided us with a large dynamic range to identify inhibitors of LCMV multiplication (Fig. 1B). We observed that rLCMV/GFP-P2A-NP exhibited an early slightly delayed growth kinetics compared to rLCMV/WT, but rLCMV/GFP-P2A-NP reached similar peak titers as rLCMV/WT (Fig. 1C). These early growth kinetic differences between rLCMV/GFP-P2A-NP and rLCMV/WT were more noticeable in A549 cells infected at a low MOI (0.01). This might reflect that rLCMV/GFP-P2A-NP had a very modest fitness decrease that was magnified in A549 cells because the presence of a fully active type I interferon pathway. We identified compound F3406 as a potent inhibitor of LCMV infection (Fig. 3), and results from studies using a multitude of cell-based functional assays for different steps of the life cycle of

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incubated in DMEM–2% FBS containing 2 or 10 ␮M F3406, or DMSO vehicle. After 30 min treatment at 37°C and 5% CO2, viral samples were diluted and used to infect BHK-21 cells (MOI ⫽ 0.1). After 90 min of adsorption, the cells were washed once, and the transfection medium was replaced with DMEM–2% FBS without compound. At 20 h p.i., the cells were fixed and stained with specific antibodies to either LCMV NP or VSV N. The numbers within parentheses indicate the percentages of LCMV NP-positive cells in cells infected with rLCMV/WT. We did not observe significant differences in the numbers of VSV N-positive cells in cells infected with rVSV/WT treated with F3406 at the indicated concentrations.

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FIG 7 Selection and characterization of F3406-resistant LCMV. (A) Serial passages of rLCMV/WT in the presence or F3406 (5 ␮M). For each passage, TCS collected at 48 h p.i. were used to infect a fresh monolayer of BHK-21 cells. Starting at P3, virus recovered from F3406-treated cells exhibited resistance to F3406 and was termed rLCMV(F3406)R. (B) Comparison of growth kinetics of rLCMV(F3406)R and LCMV/WT. P3 and P5 of both rLCMV(F3406)R and LCMV/WT were used to infect (MOI ⫽ 0.01) BHK-21 cells, and at the indicated times postinfection the TCS were collected, and virus titers were determined. (C) Partial amino acid sequence of rLCMV(F3406)R GPC. RNA was isolated from P3 of cells infected in the presence of F3406 and subjected to RT-PCR to amplify the complete GPC ORF. The PCR fragment was purified and sequenced, which revealed a single amino acid substitution, M437I, in GP2. (D) Generation and characterization of rLCMV/GPC(M437I). The rLCMV/GPC(M437I) was rescued via reverse genetics as described previously (17). BHK-21 cells were pretreated with F3406 for 2 h and infected with rLCMV/WT, rLCMV(F3406)R, or rLCMV/GPC(M437I) (MOI ⫽ 0.1). After 90 min of adsorption, the cells were washed once, and the transfection medium was replaced with DMEM–2% FBS with or without compound. At 24 h p.i., the TCS were collected, and virus titers were determined. The data represent the means ⫾ the SD of two independent experiments. Each independent experiment consisted of two replicates.

LCMV, together with results from time of addition experiments, indicated that the inhibitory activity of F4306 on LCMV infection was related to the compound’s ability to interfere with the GP2mediated pH-dependent fusion event required to release the vRNP into the cell cytoplasm to initiate replication and expression of the virus genome (Fig. 4, 5, and 6). It is worth noting that for all different cell types tested, F3406 exhibited a more potent antiLCMV activity than Rib, the current gold standard in antiarenaviral drugs, which has also been shown to have a varying antiviral efficacy depending on the cell line used (38). Selection and characterization of a LCMV variant resistant to F3406, rLCMV(F3406)R identified mutation M437I within GP2 as responsible for viral resistance to F3406, which we unequivocally confirmed by showing that rLCMV/GPC(M437I), generated via reverse genetics and containing only the M437I mutation in GP2, was resistant to F3406 (Fig. 7). M437 is located within the transmembrane domain of GP2 that has been implicated in the SSPGP2 interactions that play critical roles in maintaining the prefu-

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BHK21-cells were pretreated with the indicated F3406 concentrations for 2 h and then infected with rLCMV/LASVGPC (MOI ⫽ 0.01). After 90 min of adsorption, the cells were washed once, and DMEM–2% FBS was added with the indicated F3406 concentration. At 24 h p.i., TCS were collected, and virus titers were determined. The data represent the means ⫾ the SD of two independent experiments. Each independent experiment consisted of two replicates. (B) rLCMV/LASVGPC (2 ⫻ 105 FFU) were incubated in DMEM–2% FBS containing 2 or 10 ␮M F3406, or vehicle (DMSO) as control, for 30 min at 37°C and 5% CO2. Samples were diluted 35-fold and used to infect BHK-21 cells (MOI ⫽ 0.1). After 90 min of adsorption, the cells were washed once, and the transfection medium was replaced with DMEM–2% FBS without compound. At 20 h p.i., the cells were fixed and stained with an antibody to LCMV NP. Panels correspond to representative fields of results observed in three independent experiments. The number in parentheses indicates the percentage of NP-positive cells in each case. (C) Cells were transfected with LASV GPC and treated with F3406, or vehicle (DMSO), prior to exposure to acidified DMEM. Panels correspond to representative fields of results observed in three independent experiments.

