Article Development and Validation of a Novel Dual Luciferase Reporter Gene Assay to Quantify VP24 Inhibition of IFN Signaling

Elisa Fanunza 1 ID , Aldo Frau 1, Marco Sgarbanti 2, Roberto Orsatti 2, Angela Corona 1 ID and Enzo Tramontano 1,3,* ID

1 Department of Life and Environmental Sciences, University of Cagliari, 09124 Cagliari, Italy; [email protected] (E.F.); [email protected] (A.F.); [email protected] (A.C.) 2 Department of Infectious Diseases, Istituto Superiore di Sanità, 00161 Rome, Italy; [email protected] (M.S.); [email protected] (R.O.) 3 Genetics and Biomedical Research Institute, National Research Council, 09042 Monserrato, Italy * Correspondence: [email protected]; Tel.: +39-070-6754538

Received: 11 January 2018; Accepted: 22 February 2018; Published: 24 February 2018

Abstract: The (IFN) system is the first line of defense against viral infections. Evasion of IFN signaling by Ebola 24 (VP24) is a critical event in the pathogenesis of the infection and, hence, VP24 is a potential target for drug development. Since no drugs target VP24, the identification of molecules able to inhibit VP24, restoring and possibly enhancing the IFN response, is a goal of concern. Accordingly, we developed a dual signal firefly and Renilla luciferase cell-based drug screening assay able to quantify IFN-mediated induction of Interferon Stimulated Genes (ISGs) and its inhibition by VP24. Human Embryonic Kidney 293T (HEK293T) cells were transiently transfected with a luciferase reporter gene construct driven by the promoter of ISGs, Interferon-Stimulated Response Element (ISRE). Stimulation of cells with IFN-α activated the IFN cascade leading to the expression of ISRE. Cotransfection of cells with a plasmid expressing VP24 cloned from a virus isolated during the last 2014 outbreak led to the inhibition of ISRE , quantified by a luminescent signal. To adapt this system to test a large number of compounds, we performed it in 96-well plates; optimized the assay analyzing different parameters; and validated the system by calculating the Z0- and Z-factor, which showed values of 0.62 and 0.53 for IFN-α stimulation assay and VP24 inhibition assay, respectively, indicative of robust assay performance.

Keywords: Ebola virus VP24; innate immunity; IFN signaling; IFN inhibition; dual luciferase gene reporter assay; drug development

1. Introduction Ebola virus (EBOV) causes sporadic outbreaks of severe hemorrhagic fever in humans and nonhuman primates with fatality rates as high as 90%. The EBOV outbreak in 2014 is one of the largest viral outbreaks in history and the first in West Africa, causing over 11,000 deaths in Africa and other parts of the world [1]. EBOV is an enveloped negative-sense single-stranded RNA virus that belongs to the family of Filoviridae. Although the molecular determinants of EBOV pathogenesis remain still incompletely defined, the ability of the virus to counteract early antiviral responses, particularly the antagonism of the type I interferon (IFN-I) response, is thought to contribute to its high virulence [2–4]. The IFN system is the first line of defense against viral infections. It begins with recognition of a pathogen-associated molecular pattern (PAMP) by pattern recognition receptors (PRRs), whose activation leads to the production of IFN-I (IFN-α/β). Binding of IFN-I to the cell surface IFN-I receptor (IFNAR) initiates a signaling cascade that results in the activation and phosphorylation

Viruses 2018, 10, 98; doi:10.3390/v10020098 www.mdpi.com/journal/viruses Viruses 2018, 10, 98 2 of 12 of the Janus kinases, Jak1 and Tyk2, and the Signal Transducer and Activator of Transcriptions 1 and 2 (STAT1 and STAT2). Phosphorylated STAT1 (P-STAT1) either dimerizes or forms a heterotrimeric transcriptional complex with STAT2 and the IFN Regulatory Factor 9 (IRF9), named IFN-Stimulated Gene Factor 3 (ISGF3), and is subsequently transported to the nucleus via karyopherin-α (KPNA) where it regulates the expression of hundreds of IFN-stimulated genes (ISGs) that target specific aspects of the viral life cycle [5]. Among the seven major EBOV gene products, two viral proteins have been shown to suppress host IFN response. Viral protein 35 (VP35) blocks the IFN production cascade by binding double-stranded (ds)RNA and shielding it from recognition by host immune sensors such as the Retinoic acid-Inducible Gene I (RIG-I) and the Melanoma Differentiation-Associated protein 5 (MDA-5) [4,6,7]; in addition, it also interferes with the activation of the IFN regulatory factor 3 (IRF3) [7–11] and prevents RIG-I interaction with the Protein kinase R activator, PACT [12,13]. Viral protein 24 (VP24) inhibits signaling downstream of both IFN-α/β and IFN-γ by sequestering KPNA proteins (α1, α5, and α6) and preventing nuclear transport of activated P-STAT1 [14–16]. In addition, VP24 also binds directly to P-STAT1 [17]. EBOV evasion of IFN signaling by VP24 is a critical event in the pathogenesis of the infection. Mutations in VP24 correlated with IFN evasion are responsible for the acquisition of high virulence in animal models [18]. Being a key factor in EBOV virulence, VP24 is a potential target for the development of new drugs. In the absence of approved drugs specific for VP24, the identification of molecules able to inhibit VP24, restoring and possibly enhancing the IFN response, is a goal of concern. We describe here the development of a novel artificial dual cell-based gene reporter screening assay able to quantify IFN induction and its inhibition by VP24. Human Embryonic Kidney 293T (HEK293T) cells transiently express the human Interferon-Stimulated Response Element (ISRE) driving a luciferase reporter gene. Stimulation with human recombinant IFN-α activates the IFN signaling cascade leading to the expression of ISGs genes. This system may be a suitable cellular model to reproduce the effect of VP24 on IFN signaling and to test VP24 inhibitors. The enzymatic activity of firefly luciferase provides a susceptible, stable, and rapid means to quantify transcriptional activity of ISRE. We optimized the assay, evaluating different parameters to achieve an excellent signal. Further, the normalization with a Renilla luciferase control allows us to minimize variability between experiments, providing high reproducibility of our dual drug screening assay.

