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Journal of General Virology (2007), 88, 981–990 DOI 10.1099/vir.0.82499-0 Thioaptamer decoy targeting of AP-1 proteins influences cytokine expression and the outcome of arenavirus infections Susan M. Fennewald,1,2 Erin P. Scott,1,24 Lihong Zhang,1,2 Xianbin Yang,3 Judith F. Aronson,1,2 David G. Gorenstein,3 Bruce A. Luxon,3 Robert E. Shope,1,23 David W. C. Beasley,1,2 Alan D. T. Barrett1,2 and Norbert K. Herzog1,2 1 Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555-0609, USA Correspondence Norbert K. Herzog 2 [email protected] Center for Biodefense and Emerging Infectious Diseases, Sealy Center for Structural Biology, University of Texas Medical Branch, Galveston, TX 77555-0609, USA 3 Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555-0609, USA Received 25 August 2006 Accepted 10 November 2006 Viral haemorrhagic fever (VHF) is caused by a number of viruses, including arenaviruses. The pathogenesis is believed to involve dysregulation of cytokine production. The arenaviruses Lassa virus and Pichinde virus have a tropism for macrophages and other reticuloendothelial cells and both appear to suppress the normal macrophage response to virus infection. A decoy thioaptamer, XBY-S2, was developed and was found to bind to AP-1 transcription factor proteins. The P388D1 macrophage-like cell line contains members of the AP-1 family which may act as negative regulators of AP-1-controlled transcription. XBY-S2 was found to bind to Fra-2 and JunB, and enhance the induction of cytokines IL-6, IL-8 and TNF-a, while reducing the binding to AP-1 promoter elements. Administration of XBY-S2 to Pichinde virus-infected guinea pigs resulted in a significant reduction in Pichinde virus-induced mortality and enhanced the expression of cytokines from primary guinea pig macrophages, which may contribute to its ability to increase survival of Pichinde virus-infected guinea pigs. These data demonstrate a proof of concept that thioaptamers can be used to modulate the outcome of in vivo viral infections by arenaviruses by the manipulation of transcription factors involved in the regulation of the immune response. INTRODUCTION Members of the family Arenaviridae (genus Arenavirus) are small RNA viruses that include the prototype Lymphocytic choriomeningitis virus (LCMV), Pichinde virus (PICV) that is non-pathogenic for humans, and highly pathogenic viruses that cause viral haemorrhagic fevers (VHF), including Lassa fever and Argentine, Venezuelan and Bolivian haemorrhagic fevers. Although arenaviruses infect macrophages and dendritic cells, the immune response to arenavirus infections is poorly understood and, as with many of the viruses causing VHF, it is unclear how these viruses evade the immune system or cause death with only limited cellular damage. Our laboratory has been using PICV to study the effects on the immune response of both lethal and nonlethal arenavirus infection of guinea pigs. Increases in TNF-a 3Dr Robert E. Shope died 19 January 2004 in Galveston. 4Present address: Department of Microbiology, University of Pennsylvania, Philadelphia, PA 19104, USA. 0008-2499 G 2007 SGM have been associated with fatal PICV infection of guinea pigs (Aronson et al., 1995), but only in the late stages of infection. Alternatively, there is evidence that swift elaboration of proinflammatory cytokines and early engagement of the innate immune response may help protect an infected host from lethal haemorrhagic fever (Baize et al., 1999; Peters et al., 1987; Leroy et al., 2000; Fisher-Hoch et al., 1988). We have been interested in developing strategies to interfere with the pathogenic sequence, or even boost early protective innate immune responses. Members of the AP-1 family of transcription factors are key regulators of a wide range of cellular processes including cell proliferation, cell death, cell differentiation, oncogenesis, inflammation and innate immune responses (Adcock, 1997). While not as strongly associated with the immune response as the NF-kB family of transcription factors, it is still involved in the transcriptional regulation of T-cell receptor alpha (Giese et al., 1995), beta-interferon (IFN-b) (Du et al., 1993; Merika et al., 1998; Thanos & Maniatis, Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 03 Aug 2017 10:24:16 Printed in Great Britain 981 S. M. Fennewald and others 1995), TNF-a (Falvo et al., 2000; Tsai et al., 2000) and other genes important to an antiviral immune response (Chinenov & Kerppola, 2001). Similar to the NF-kB family, the AP-1 transcription factors are composed of various dimers which can act as positive or negative regulators of transcription. The subunits are basic region–leucine zipper proteins of the Jun, Fos, Maf, ATF and cAMP response element-binding (CREB) protein subfamilies. The best-studied activator dimers are the Fos/Jun dimers, which bind to the canonical AP-1 site, and the CREB dimers, which bind to the similar CRE sites with some level of cross-binding. The significance of the various dimer combinations and binding sites are not fully understood at this time (Shaulian & Karin, 2001, 2002; Karin & Shaulian, 2001; De Cesare et al., 1999). The c-Jun, c-Fos and FosB proteins contain transactivation domains, while Fra-1, Fra-2 and some splice variants of FosB do not (Chinenov & Kerppola, 2001) and can act as negative regulators at AP-1binding sites (Suzuki et al., 1991; Sonobe et al., 1995). The regulation of AP-1 activity is complex and occurs through: (i) changes in jun and fos transcription and mRNA turnover, (ii) Fos and Jun protein turnover, (iii) post-translational modifications of both Fos and Jun