SUPPLEMENT ARTICLE. Valeria Cannas, Gian Luca Daino, Angela Corona, Francesca Esposito, and Enzo Tramontano

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1 SUPPLEMENT ARTICLE A Luciferase Reporter Gene Assay to Measure Ebola Virus Viral Protein 35 Associated Inhibition of Double-Stranded RNA Stimulated, Retinoic Acid Inducible Gene 1 Mediated Induction of Interferon β Valeria Cannas, Gian Luca Daino, Angela Corona, Francesca Esposito, and Enzo Tramontano Department of Life and Environmental Sciences, University of Cagliari, Italy During Ebola virus(ebov) infection, the type I interferon α/β (IFN-α/β) innate immune response is suppressed by EBOV viral protein 35 (VP35), a validated drug target. Identification of EBOV VP35 inhibitors requires a cellular system able to assess the VP35-based inhibitory functions of viral double-stranded RNA (dsrna) IFN-β induction. We established a miniaturized luciferase gene reporter assay in A549 cells that measures IFN-β induction by viral dsrna and is dose-dependently inhibited by VP35 expression. When compared to influenza A virus NS1 protein, EBOV VP35 showed improved inhibition of viral dsrna based IFN-β induction. This assay can be used to screen for EBOV VP35 inhibitors. Keywords. Ebola virus; VP35; NS1; luciferase reporter gene assay; IFN-β induction; IFN-β inhibition; drug development; innate immunity; RIG-I activation; dsrna recognition. Ebola virus disease (EVD), previously known as Ebola hemorrhagic fever, is a rare and deadly disease caused by infection with a negative-sense single-stranded RNA (ssrna) virus of the family Filoviridae, genus Ebolavirus (EBOV), for which no drug is currently available. In 2014, the largest known outbreak of EVD beganin4westafricacountries,with>7500deaths and > reported cases at the time of writing, confirming that EVD epidemics are highly unpredictable and that identification of small molecules to counteract EBOV infection is a global health priority [1]. The high lethality of EVD has been attributed to the ability of the virus to efficiently suppress the host innate antiviral response [2], which begins with the recognition Presented in part: 2nd Innovative Approaches for Identification of Antiviral Agents Summer School, Cagliari, Italy, 28 September 3 October Correspondence: Enzo Tramontano, PhD, Department of Life and Environmental Sciences, University of Cagliari, Cittadella di Monserrato, Monserrato (Cagliari), Italy (tramon@unica.it). The Journal of Infectious Diseases 2015;212:S The Author Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please journals.permissions@oup.com. DOI: /infdis/jiv214 of viral dsrna, as a replicative intermediate, by the cytoplasmic pattern-recognition receptor retinoic acid inducible gene 1 (RIG-I) that induces a type I interferon α/β (IFN-α/β) response. This induction is prevented by EBOV viral protein 35 (VP35), a multifunctional essential viral component of the EBOV RNA polymerase complexandnucleocapsidassemblyfactor[2 7]thatalso(1) binds viral dsrna, mimicking RIG-I recognition of 5 - triphosphorylated dsrna [8]; (2) interferes with the activation of IRF-3 [9]; and (3) inhibits PACT-induced RIG-I ATPase activity by preventing RIG-I interaction with PACT [10]. Site-directed mutagenesis studies demonstrated that EBOV VP35 is a validated drug target [5, 11]. To characterize the role of EBOV VP35 in inhibiting the RIG-I signaling cascade that leads to IFN-β production, and to identify small molecules that could interfere with VP35 properties, a cell-based system able to quantify dsrna IFN-β induction and its inhibition by EBOV VP35 is essential. Influenza A virus (IAV) NS1 protein also strongly inhibits RIG-I mediated IFN-β induction, and a luciferase reporter gene assay has been used to assess its properties [12], even though it was not suitable for drug development. EBOV VP35 Inhibition of IFN-β Activation JID 2015:212 (Suppl 2) S277