2. Materials and Methods

2.1. Cells and Reagents HEK293T cells (ATCC® CRL-3216™) were grown in Dulbecco’s modified Eagle’s medium (Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/ streptomycin (Sigma, St. Louis, MO, USA). Cells were incubated at 37 ◦C in a humidified 5% CO2 atmosphere. Plasmid pISRE-luc was kindly provided by Prof. Ian Goodfellow. Plasmid pcDNA3-V5-VP24 was a kind gift of Dr. St Patrick Reid. pcDNA3-NS1 was kindly given by Prof. Stephan Ludwig. pRL-TK was purchased from Promega (Madison, WI, USA). T-Pro P-Fect Transfection Reagent was from T-Pro Biotechnology (Taipei, Taiwan R.O.C.). Human Recombinant IFN-α was purchased from PeproTech (London, UK). Mouse monoclonal anti-FLAG M2 was obtained from Sigma, and rabbit monoclonal anti-GAPDH and HRP-linked anti-rabbit IgG were purchased from Cell Signaling (Danvers, MA, USA). HRP-linked anti-mouse IgG was provided by Invitrogen (Carlsbad, CA, USA). Pierce ECL Western Blotting Substrate was from Thermo Fisher Scientific (Waltham, MA, USA).

2.2. Construction of EBOV VP24 Mammalian Expression Plasmid The EBOV VP24 cDNA was obtained by de novo gene synthesis (GenScript USA Inc. Piscataway, NJ, USA) using the sequence of VP24 from the isolate H.sapiens-wt/ GIN/2014/Gueckedou-C05, Sierra Leone/Guinea. The FLAG epitope, present in frame at the NH2 terminus of VP24 sequence, and the HindIII and EcoRV restriction sites at both ends, used for the Viruses 2018, 10, 98 3 of 12 subcloning in the pcDNA3.1(+) mammalian expression vector (Invitrogen), were also part of the entire sequence obtained by de novo gene synthesis.

2.3. Luciferase Reporter Gene Assay HEK293T cells were transfected using T-Pro P-Fect Transfection Reagent, according to the manufacturer’s protocol. Cells (1.5 × 104 cells/well) were seeded in 96-well plates 24 h before transfection. Plasmids pISRE-luc, pRL-TK (Promega) were mixed in reduced serum medium Optimem (Gibco) and incubated with transfection reagent for 20 min at room temperature. Transfection complexes were then gently added into individual wells of the 96-well plate. Twenty-four hours after transfection, cells were stimulated with Human Recombinant IFN-α and incubated for 8 h at 37 ◦C in 5% CO2. Cells were harvested with 50 µL of lysis buffer (50 mM Na-2-(N-Morpholino)ethanesulfonic acid(MES) (pH 7.8), 50 mM Tris-HCl (pH 7.8), 1 mM dithiothreitol, and 0.2% Triton X-100). To the lysates were added 50 µL of luciferase assay buffer (125 mM Na-MES (pH 7.8), 125 mM Tris-HCl (pH 7.8), 25 mM magnesium acetate, and 2.5 mg/mL ATP). Immediately after addition of 50 µL of 1 mM D-luciferin, the firefly luminescence was measured in a Victor3 luminometer (Perkin Elmer, Waltham, MA, USA). An equal volume, 50 µL, of coelenterazine assay buffer (125 mM Na-MES (pH 7.8), 125 mM Tris-HCl (pH 7.8), 25 mM magnesium acetate, 5 mM KH2PO4, 10 µM coelenterazine) was added and the bioluminescence was read. The relative light units (RLU) were normalized as the fold induction over unstimulated controls. Each assay was performed in triplicate.

2.4. EBOV VP24 Inhibition Assay EBOV VP24 inhibition assay was performed in 96-well plates. The construct pISRE-luc was cotransfected with pcDNA3.1-FLAG-VP24 or pcDNA3-V5-VP24. Empty vector pcDNA3.1(+) was used as the negative control and Influenza type A Virus (IAV) nonstructural protein 1 (NS1) expression plasmid as the positive control of inhibition. pRL-TK (Promega) was used as the internal control for transfection efficiency. Concentrations of plasmids are indicated in the figures’ legends. Luciferase activity was expressed as the percentage of EBOV VP24 transfected samples values normalized to the empty vector (indicated as 100%).

2.5. Immunoblot Analysis To detect EBOV VP24 expression levels, HEK293T cells were seeded in 6-well plates (4 × 105 cell/mL) and transfected with pISRE-luc and increasing concentrations of EBOV FLAG-VP24 plasmid. Twenty-four hours post-transfection, the medium was replaced with IFN-α. Eight hours post-IFN addition, cells were harvested and lysed in radioimmunoprecipitation (RIPA) buffer (50 mM Tris-HCl (pH 8), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mM phenylmethylsulfonyl fluoride, 1× Roche complete protease inhibitor cocktail, 1 mM sodium orthovanadate). Total cell extracts were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene difluoride (PVDF) membrane by standard methods. Membranes were blocked with 3% nonfat dry milk in Tris Buffered Saline (50 mM Tris-HCl, 0.138 M NaCl, 2.7 mM KCl, pH 8.0) and probed with mouse monoclonal anti-FLAG M2 and rabbit monoclonal anti-GAPDH. Secondary were HRP-linked anti-mouse IgG and HRP-linked anti-rabbit IgG. Detection was performed using Pierce ECL Western Blotting Substrate and the Chemidoc MP Imaging System (Bio-Rad, Hercules, CA, USA).

2.6. Data Analysis To determine the concentration of IFN-α that provides the maximal activation of the JAK-STAT pathway and to calculate the concentration of EBOV VP24 plasmid required to inhibit 50% of IFN-α stimulation (half maximal inhibitory concentration, IC50), we used a three-parameter concentration–response curve as described previously [19] using the log agonist concentration versus Viruses 2018, 10, 98 4 of 12 response, variable slope algorithm in GraphPad Prism software (GraphPad Prism 6, La Jolla, CA, USA),Viruses where: 2018, 10, 98 4 of 12  Y = Bottom + (Top − Bottom)/ 1 + 10^ LogIC50 (1)