sion state of GPC and its response to acidic pH (1, 36, 37, 39–41). LCMV GPC-transfected cells transiently treated with F3406 prior treatment with acidified medium did not form syncytia (Fig. 5), and cell-free virions treated with F3406 lost the ability to infect cells (Fig. 6). Because F3406 was washed out prior to the low pH pulse, these findings suggest that F3406 inhibits LCMV cell entry by binding tightly to GP2 and stabilizing a prefusion state of GPC within the acidic environment of the endosome. F3406 differs chemically from several distinct classes of compounds that have been previously shown to inhibit arenavirus GPC-mediated membrane fusion by binding to a common site on GPC and stabilizing the prefusion GPC complex (39, 42–45). Interestingly, previous work by Larson et al. showed that arenavirus sensitivity or resistance to the cell entry inhibitor ST-193 correlated with the presence of V or M residues, respectively, at a GP2 position corresponding to M437 in LCMV (43). Thus, mutation V421A in TCRV GP2 (the equivalent position to LCMV GP2 M437) resulted in a 2,000-fold increase in the IC50 of ST-193 for TCRV GPC, and mutation of V to M in the equivalent LASV GP2 residue resulted in a 850-fold increase in the IC50 of ST-193 for LASV GPC (43). Intriguingly, we have observed a reversed correlation for compound F3406. The elucidation of the molecular and atomic requirements for the distinct effects of ST-193 and F3406 on GPC-mediated membrane fusion, which has been shown to be dependent on the amino acid residue present at LCMV GP2 437, remain to be determined and will contribute to a better understanding of arenavirus GPC-mediated membrane fusion. This knowledge would facilitate the development of potent and broadspectrum cell entry inhibitors that will help combat humanpathogenic arenaviruses. We have generated and characterized rLCMV expressing the GPC of LASV (24) and the live attenuated Candid1 strain of JUNV

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(46). Therefore, a similar strategy to the one we described here using rLCMV/GFP-P2A-NP can be implemented for large HTS aimed at identifying inhibitors of LASV and JUNV GPC-mediated cell entry. The rLCMV expressing the arenavirus GPC of interest can be easily expanded to generate large quantities of high titer stocks, which represents an advantage over the use of pseudotyped retroviral particles. In addition, sharing the same LCMV replication machinery would facilitate the corresponding counterscreen to distinguish between inhibitors that specifically inhibit LASV or JUNV, or both, GPC-mediated cell entry from those that inhibit LCMV RNA replication and gene expression. ACKNOWLEDGMENTS This research was supported by NIH/NIAID grants AI047140 and AI077719 (J.C.D.L.T.) and by the Japan Society for the Promotion of Science, the Daiichi Sankyo Foundation of Life Science, and the KANAE Foundation for the Promotion of Medical Science (M.I.).

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35. Radoshitzky SR, Abraham J, Spiropoulou CF, Kuhn JH, Nguyen D, Li W, Nagel J, Schmidt PJ, Nunberg JH, Andrews NC, Farzan M, Choe H. 2007. Transferrin receptor 1 is a cellular receptor for New World haemorrhagic fever arenaviruses. Nature 446:92–96. http://dx.doi.org/10.1038 /nature05539. 36. Eschli B, Quirin K, Wepf A, Weber J, Zinkernagel R, Hengartner H. 2006. Identification of an N-terminal trimeric coiled-coil core within arenavirus glycoprotein 2 permits assignment to class I viral fusion proteins. J Virol 80:5897–5907. http://dx.doi.org/10.1128/JVI.00008-06. 37. Igonet S, Vaney MC, Vonrhein C, Bricogne G, Stura EA, Hengartner H, Eschli B, Rey FA. 2011. X-ray structure of the arenavirus glycoprotein GP2 in its postfusion hairpin conformation. Proc Natl Acad Sci U S A 108:19967–19972. http://dx.doi.org/10.1073/pnas.1108910108. 38. Shah NR, Sunderland A, Grdzelishvili VZ. 2010. Cell type mediated resistance of vesicular stomatitis virus and Sendai virus to ribavirin. PLoS One 5:e11265. http://dx.doi.org/10.1371/journal.pone.0011265. 39. York J, Dai D, Amberg SM, Nunberg JH. 2008. pH-induced activation of arenavirus membrane fusion is antagonized by small-molecule inhibitors. J Virol 82:10932–10939. http://dx.doi.org/10.1128/JVI.01140-08. 40. York J, Nunberg JH. 2006. Role of the stable signal peptide of Junin arenavirus envelope glycoprotein in pH-dependent membrane fusion. J Virol 80:7775–7780. http://dx.doi.org/10.1128/JVI.00642-06. 41. York J, Nunberg JH. 2009. Intersubunit interactions modulate pHinduced activation of membrane fusion by the Junin virus envelope

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