2 We have established a luciferase reporter gene assay to assess EBOV VP35 inhibition of RIG-I mediated IFN-β induction. We miniaturized and characterized the assay that can be used to evaluate the properties of EBOV VP35, as well as the efficacy of small molecules that can act as VP35 inhibitors. MATERIALS AND METHODS Cell Line A549 cells were grown in Dulbecco s modified Eagle s medium supplemented with 10% fetal bovine serum and 1% penicillinstreptomycin. Cells were incubated at 37 C in a humidified 5% CO 2 atmosphere. Construction of EBOV VP35 Mammalian Expression Plasmid To introduce the EBOV VP35 gene into a mammalian expression vector, we amplified the EBOV VP35 gene Zaire species (1976 Yambuku-Mayinga strain), previously cloned in the pet45b-zebov-vp35 vector [13], by polymerase chain reaction (PCR). Two primers were designed to amplify the gene and introduce it into the mammalian expression vector construct by BamHI and NotI restriction enzymes. Primer sequences were 5 -TCAGCAGAGGATCCGATAATGCATCACCACCACCAT- CAC-3 and 5 -GTACTAATATGCGGCCGCTCAAATTTT- GAGTCCAAGTGT-3. PCR was performed in a mixture containing pet45b-zebov-vp35 plasmid (100 ng), each primer (400 μm), MgCl 2 (1.5 mm), each dntp (0.2 mm), and μl FideliTaq DNA polymerase (Usb). The PCR mixtures were filled with nuclease-free water to a final volume of 50 μl, and the PCR cycle consisted of initial denaturation at 94 C for 2 minutes, 35 cycles of denaturation at 94 C for 30 seconds, annealing at 55 C for 30 seconds, extension at 68 C for 2 minutes, and a final extension at 68 C for 5 minutes. The amplified EBOV VP35 gene and the pcdna3 plasmid (Invitrogen) were digested by BamHI and NotI (New England BioLabs), linear pcdna3 plasmid was dephosphorylated with Antarctic Phosphatase (New England BioLabs), and fragments (50 ng of linear pcdna3 and 20 ng of insert VP35) were ligated by T4 DNA ligase (New England BioLabs) to obtain the pcdna3- ZEBOV-VP35 plasmid that was transformed to the Escherichia coli Top10 strain by a standard heat shock protocol at 42 C for 90 seconds. Plasmid was extracted and sequenced for control. IAV RNA Extraction For production of viral RNA (vrna), A549 cells were infected with IAV/Puerto-Rico/8/34 (IAV PR8) strain with a multiplicity of infection of 5. Five hours after infection, total RNA was isolated using the RNeasy Kit (Qiagen). Luciferase Reporter Gene Assay A549 cells ( cells/well) were transfected in 48-well plates with T-Pro P-Fect Transfection Reagent (T-Pro Biotechnology) with the construct pgl IFN-β luc (kindly provided by Prof Stephan Ludwig, Institute of Molecular Virology, University of Münster, Germany). Twenty-four hours after transfection, cells were additionally transfected with IAV PR8 RNA and incubated for further 6 hours at 37 C in 5% CO 2. Cells were harvestedwithlysisbuffer(50mmna-mes[ph7.8],50mm Tris-HCl [ph 7.8], 1 mm dithiothreitol, and 0.2% Triton X-100). The crude cell lysates were cleared by centrifugation, and 50 µl of cleared lysates were added to 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) in a white 96-well plate. Immediately after addition of 50 µl 1mMD-luciferin into each well, the luminescence was measured in Victor 3 luminometer (Perkin Elmer). The relative light units (RLU) were normalized as the fold activity of the unstimulated control. Each assay was performed in triplicate. EBOV VP35 Luciferase Reporter Gene Inhibition Assay The luciferase reporter gene assay described above was used with the cotransfection of the pgl IFN-β luc plasmid with pcdna3-zebov-vp35. When IAV NS1 was used as a control, the mammalian expression plasmid pcdna3-ns1 (kindly provided by Prof Stephan Ludwig) was cotransfected with the pgl IFN-β luc plasmid. Inhibition of luciferase expression was indicated as