Y Bottom Top Bottom/1 10ˆLogIC (1) The IC50 value was calculated based on three independent experiments performed in triplicate. 0 The Z- andThe Z -factorIC50 value values was werecalculated calculated based fromon three the independent following equations experiments developed performed by Zhangin triplicate. et al. [20]: The Z‐ and Z′‐factor values were calculated from the following equations developed by Zhang et al. [20]: (3 × SD + 3 × SD ) Z = 1 − s c (2) |M − M | 3 SDs 3SDc Z1 (2) |M M| (3 × SD + + 3 × SD −) Z0 = 1 − c c (3) 3|M SDc+ −3SDMc−| Z 1 (3) | | where SD and M are the standard deviation and mean,M sM and c represent sample and control, and c+ and c− representwhere SD positive and M are and the negative standard controls, deviation respectively. and mean, s and For c ISRE represent activation sample assay and control, we calculated and c+ the Z0-factor,and c considering− represent positive c+ and and c− negativeas the stimulated controls, respectively. control and For the ISRE not-stimulated activation assay control, we calculated respectively. For VP24the Z assay′‐factor, we considering considered c+ the and Z-factor, c− as the where stimulated s are samplescontrol and cotransfected the not‐stimulated with pISRE-luc control, and respectively. For VP24 assay we considered the Z‐factor, where s are samples cotransfected with VP24 and c are cells cotransfected with pISRE-luc and empty vector. All experiments were repeated at pISRE‐luc and VP24 and c are cells cotransfected with pISRE‐luc and empty vector. All experiments least three times. Quantitative data were expressed as the mean ± SD. were repeated at least three times. Quantitative data were expressed as the mean ± SD. 3. Results 3. Results 3.1. Establishment of a Miniaturized Cell-Based Assay for Evaluating VP24 Inhibition of JAK/STAT 3.1. Establishment of a Miniaturized Cell‐Based Assay for Evaluating VP24 Inhibition of JAK/STAT Cascade Cascade Activation Activation To monitorTo monitor the the activation activation status status of of IFN IFN signaling, signaling, we we transfected transfected HEK293T HEK293T cells cells with with pISRE pISRE-luc,‐luc, a reportera reporter plasmid plasmid that that is widelyis widely used used to to evaluate evaluate the transcription effect effect of ofactivated activated JAK/STAT JAK/STAT pathwaypathway by type by Itype IFNs I [ 21IFNs,22 ].[21,22]. Treatment Treatment with human with human recombinant recombinant IFN-α IFNof the‐α transientlyof the transiently transfected HEK293Ttransfected cells activatesHEK293T the cells IFN activates signaling the IFN pathway signaling leading pathway to a leading luminescent to a luminescent signal. To signal. use this To assay to testuse a this considerable assay to test number a considerable of compounds, number of we compounds, performed we it performed in 96-well it in plates. 96‐well For plates. initial For assay development,initial assay we development, analyzed the we analyzed concentration the concentration of luciferase of luciferase plasmid plasmid to use to for use transfection. for transfection. Hence, HEK293THence, cells HEK293T were transfectedcells were transfected with various with amountsvarious amounts of plasmid of plasmid per well. per well. Results Results showed showed that the that the optimal concentration for the reporter vector was 60 ng/well (Figure 1a). To assess the optimal optimal concentration for the reporter vector was 60 ng/well (Figure1a). To assess the optimal time time of stimulation, we performed a time‐course analysis. HEK293T cells exhibited an IFN‐α of stimulation, we performed a time-course analysis. HEK293T cells exhibited an IFN-α treatment treatment time for maximal signal between 12 and 24 h (Figure 1b). We chose the 8 h time point to time forallow maximal strong stimulation, signal between with 12luciferase and 24 hactivity (Figure increased1b). We by chose 27‐fold the 8compared h time point with toin allowcells not strong stimulation,treated with with IFN. luciferase activity increased by 27-fold compared with in cells not treated with IFN.

(a) (b)

Figure 1. Miniaturized luciferase reporter gene assay in 96‐well plates. (a) Human embryonic kidney Figure 1. Miniaturized luciferase reporter gene assay in 96-well plates. (a) Human embryonic kidney 293T (HEK293T) cells were transfected with 15, 30, 60, and 240 ng of pISRE‐luc per well. Twenty‐four 293T (HEK293T) cells were transfected with 15, 30, 60, and 240 ng of pISRE-luc per well. Twenty-four hours after transfection, cells were stimulated (S) or not (NS) with interferon‐α (IFN‐α) for IFN α α hourssignaling after transfection, pathway activation. cells were After stimulated 8 h, cells (S) were or not lysed (NS) and with luciferase interferon- activity(IFN- was )measured; for IFN signaling (b) pathwayHEK293T activation. cells were After transfected 8 h, cells with were 60 ng/well lysed and of pISRE luciferase‐luc. Twenty activity‐four was hours measured; after transfection, (b) HEK293T cells werecells were transfected stimulated with with 60 IFN ng/well‐α. After of 4, pISRE-luc. 8, 12, 24, 48 Twenty-four h, cells were hoursharvested after and transfection, luciferase activity cells were stimulatedwas measured. with IFN- Resultsα. After are 4, 8,shown 12, 24, as 48 pISRE h, cells‐luc were fold harvested induction andof stimulated luciferase cells activity over was the measured. not‐ Resultsstimulated are shown control. as pISRE-lucError bars indicate fold induction the mean of± SD. stimulated cells over the not-stimulated control. Error bars indicate the mean ± SD.

Viruses 2018, 10, 98 5 of 12

The ability of EBOV VP24 to inhibit the IFN signaling pathway has been demonstrated [14–16]. GivenThe the ability contribution of EBOV of VP24VP24 to inhibitthe EBOV the virulence IFN signaling [18,23], pathway the protein has been could demonstrated be a very important [14–16]. targetGiven for the contributiondrug development. of VP24 For to the this EBOV reason, virulence we wanted [18,23 to], the evaluate protein its could inhibitory be a very effect important in our system,target for to drugprovide development. a suitable model For thisfor screening reason, we a large wanted quantity to evaluate of potential its inhibitory inhibitors. effect HEK293T in our weresystem, cotransfected to provide awith suitable pISRE model‐luc reporter for screening and empty a large vector quantity pcDNA3.1(+), of potential or inhibitors. expression HEK293T plasmid forwere the cotransfected FLAG‐ or V5 with‐tagged pISRE-luc VP24. We reporter also cotransfected and empty vector cells pcDNA3.1(+),with the expression or expression plasmid plasmidfor IAV NS1,for the a FLAG-previously or V5-tagged demonstrated VP24. inhibitor We also of cotransfected IFN signaling, cells as with a positive the expression control plasmid[24]. Cells for were IAV stimulatedNS1, a previously with IFN demonstrated‐α for 8 h. A 30 inhibitor‐fold induction of IFN signaling, in ISRE activity as a positive was observed control in [24 IFN]. Cells‐α treated were samplesstimulated transfected with IFN- withα for pISRE 8 h. A‐luc 30-fold with or induction without in empty ISRE vector. activity This was activation observed inwas IFN- inhibitedα treated in cellssamples expressing transfected either with FLAG pISRE-luc‐ or V5 with‐tagged or without VP24 empty protein vector. and Thisin cells activation expressing was inhibited NS1, but in with cells differentexpressing percentages either FLAG- (Figure or V5-tagged 2a). In VP24order protein to demonstrate and in cells that expressing the inhibitory NS1, but effect with differentof ISRE transcriptionpercentages (Figure was due2a). effectively In order to to demonstrate VP24 expression that theat the inhibitory protein effect level, of we ISRE performed transcription a Western was blotdue effectivelyof the samples to VP24 transfected expression with at EBOV the protein VP24‐ level,FLAG we or performedempty vector a Western treated blotor not of thewith samples IFN‐α and,transfected in parallel, with EBOV assayed VP24-FLAG the luciferase or empty activity. vector Results treated orclearly not with showed IFN- αthatand, higher in parallel, amounts assayed of expressedthe luciferase VP24 activity. corresponded Results clearly to higher showed inhibition that higher of the amounts pathway of expressed(Figure 2b), VP24 demonstrating corresponded that to thehigher dose inhibition‐dependent of theinhibition pathway of (Figurethe luciferase2b), demonstrating activity was thatdue theto the dose-dependent dose‐dependent inhibition expression of the of VP24.luciferase activity was due to the dose-dependent expression of VP24.