the percentage of induced control. Each assay was performed in triplicate. RESULTS When viral 5 -triphosporylated dsrna is accumulated into the cytoplasm of infected cells, its presence is recognized by RIG-I, which induces a signaling cascade leading to the expression of IFN-β. This cytokine acts then in both an autocrine and paracrine manner to induce the expression of a number of proteins coded by the IFN-stimulated genes that, in turn, suppress viral propagation. EBOV VP35 inhibits RIG-I mediated IFN-β induction [2], and this ability has been measured with a luciferase reporter gene assay in which IFN-β induction was stimulated by Sendai virus (SeV) infection [5], an aspect that strongly limits its range of use. We wanted to establish a more suitable assay that could mimic the viral dsrna IFN-β induction and could be used for drug screening. We used human lung epithelial A549 cells, characterized by a strong IFN-response due to viral stimulation, that were transiently transfected with a luciferase reporter gene construct (pgl IFN-β luc) driven by the IFN-β enhanceosome, a promoter element that contains all principal transcription factor binding sites of the IFN-β promoter. In this assay, the transfection of IAV PR8 RNA into A549 cells containing pgl IFN-β luc plasmid activates the RIG-I signaling cascade leading to a luminescent signal. Such a method has been previously reported [12, 14], but we viewed the use of S278 JID 2015:212 (Suppl 2) Cannas et al

3 Figure 1. Miniaturization of the luciferase reporter gene assay in 48-well plates. A, A549 cells were transfected with pgl interferon β (IFN-β) luc. Twenty-four hours after transfection, cells were additionally transfected with influenza A virus (IAV) RNA. Cells were lysed after 1, 2, 3, 4, 5, and 6 hours, and the luciferase activity was measured. B, A549 cells were transfected with pgl IFN-β luc. Twenty-four hours after transfection, cells were additionally transfected with IAV RNA. The medium was changed after 1, 2, 3, 4, 5, and 6 hours, and luciferase activity was measured. C, A549 cells were transfected with 125, 250, and 500 ng of pgl IFN-β luc. Twenty-four hours after transfection, cells were additionally transfected with 75, 125, 250, and 500 ng of IAV RNA. Six hours after transfection, cells were lysed, and luciferase activity was measured. 12-well plates as unsuitable for large-scale drug screening efforts. To adapt this system to test large numbers of compounds against EBOV VP35 inhibitory functions, we looked to miniaturize the format. First, we evaluated the IFN-β activation by vrna transfection in 48-well plates and performed a timecourse analysis to assess the timing of optimal RLU signaling (Figure 1A). Results showed that a minimum of 6 hours was needed to obtain a sufficiently strong RLU signal. Second, we investigated the optimal timing for an efficient vrna transfection and performed a vrna transfection time-course analysis, observing that the minimum time for an efficient transfection was 3 hours (Figure 1B). Third, to further optimize the assay, we investigated the optimal amounts of luciferase plasmid and vrna to be transfected into each 48-well plate. Hence, A549 cells were initially transfected with various amount of pgl IFN-β luc (125, 250, and 500 ng/well), and after 24 hours cells were additionally transfected with various amount of IAV PR8 RNA (75, 125, 250, and 500 ng/well). Luciferase activity was measured 6 hours after transfection. Results showed that optimal concentrations for both reporter vector and vrna were 250 ng (Figure 1C). We also tried to further miniaturize EBOV VP35 Inhibition of IFN-β Activation JID 2015:212 (Suppl 2) S279

Figure 2. The inhibitory effect of Ebola virus (EBOV) viral protein 35 (VP35) and influenza A virus (IAV) NS1 in the luciferase reporter gene assay in 48-well plates.