(a) (b)

Figure 2. Ebola virus (EBOV)(EBOV) VP24VP24 inhibition inhibition assay. assay. ( a()a HEK293T) HEK293T cells cells were were transfected transfected with with pISRE-luc pISRE‐ lucand and 60 ng/well 60 ng/well of empty of empty vector vector pcDNA3.1(+), pcDNA3.1(+), or expression or expression plasmid plasmid for EBOV for EBOV FLAG- FLAG or V5-tagged‐ or V5‐ taggedVP24 and VP24 IAV and NS1. IAV Twenty-four NS1. Twenty hours‐four after hours transfection, after transfection, cells were cells stimulated were stimulated with 10 ng/mL with 10 of ng/mL IFN-α ofor IFN left untreated‐α or left untreated (NS). After (NS). 8 h, cells After were 8 h, lysed cells andwere luciferase lysed and activity luciferase was measured.activity was (b )measured. Expression (b of) ExpressionEBOV VP24 of at EBOV protein VP24 level. at HEK293T protein level. were HEK293T seeded in were a 6-well seeded plate. in After a 6‐well 24 h, plate. cells wereAfter transfected 24 h, cells werewith pISRE-luctransfected and with pcDNA3.1 pISRE‐luc (1 µandg/well) pcDNA3.1 and 0.25 (1 and μg/well) 1 µg/well and 0.25 of EBOV and 1 VP24-FLAG. μg/well of EBOV The day VP24 after,‐ FLAG.cells were The incubated day after, or cells not were with IFN-incubatedα for 8or h not and with subsequently IFN‐α for lysed 8 h and and subsequently examined for lysed luciferase and examinedactivity and for protein luciferase expression activity byand Western protein blotting. expression Results by Western are shown blotting. as percentage Results are of pISRE-lucshown as percentageactivation in of VP24 pISRE transfected‐luc activation cells overin VP24 empty transfected vector transfected cells over controls. empty vector Each experimenttransfected wascontrols. done Eachin triplicate. experiment Error was bars done indicate in triplicate. the mean Error± SD. bars indicate the mean ± SD.

3.2. Optimization of Stimulation Conditions and Evaluation of VP24 IC50 3.2. Optimization of Stimulation Conditions and Evaluation of VP24 IC50 Normalization with an internal control is crucial to reduce variability between experiments and toto develop a drug screening assay. In order to assess the optimal concentration of IFN IFN-‐α α toto use for quantifying the dose-dependentdose‐dependent effecteffect ofof VP24VP24 inin ourour inhibition inhibition assay, assay, we we performed performed a a curve curve of of IFN- IFNα‐ α usingusing Renilla Renilla luciferase luciferase as as internal internal control control for for transfection. transfection. As As shownshown inin FigureFigure3 3a,a, IFN- IFN‐α α activated thethe ISRE ISRE reporter reporter gene gene expression expression concentration concentration dependently. dependently. For For inhibition inhibition assay, assay, the concentration of agonist required to achieve a “maximum” signal response corresponds to the 80% of stimulation,

Viruses 2018, 10, 98 6 of 12

Viruses 2018, 10, 98 6 of 12 of agonist required to achieve a “maximum” signal response corresponds to the 80% of stimulation, accordingly with acceptance criteria for assay validation [25]. Hence, we used the concentration at accordingly with acceptance criteria for assay validation [25]. Hence, we used the concentration at the the initial inflexion of the IFN curve (1 ng/mL) to perform the VP24 dose–response assay. initial inflexion of the IFN curve (1 ng/mL) to perform the VP24 dose–response assay. Cotransfection of Cotransfection of HEK293T with concentrations between 0.5 and 120 ng/well were utilized to create HEK293T with concentrations between 0.5 and 120 ng/well were utilized to create the dose-dependence the dose‐dependence inhibition curve (Figure 3b) and the 50% inhibitory concentration (IC50) was inhibitioncalculated curve (30 (Figureng/well).3b) Thus, and the we 50% chose inhibitory this concentration concentration for (IC further50) was drug calculated screening (30 ng/well). assay Thus,development. we chose this concentration for further drug screening assay development.

(a) (b)