4 Figure 2. The inhibitory effect of Ebola virus (EBOV) viral protein 35 (VP35) and influenza A virus (IAV) NS1 in the luciferase reporter gene assay in 48-well plates. A549 cells were cotransfected with 250 ng of pgl interferon β (IFN-β) luc and various amounts (10, 45, 180, 750, and 3000 ng) of pcdna3-vp35 or pcdna3-ns1. Twenty-four hours after transfection, cells were additionally transfected with 250 ng of IAV RNA. After an additional 6 hours, cells were lysed, and luciferase activity was measured. the assay by using 96-well plates, but RLU signal readings were found to be too inconsistent to provide reproducible results (data not shown). Next, we asked whether this 48-well assay could be used to evaluate the ability of EBOV VP35 to inhibit the RIG-I mediated IFN-β induction. We subcloned the EBOV Zaire strain VP35 gene from the bacterial expression vector pet45b-zebov-vp35 [13] into the mammalian expression vector pcdna3. Since it is known that IAV NS1 protein strongly inhibits RIG-I mediated IFN-β production [12], we used NS1 as a control. The reporter vector was cotransfected with various amounts of pcdna3- VP35 or pcdna3-ns1 (10, 45, 180, 750, and 3000 ng), leading to different levels of viral protein expressions inside the cells (data not shown). Results showed a dose-dependent inhibition of dsrna-stimulated, RIG-I mediated IFN-β production by both EBOV VP35 and IAV NS1 (Figure 2). It is worthwhile to note that, at all plasmid concentrations, EBOV VP35 showed an inhibitory effect higher than the one shown by IAV NS1, suggesting that EBOV VP35 could be more efficient than IAV NS1 in inhibiting innate immune activation. DISCUSSION In 2014, the largest EVD outbreak in West Africa began, resulting in tens of thousands of cases and raising great concerns as it compromised public health systems and led to social disruptions. Cases of EVD have also been reported in both the United States and Europe, further increasing the level of public awareness of the threat of EVD diffusion. Development of drugs that can inhibit EBOV replication and counteract EVD progression is therefore a global health priority. EBOV VP35 is a valid drug target, since it is one of the most potent weapons that EBOV uses to evade the innate immune antiviral response [4 6, 9]. In fact, EBOV VP35 interferes at various levels of the RIG-I mediated signaling cascade that leads to type I IFN production, allowing undisturbed viral multiplication. We previously obtained a full-length recombinant version of EBOV VP35 [13] and characterized its dsrna binding function, using a newly established in vitro magnetic pull-down dsrna binding assay [15]. However, the need to assess the specific role that each VP35 function has in inhibiting the RIG-I mediated signaling cascade and to provide a means of screening potential inhibitors required the development of a robust and reproducible cellular assay that could measure dsrna-induced activation of the RIG- I signaling pathway. Until now, methods used to analyze the effects of VP35 on IFN-β promoter activation used cellular infection with SeV as an innate immunity stimulus [5]. This technique requires up to 24 hours of stimulation to measure the luminescence signal and a more complex experimental approach, including virus manipulation. In addition, SeV infection does not activate only the RIG-I mediated signaling pathway, which is the major VP35 target, eventually leading to IFN-β promoter activation through other pathways. An alternate, more-manageablemethodusediavdsrna transfection as a stimulus for the activation of the RIG-I signaling pathway that culminates in IFN-β production [14]. Therefore, we modified this assay by miniaturizing it from 12-well to 48- well plates, a format more suitable for small molecules screening, and optimized the assay condition, which only requires 6 hours of