Figure 3. (a) HEK293T cells were transfected with pISRE‐luc (60 ng/well), RL‐TK (10 ng/well), and Figure 3. (a) HEK293T cells were transfected with pISRE-luc (60 ng/well), RL-TK (10 ng/well), and pcDNA3.1 (30 ng/well). Twenty‐four hours after transfection, cells were stimulated with different pcDNA3.1 (30 ng/well). Twenty-four hours after transfection, cells were stimulated with different concentrations of IFN‐α between 0.3 and 1000 ng/mL or left unstimulated. The medium was changed concentrations of IFN-α between 0.3 and 1000 ng/mL or left unstimulated. The medium was changed after 8 h, and luciferase activity was measured. Results are shown as pISRE‐luc fold induction of after 8 h, and luciferase activity was measured. Results are shown as pISRE-luc fold induction of stimulated cells over not stimulated control. Firefly luciferase activity is normalized to the Renilla stimulated cells over not stimulated control. Firefly luciferase activity is normalized to the Renilla luciferase internal control. Error bars indicate the mean ± SD; (b) Regression curve of VP24 inhibition luciferase internal control. Error bars indicate the mean ± SD; (b) Regression curve of VP24 inhibition was obtained transfecting HEK293T cells with reporter vector at the fixed concentration of 60 ng/well, was obtained transfecting HEK293T cells with reporter vector at the fixed concentration of 60 ng/well, RL‐TK (10 ng/well), and EBOV VP24‐FLAG or empty vector in concentrations between 0.5 and 120 RL-TKng/well. (10 Results ng/well), are shown and EBOV as percentage VP24-FLAG of pISRE or‐ emptyluc activation vector in in VP24 concentrations transfected cells between over empty 0.5 and 120vector ng/well. transfected Results controls. are shown Firefly as percentage luciferase activity of pISRE-luc is normalized activation to the in VP24Renilla transfected luciferase internal cells over emptycontrol. vector Error transfected bars indicate controls. the mean Firefly ± SD. luciferase activity is normalized to the Renilla luciferase internal control. Error bars indicate the mean ± SD. 3.3. Luciferase Assay Signal Stability 3.3. Luciferase Assay Signal Stability The stability of the luciferase signal is a relevant factor supporting drug screening applications. TheThe time stability‐dependent of the decay luciferase of RLU signal generated is a relevant from our factor firefly supporting luciferase activity drug screening was explored. applications. After Theadding time-dependent D‐luciferin, the decay luminescent of RLU signal generated generated from from our cells firefly transfected luciferase with activity ISRE alone was and explored. with AfterISRE adding and VP24D-luciferin, was recorded the luminescent every 5 min. signal As it is generated shown by fromthe luminescence cells transfected decay with curve ISRE (Figure alone and4a) with the ISREmaximum and VP24 level of was bioluminescence recorded every was 5 min. reached As it 5 is min shown after bysubstrate the luminescence addition, followed decay curve by (Figureslow4 decaya) the over maximum 1 h. Because level ofreading bioluminescence a 96‐well plate was requires reached the 5 readout min after signal substrate to be stable addition, for about followed 20 bymin slow (10 decay s/well), over to minimize 1 h. Because timing reading effects we a 96-well decided plate to read requires lines two the by readout two, avoiding signal the to problem be stable of for aboutdecay 20 of min the (10signal. s/well), The time to minimize needed by timingthe luminometer effects we to decided read two to lines read is 4.30 lines min, two time by two,in which avoiding the signal is perfectly stable. We wanted to verify that the addition of luciferase substrate at 0, 5, 10, and 15 the problem of decay of the signal. The time needed by the luminometer to read two lines is 4.30 min, min after the incubation with luciferase assay buffer for Lines 1 and 2, Lines 3 and 4, Lines 5 and 6, and time in which the signal is perfectly stable. We wanted to verify that the addition of luciferase substrate Lines 7 and 8, respectively, does not affect the intensity of signal obtained. In Figure 4b we show that even at 0, 5, 10, and 15 min after the incubation with luciferase assay buffer for Lines 1 and 2, Lines 3 if harvesting and luciferase assay buffer are present for longer times in the other lines compared to the and 4, Lines 5 and 6, and Lines 7 and 8, respectively, does not affect the intensity of signal obtained. first two, the intensity of the signal does not change. The luminescence signal is expressed as percentage In Figure4b we show that even if harvesting and luciferase assay buffer are present for longer times in over the value (taken to be 100%) at time t = 0 min (t0) that represents the lines 1 and 2 where D‐luciferin is theimmediately other lines comparedadded. to the first two, the intensity of the signal does not change. The luminescence signal is expressed as percentage over the value (taken to be 100%) at time t = 0 min (t0) that represents the lines 1 and 2 where D-luciferin is immediately added.

Viruses 2018, 10, 98 7 of 12 Viruses 2018, 10, 98 7 of 12

(a) (b)

Figure 4. Time‐dependent decay of luciferase activity. HEK293T cells cotransfected with pISRE‐luc Figure 4. Time-dependent decay of luciferase activity. HEK293T cells cotransfected with pISRE-luc and and empty vector or pISRE‐luc and VP24 were lysed. (a) After addition of D‐luciferin, luciferase signal empty vector or pISRE-luc and VP24 were lysed. (a) After addition of D-luciferin, luciferase signal was was read over 1320 min. The relative decay is reported by plotting relative light units (RLU) as a read over 1320 min. The relative decay is reported by plotting relative light units (RLU) as a proportion proportion of the RLU at t0 (100%); (b) Addition of D‐luciferin was performed at 0, 5, 10, 15 min after of theincubation RLU at t0with(100%); luciferase (b) Addition assay buffer. of D-luciferin The luciferase was performed signal is reported at 0, 5, 10, by 15 plotting min after RLU incubation as a withproportion luciferase of assay the RLU buffer. at t = The 0 min luciferase after addition signal of is luciferase reported buffer by plotting (100%). RLU as a proportion of the RLU at t = 0 min after addition of luciferase buffer (100%). 3.4. Assay Validation 3.4. Assay Validation To achieve optimization, coefficients of variation (CV) were calculated for the not‐stimulated controlsTo achieve (Min optimization, signal), for cells coefficients cotransfected of variationwith pISRE (CV)‐luc wereand pcDNA3.1 calculated (Max for thesignal) not-stimulated or VP24 controls(IC50) (Min (Mid signal), signal) forafter cells stimulation cotransfected with the with maximum pISRE-luc signal and pcDNA3.1response concentration (Max signal) of or IFN VP24‐α (1 (IC 50) (Midng/mL). signal) Representative after stimulation results with are the provided maximum in signalTable 1. response CVs for concentration each signal are of less IFN- thanα (1 20%, ng/mL). Representativeindicating achieved results acceptance are provided criteria in [25]. Table 1. CVs for each signal are less than 20%, indicating achieved acceptance criteria [25]. Table 1. Well‐to‐well reproducibility of viral protein 24 (VP24) inhibition assay. Table 1. Well-to-well reproducibility of viral protein 24 (VP24) inhibition assay. Signal Coefficients of Variation, CV (%) Min 6.1 Signal Coefficients of Variation, CV (%) Mid 4.6 Min 6.1 Max 3.3 Mid 4.6 Max 3.3 In order to assess the plate uniformity and to evaluate the signal variability between different days, we performed inter‐plate and inter‐day tests. Plate‐to‐plate or day‐to‐day variation in 50% In order to assess the plate uniformity and to evaluate the signal variability between different activity needs to be assessed during validation of a drug screening assay [25]. Thus, we performed days, we performed inter-plate and inter-day tests. Plate-to-plate or day-to-day variation in 50% the tests evaluating as the midpoint the signal obtained transfecting cells with the IC50 of VP24 activityplasmid needs (30 to ng/well). be assessed In Table during 2 are validation indicated ofthe a percentages drug screening of ISRE assay expression [25]. Thus, in the we presence performed of the testsVP24 evaluating (IC50). The as thefold midpoint shift is not the higher signal than obtained 2 for both transfecting the inter‐day cells and with inter‐ theplate IC tests,50 of confirming VP24 plasmid (30 ng/well).acceptance In criteria Table 2for are validation. indicated the percentages of ISRE expression in the presence of VP24 (IC 50). The foldUsing shift isthe not optimized higher than reaction 2 for bothconditions the inter-day previously and calculated, inter-plate the tests, assay confirming procedures acceptance were criteriavalidated for validation. in a Z‐factor experiment. The Z‐factor is a measure of assay quality that takes into account theUsing standard the optimizeddeviation, the reaction dynamic conditions range between previously positive calculated, and negative the controls, assay and procedures the signal were validatedvariation in aamongst Z-factor replicates. experiment. A scale The of Z-factor0–1 is used, is awith measure values of greater assay than quality or equal that to takes 0.5 indicative into account theof standard an excellent deviation, assay. theFor dynamicISRE activation range assay between we positivecalculated and the negativeZ′‐factor using controls, HEK293T and the cells signal untreated or treated with IFN‐α as negative and positive controls, respectively. For inhibition assay, variation amongst replicates. A scale of 0–1 is used, with values greater than or equal to 0.5 indicative of the Z‐factor was measured considering the difference in signal between cells cotransfected with an excellent assay. For ISRE activation assay we calculated the Z0-factor using HEK293T cells untreated pISRE‐luc and empty vector or pISRE‐luc and VP24 expression plasmid. As shown in Figure 5, the α or treatedZ′‐ and with Z‐value IFN- for aseach negative assay plate and positivewere 0.62 controls, for stimulation respectively. assay Forand inhibition 0.53 for inhibition assay, the assay, Z-factor wasindicating measured the considering robustness of the the difference system. in signal between cells cotransfected with pISRE-luc and empty vector or pISRE-luc and VP24 expression plasmid. As shown in Figure5, the Z 0- and Z-value for each assay plate were 0.62Table for 2. stimulation Inter‐plate and assay Inter‐ andday tests 0.53 for for VP24 inhibition inhibition assay, assay. indicating the robustness of the system.Inter‐Plate Test pISRE‐luc Activation (%) Inter‐Day Test pISRE‐luc Activation(%) Plate 1 52.4 Plate 1 49.3 Plate 2Table 2. Inter-plate50.8 and Inter-day testsPlate for VP242 inhibition assay.49.0