stimulation. Subsequently, we showed that EBOV VP35 expression can inhibit dsrna-induced RIG-I mediated IFN-β activation dose dependently, demonstrating that the assay can be used to identify EBOV VP35 inhibitors. Of note, compared with NS1, the inhibitory efficacy of EBOV VP35 was greater at higher transfected plasmid concentrations. More studies, however, should be performed to assess whether EBOV can suppress activation of innate immunity more effectively than IAV. In summary, we described the establishment of a new cellbased method to characterize EBOV VP35 properties related to its inhibition of the RIG-I mediated IFN-β induction and to identify small molecules that can interfere with these properties. Notes Acknowledgments. We thank Prof Stephan Ludwig and his staff at the Institute of Molecular Virology, University of Münster, for the kind support and insider tips. Potential conflicts of interest. All authors: No reported conflicts. S280 JID 2015:212 (Suppl 2) Cannas et al

5 All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. References 1. Centers for Disease Control and Prevention (CDC) Ebola outbreak in West Africa. Atlanta: CDC, ebola/outbreaks/2014-west-africa/index.html. Accessed 31 December Basler CF, Wang X, Mühlberger E, et al. The Ebola virus VP35 protein functions as a type I IFN antagonist. Proc Natl Acad Sci U S A 2000; 97: Cárdenas WB, Loo Y-M, Gale M, et al. Ebola virus VP35 protein binds double-stranded RNA and inhibits alpha/beta interferon production induced by RIG-I signaling. J Virol 2006; 80: Basler CF, Mikulasova A, Martinez-Sobrido L, et al. The Ebola virus VP35 protein inhibits activation of interferon regulatory factor 3. J Virol 2003; 77: Prins KC, Binning JM, Shabman RS, Leung DW, Amarasinghe GK, Basler CF. Basic residues within the ebolavirus VP35 protein are required for its viral polymerase cofactor function. J Virol 2010; 84: Leung DW, Prins KC, Basler CF, Amarasinghe GK. Ebolavirus VP35 is a multifunctional virulence factor. Virulence 2010; 1: Zinzula L, Tramontano E. Strategies of highly pathogenic RNA viruses to block dsrna detection by RIG-I-like receptors: hide, mask, hit. Antiviral Res 2013; 100: Schlee M, Roth A, Hornung V, et al. Recognition of 5 triphosphate by RIG-I helicase requires short blunt double-stranded RNA as contained in panhandle of negative-strand virus. Immunity 2009; 31: Hartman AL, Bird BH, Towner JS, Antoniadou Z-A, Zaki SR, Nichol ST. Inhibition of IRF-3 activation by VP35 is critical for the high level of virulence of Ebola virus. J Virol 2008; 82: Luthra P, Ramanan P, Mire CE, et al. Mutual antagonism between the Ebola virus VP35 protein and the RIG-I activator PACT determines infection outcome. Cell Host Microbe 2013; 14: Mitchell WM, Carter WA. The quest for effective Ebola treatment: Ebola VP35 is an evidence-based target for dsrna drugs. Emerg Microbes Infect 2014; 3:e Rückle A, Haasbach E, Julkunen I, Planz O, Ehrhardt C, Ludwig S. The NS1 protein of influenza A virus blocks RIG-I-mediated activation of the noncanonical NF-κB pathway and p52/relb-dependent gene expression in lung epithelial cells. J Virol 2012; 86: Zinzula L, Esposito F, Mühlberger E, et al. Purification and functional characterization of the full length recombinant Ebola virus VP35 protein expressed in E. coli. Protein Expr Purif 2009; 66: Hillesheim A, Nordhoff C, Boergeling Y, Ludwig S, Wixler V. β-catenin promotes the type I IFN synthesis and the IFN-dependent signaling response but is suppressed by influenza A virus-induced RIG-I/NF-κB signaling. Cell Commun Signal 2014; 12: Zinzula L, Esposito F, Pala D, Tramontano E. dsrna binding characterization of full length recombinant wild type and mutants Zaire ebolavirus VP35. Antiviral Res 2012; 93: EBOV VP35 Inhibition of IFN-β Activation JID 2015:212 (Suppl 2) S281

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