Inter-Plate Test pISRE-luc Activation (%) Inter-Day Test pISRE-luc Activation (%) Plate 1 52.4 Plate 1 49.3 Plate 2 50.8 Plate 2 49.0 VirusesViruses 20182018,, 1010,, 98 98 88 ofof 1212 Viruses 2018, 10, 98 8 of 12

(a) (b) (a) (b) Figure 5. Z′‐ and Z‐factor determination for IFN‐α stimulation and VP24 inhibition assays. Firefly Figure 5. 0Z′‐ and Z‐factor determination for IFN‐α stimulation and VP24 inhibition assays. Firefly Figureluciferase 5. Zactivity- and Z-factor is normalized determination to the Renilla for IFN- luciferaseα stimulation internal and control. VP24 inhibitionResults are assays. expressed Firefly as luciferase activity is normalized to the Renilla luciferase internal control. Results are expressed as luciferasepISRE‐luc activityfold induction is normalized over the to theunstimulated Renilla luciferase control. internal The Z′‐ control.and Z‐factor Results were are expressedcalculated asas pISRE‐luc fold induction over the unstimulated control. The Z′‐ and Z‐factor were calculated as pISRE-luc fold induction over the unstimulated control. The Z0- and Z-factor were calculated as describeddescribed in in methods. methods. ( a()a )HEK293T HEK293T cells cells werewere seeded inin aa 9696‐‐wellwell plate plate and and transfected transfected with with pISRE pISRE‐ ‐ described in methods. (a) HEK293T cells were seeded in a 96-well plate and transfected with pISRE-luc lucluc (60 (60 ng/well) ng/well) and and pcDNA3.1 pcDNA3.1 (30 (30 ng/well). ng/well). Twenty‐‐fourfour hourshours post post‐transfection,‐transfection, 48 48 wells wells (S) (S) were were (60 ng/well) and pcDNA3.1 (30 ng/well). Twenty-four hours post-transfection, 48 wells (S) were incubatedincubated with with 1 1ng/mL ng/mL of of IFN IFN‐α ‐α andand 4848 wellswells were leftleft untreateduntreated (NS). (NS). ( b(b) HEK293T) HEK293T cells cells seeded seeded in in incubated with 1 ng/mL of IFN-α and 48 wells were left untreated (NS). (b) HEK293T cells seeded in 48 48wells wells were were transfected transfected with with pISRE pISRE‐‐lucluc (60(60 ng/well) andand pcDNA3.1pcDNA3.1 (30 (30 ng/well), ng/well), and and those those in inthe the 48 wells were transfected with pISRE-luc (60 ng/well) and pcDNA3.1 (30 ng/well), and those in the remainingremaining 48 48 wells wells were were transfected transfected with with pISREpISRE‐luc (60 ng/well)ng/well) and and expression expression plasmid plasmid for for VP24 VP24 (30 (30 remaining 48 wells were transfected with pISRE-luc (60 ng/well) and expression plasmid for VP24 ng/well).ng/well). Thirty Thirty‐five‐five wells wells for for each each sample sample werewere stimulated withwith 1 1 ng/mL ng/mL of of IFN IFN‐α ‐α andand 12 12 were were not not α (30stimulated.stimulated. ng/well). Thirty-five wells for each sample were stimulated with 1 ng/mL of IFN- and 12 were not stimulated. 3.5.3.5. Designing Designing the the Drug Drug Screening Screening Assay Assay 3.5. Designing the Drug Screening Assay ForFor drug drug screening screening assay, assay, HEK293T HEK293T cellscells (1.5 × 1044 cells/well)cells/well) are are cotransfected cotransfected with with pISRE pISRE‐luc‐luc 4 andand Forempty empty drug vector vector screening or or VP24 VP24 assay, expression expression HEK293T plasmidplasmid cells (1.5 (IC×5010).). AtAtcells/well) 2424 hh postpost aretransfection, transfection, cotransfected the the cells with cells are pISRE-luc are mock mock andtreatedtreated empty or or treated vector treated orwith with VP24 IFN IFN expression‐α ‐α inin thethe plasmidpresencepresence (IC of 50compound). At 24 h at postat threethree transfection, different different concentrations theconcentrations cells are mock or or treateddimethyldimethyl or sulfoxide treated sulfoxide with (DMSO). (DMSO). IFN- αA A inconcentration concentration the presence of ofIFN compound‐α ‐α (0.3(0.3 ng/mL)ng/mL) at three in in the the different linear linear range range concentrations of ofthe the IFN IFN‐α or‐α dimethyldose–responsedose–response sulfoxide curve curve (DMSO). is is used. used. A At concentrationAt 88 hh postpost‐‐treatment,treatment, of IFN-α (0.3cellscells ng/mL) areare harvested,harvested, in the linearand and firefly rangefirefly and of and the renilla IFN-renilla α luciferase activities are measured. Positive controls of inhibition are cells cotransfected with pISRE‐ dose–responseluciferase activities curve are is used. measured. At 8 h Positive post-treatment, controls cells of inhibition are harvested, are cells and fireflycotransfected and renilla with luciferase pISRE‐ luc and VP24 in presence of DMSO, while negative controls of inhibition are cells cotransfected with activitiesluc and VP24 are measured. in presence Positive of DMSO, controls while of negative inhibition controls are cells of cotransfected inhibition are with cells pISRE-luc cotransfected and VP24 with pISRE‐luc and empty vector in presence of DMSO. Luciferase activity is calculated as folds induction inpISRE presence‐luc and of DMSO, empty vector while negative in presence controls of DMSO. of inhibition Luciferase are activity cells cotransfected is calculated with as folds pISRE-luc induction and of stimulated versus not‐stimulated controls as well as percentage of pISRE‐luc activation in VP24 emptyof stimulated vector inversus presence not‐ ofstimulated DMSO. Luciferase controls as activity well as is percentage calculated asof foldspISRE induction‐luc activation of stimulated in VP24 transfected cells over empty vector transfected controls. A plate map illustrating the layout of the versus not-stimulated controls as well as percentage of pISRE-luc activation in VP24 transfected cells transfectedscreening cellsplate, over including empty wellvector locations transfected of compounds controls. A and plate positive map illustrating and negative the controls,layout of is the over empty vector transfected controls. A plate map illustrating the layout of the screening plate, screeningrepresented plate, in Figureincluding 6. well locations of compounds and positive and negative controls, is includingrepresented well in locationsFigure 6. of compounds and positive and negative controls, is represented in Figure6.

Figure 6. Representative scheme of 96‐well plate for drug screening.

3.6. IFN‐α PartiallyFigure Reverts 6. VP24Representative Inhibition schemeof JAK/STAT of 96-well96‐ wellCascade plateplate forfor drugdrug screening.screening.

3.6. IFNThe‐α Partially lack of inhibitorsReverts VP24 for VP24Inhibition inhibition of JAK/STAT of the JAK/STAT Cascade cascade does not allow a positive control of restoration of the JAK/STAT cascade activation in presence of VP24. However, to The lack of inhibitors for VP24 inhibition of the JAK/STAT cascade does not allow a positive control of restoration of the JAK/STAT cascade activation in presence of VP24. However, to

Viruses 2018, 10, 98 9 of 12

3.6. IFN-α Partially Reverts VP24 Inhibition of JAK/STAT Cascade

VirusesThe 2018 lack, 10, 98 of inhibitors for VP24 inhibition of the JAK/STAT cascade does not allow a positive9 of 12 control of restoration of the JAK/STAT cascade activation in presence of VP24. However, to investigate theinvestigate suitability the ofsuitability the assay of for the drug assay screening, for drug screening, we evaluated we evaluated the reversion the reversion of the effect of the of VP24 effect on of pISRE-lucVP24 on pISRE activation‐luc activation mediated mediated by IFN-α. by HEK293T IFN‐α. HEK293T cells were cells cotransfected were cotransfected with pISRE-luc with andpISRE empty‐luc vectorand empty or VP24 vector expression or VP24 expression plasmid at plasmid 30 ng/mL at (IC30 ng/mL50). In addition (IC50). In toaddition the standard to the standard amount of amount IFN-α (0.3of IFN ng/mL),‐α (0.3 weng/mL), added we increasing added increasing concentrations concentrations of IFN-α of(0.7, IFN 3,‐α 9.7(0.7, ng/mL). 3, 9.7 Asng/mL). shown As in shown Figure 7in, IFN-Figureα is7, ableIFN‐α to partiallyis able to revertpartially the revert ISRE expression the ISRE expression in presence in of presence VP24, demonstrating of VP24, demonstrating its inhibitory its effectinhibitory against effect the against viral protein. the viral protein.

Figure 7. Effect of IFN IFN-‐α α on VP24 inhibition of IFN signaling. HEK293T cells were cotransfected with pISRE-lucpISRE‐luc (60 ng/well),ng/well), RL RL-TK‐TK (10 ng/well), and and pcDNA3.1 pcDNA3.1 or or VP24 VP24 expression plasmid (30 ng/well). Twenty-fourTwenty‐four hourshours afterafter transfection,transfection, cells cells were were stimulated stimulated with with 0.3 0.3 ng/mL ng/mL of IFN IFN-‐αα. Then, increasing increasing concentrations of IFN-IFN‐α α were added. After 8 h of stimulation, stimulation, the medium was changed and luciferase activity was measured. Results are shown as percentage of ISRE expression in VP24 transfected cells over empty vector transfected control. Firefly Firefly luciferase activity is normalized to the Renilla luciferase internal control. Error bars indicate the mean ± SD.SD.

4. Discussion Ebola virus has evolvedevolved multiplemultiple strategiesstrategies toto antagonizeantagonize thethe IFN-IFN‐αα//β β responsesresponses in target cells such as macrophages, monocytes, and dendritic cells, resulting in total impairment of the . An important contribution to EBOV virulence is given by the protein VP35 through its suppression of IFN production [6–9,12], [6–9,12], but a crucial role is is exerted exerted by by another another viral viral protein, protein, VP24, VP24, that disablesdisables cells cells for for viral viral replication replication and and propagation propagation by interfering by interfering with IFN with signaling IFN signaling [14–18,26 [14–,27]. Since18,26,27]. it is Since clear thatit is theclear early that control the early of viremia control isof one viremia of the is keys one forof the survival, keys for blocking survival, EBOV blocking VP24 actionEBOV onVP24 the action cellular on immune the cellular response immune is an important response subjectis an important for investigation subject and,for investigation hence, a validated and, pharmacologicalhence, a validated target. pharmacological target. Much work has been done in the last years to elucidate the function of EBOV VP24 in IFN antagonism. These efforts have been conducted employing different methods. ImmunofluorescenceImmunofluorescence analysis with with multiple multiple epitope epitope tags, tags, such such as phospho as phospho-specific‐specific antibodies antibodies for P‐ forSTAT1 P-STAT1 or STAT1 or STAT1 fused fusedto green to greenfluorescent fluorescent protein protein (GFP), (GFP), was used was to used demonstrate to demonstrate the ability the ability of VP24 of VP24to inhibit to inhibit IFN‐ IFN-inducedinduced nuclear nuclear localization localization and tyrosine and tyrosine phosphorylation phosphorylation of human of human STAT1. STAT1. An equivalent An equivalent effect effectwas confirmed was confirmed after EBOV after EBOV infection infection of cells of [14–16]. cells [14 –Co16‐].immuonoprecipitation Co-immuonoprecipitation studies studies revealed revealed that thatVP24 VP24 selectively selectively interacts interacts with KPNA with KPNA (α1, α5, (α and1, α α5,6), and competingα6), competing with P‐STAT1 with P-STAT1 for the same for the binding same binding[14,15], and [14, 15in], vitro and inELISA vitro assaysELISA were assays used were to used show to that show purified that purified EBOV EBOV VP24 VP24could could also bind also binddirectly directly to purified to purified truncated truncated STAT1 STAT1 [17]. [17 Subsequently,]. Subsequently, characterization characterization of of the the binding binding of of EBOV VP24 to KPNA was performed by isothermal titration calorimetry (ITC), in vitro pull down assay with recombinant expressed proteins, and computational analysis [28]. In addition, X‐ray crystallization analysis allowed the determination of the complex structure [28]. Affinity tagging coupled to label‐free quantitative mass spectrometry was used to identify potential interacting partners of VP24 and confirmed the binding with KPNA [29]. Reverse genetics was used to identify the mutations correlated with the ability to evade type I IFN responsible for the acquisition of the

Viruses 2018, 10, 98 10 of 12

VP24 to KPNA was performed by isothermal titration calorimetry (ITC), in vitro pull down assay with recombinant expressed proteins, and computational analysis [28]. In addition, X-ray crystallization analysis allowed the determination of the complex structure [28]. Affinity tagging coupled to label-free quantitative mass spectrometry was used to identify potential interacting partners of VP24 and confirmed the binding with KPNA [29]. Reverse genetics was used to identify the mutations correlated with the ability to evade type I IFN responsible for the acquisition of the high virulence of the adapted Ebolavirus Mayinga strain in mice [18]. Among the variety of techniques, the use of luciferase reporter assays to measure viral antagonist activity against the IFN system has long been established to be robust and biologically relevant [14–16]. Our group has previously developed a luciferase assay gene reporter able to quantify the inhibition of the IFN system by EBOV VP35 that is used for testing compounds against this protein [30]. Since with such a system we could study the inhibition of the first cascade of IFN production, the RIG-I pathway, we decided to develop a new miniaturized luciferase assay that could be used to study the second cascade of the IFN system, the JAK-STAT pathway. We report here its use to measure the EBOV VP24 inhibition of the IFN signaling in HEK293T cells, to be used as a screening system to identifying EBOV VP24 inhibitors as well as to dissect the interaction between EBOV VP24 and its cellular partners. Experiments were carried out in 96-well plates, always at least in triplicate, allowing good statistical evaluation of the results and showing good reproducibility. In addition, the use of 96-well plates allows us to perform the entire assay in only one plate, avoiding a more complex experimental approach. Luciferase assays done in 6-, 12-, 24-, and 48-well plates imply that cell lysates are moved from a big plate to another with more passages and risk losing material. One advantage of this assay is that the luminescence signal derives effectively from all cells in the well and that the technique is absolutely faster than traditional methods. Various parameters of the microtransfection method were optimized: amount of plasmids, amount of IFN-α and stimulation time. Measurements of luciferase activity performed at various times yielded optimal results between 8 and 24 h post transfection. Thus, a shorter 8 h incubation was chosen. In literature, luciferase assay performed to evaluate EBOV VP24 inhibition requires at least 16 h of stimulation to measure the luminescence signal [14,15,23,31,32]. The present method allows data to be obtained more rapidly. Conventional transfection assays also require the use of large quantities of plasmids and reagents [14,15,23], while in the present system, as little as 60 ng/well for plasmid reporter can be used, compared to 0.5–1 µg/well in a 6-well plate [14,23]. Hence, the present method appears to be extremely sensitive and allows considerable economy of all reagents. Moreover, the use of homemade reagents for reading luciferase activity is a convenient and ultra-low-cost system compared to the use of highly expensive commercial kits. Our assay provides a convenient tool for large-scale screening studies to identify selective EBOV VP24 inhibitors as well as compounds subverting the VP24 block of the IFN cascade. In fact, all acceptance criteria for drug screening validation, including the Z’-factor analysis, were satisfied. In addition, the expression of Renilla luciferase offers an internal control value with which the firefly luciferase reporter gene is normalized, providing a decrease in variability and an increase in consistency. We confirmed the feasibility of the present assay to be used for a rapid and quantitative evaluation of anti-EBOV VP24 agents. Indeed, the experiment with the increasing concentrations of IFN-α suggested that it is possible to override the inhibitory effect of VP24 and partially restore the IFN signaling in our system. In conclusion, the development of a new dual cell-based assay able to quantify the inhibition of IFN signaling by EBOV VP24 could have important implications for future studies since it represents a suitable model for screening compounds against a viral target for which no drugs are currently approved. The enzymatic activity of luciferase and its normalization with the Renilla internal control allows us to achieve robust assay performance as the Z- and Z0-factor calculations demonstrate. Overall, the present method offers multiple advantages such as 100- to 1000-fold reduction in cells, plasmids, Viruses 2018, 10, 98 11 of 12

and reagents required, shorter time assay, reduced costs, and rapid detection of molecules that are anti-EBOV VP24.

Acknowledgments: Elisa Fanunza, Aldo Frau, Angela Corona and Enzo Tramontano were supported by RAS LR 07/2007 grant n. CRP-78711/F72I15000900002. VP24 expression vector from isolate H.sapiens-wt/GIN/ 2014/Gueckedou-C05, Sierra Leone/Guinea and other reagents and tools provided by Marco Sgarbanti were obtained through the project “Search for New Drugs for Neglected and Rare Diseases of Poverty”-SP1 Activity: “Neglected and Poverty Diseases” 2014 from the Italian National Collection of Compounds and Chemical Screening Center (CNCCS) consortium. Author Contributions: Elisa Fanunza and Enzo Tramontano conceived and designed the experiments; Elisa Fanunza performed the experiments; Elisa Fanunza, Aldo Frau and Angela Corona analyzed the data; Marco Sgarbanti and Roberto Orsatti contributed reagents and material tools; Elisa Fanunza and Enzo Tramontano wrote the paper. Conflicts of Interest: The authors declare no conflict of interest.

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