proteins that modulate their activities and (iv) interactions with other transcription factors. Transcription factors such as AP-1 and NF-kB can be targeted for inhibition using RNA and DNA oligonucleotides acting as decoy ‘aptamers’, which bind and act as direct in vivo inhibitors. Various studies have demonstrated the potential of using specific decoy oligodeoxynucleotides (ODNs) as therapeutic or diagnostic reagents, and to dissect the specific roles of particular transcription factors in regulating the expression of various genes (Bielinska et al., 1990; Cho-Chung et al., 1999; Eleouet et al., 1998; Jin & Howe, 1997; Mann, 1998; Morishita et al., 1995, 1998; Osborne et al., 1997; Tomita et al., 1999; Park et al., 1999). Dithiophosphate oligonucleotides (S2-ODNs) and monothiophosphate oligonucleotides (S-ODNs), are taken up efficiently by cells and have enhanced binding to proteins (Marshall & Caruthers, 1993; Yang et al., 2002a; King et al., 2002). We previously developed thiophosphate-backbone modified aptamers (‘thioaptamers’) targeting NF-kB p50 and RelA p65 (Yang et al., 1999). Inc. Duplex oligonucleotides were annealed at 1.75 mM in 10 mM Tris/HCl (pH 7.6), 2 mM MgCl2, 50 mM NaCl, 1 mM EDTA by heating briefly at 95 uC and allowing them to cool slowly. Oligonucleotides were radiolabelled with T4 polynucleotide kinase (Promega) and [c-32P]ATP (DuPont NEN) under standard reaction conditions. Oligonucleotides containing phosphorodithioates (XBYS2, XBY-S1 and XBY-6) were synthesized using phosphorothioamidite chemistry as previously described (Yang et al., 1999). The thiophosphite triesters were oxidized with the sulfurization reagent 3-ethoxy-1,2,4-dithiazoline-5-one (Xu et al., 1996). After removal of the dimethoxytrityl group, the crude oligonucleotides were purified on a Mono Q HR 10/10 FPLC column (Pharmacia) (Yang et al., 2002b). The purified oligonucleotides were desalted and concentrated by ultrafiltration using Centricon-3 (Amicon) centrifugal concentrators. Equal molar quantities of complementary strands were mixed, annealed at 90 uC and slowly cooled to form the duplexes. Antibodies and recombinant proteins. Antibodies specific for ATF-2 (#9222), phosphorylated ATF-2 (#9221), c-Jun (c-JunB; #9162), phosphorylated c-Jun (#9261S), CREB (#9192) and phosphorylated CREB (#9191) were purchased from Cell Signaling Technology. Antibodies specific for c-Fos (210-128), FosB (210114), JunD (210-111) and JunB (210-112) were purchased from Alexis Biochemicals. Antibodies directed against Fra-1 (SC183X), Fra-2A (SC171X), Fra-2B (SC13017X), cAMP-responsive element modulator (CREM), JunB (SC73X), JunD (SC74X) and FosB (SC703X) were purchased from Santa Cruz Biotechnology. Antibody against c-Fos (c-FosA) was purchased from BD Biosciences and antibody to c-Jun (c-JunA) was purchased from Delta Biolabs. Human recombinant c-Jun was purchased from Promega. Cell lines and culture conditions. 70Z/3 (murine pre-B lymphocyte) cells were maintained in RPMI medium supplemented with 5 % fetal bovine serum (FBS), 1 % b-mercaptoethanol and 2 mM glutamine. They were treated with 10 mg Salmonella enterica serovar Typhosa LPS (W0901; Difco) ml21 for 6 h prior to extraction. P388D1 (murine monocyte-like) cells were maintained in RPMI medium supplemented with 5 % FBS and 2 mM glutamine. When indicated, they were treated with 0.1 mg LPS ml21 prior to processing for extracts. Alternatively, P388D1 cells were stimulated with poly(I : C) (25 mg ml21; Sigma-Aldrich) in Aim V medium (Invitrogen). Thioaptamer treatment of P388D1 cells. P388D1 cells were plated in T25 flasks in Aim V medium (Invitrogen). Oligonucleotide (25 mg) was mixed with 50 ml liposomes (Tfx-50, Promega) in 500 ml Aim V medium and added to the cells. Following a 2 h incubation, poly(I : C) (Sigma-Aldrich) was added to a final concentration of 25 mg ml21 and the cells were incubated for an additional 18 h before harvesting for nuclear extract preparation. Culture supernatants were used for cytokine measurements. Electrophoretic mobility shift assay (EMSA). Nuclear extracts In this study, we identify the binding specificity of a thioaptamer oligonucleotide, XBY-S2, that recognizes AP-1 and can be used to modulate the basal levels of macrophage cytokine expression as well as the expression of cytokines in response to stimulation with LPS. This thioaptamer alters cytokine gene expression in macrophage cell lines as well as primary macrophages and we show that it protects animals from a potentially lethal PICV infection. METHODS Preparation and purification of oligonucleotides. Single- stranded oligonucleotides were synthesized to order by Bio-Synthesis 982 were prepared following standard procedures as described previously (Dyer et al., 1993). For EMSA reactions, 1–5 mg nuclear extract was incubated with 0.1 pmol radiolabelled oligonucleotide in a 15 ml volume under standard reaction conditions [20 mM HEPES (pH 7.5), 50 mM KCl, 2.5 mM MgCl2, 20 mM dithiothreitol (DTT), 10 % glycerol, plus 50 mg poly(dI : C) and 0.1 mg BSA ml21] (Dyer & Herzog, 1995). For competition experiments, a 35-fold excess of unlabelled oligonucleotide was also added. For supershift experiments, nuclear extracts were incubated with antibody overnight at 4 uC in a 15 ml volume under standard reaction conditions in buffer lacking poly(dI : C) prior to the addition of the radiolabelled oligonucleotide and 50 mg poly(dI : C). Microaffinity isolation assay. Microaffinity purification (MAP) of proteins binding to the AP-1 and XBY-S2 oligonucleotides was Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 03 Aug 2017 10:24:16 Journal of General Virology 88 AP-1 inhibition and arenavirus infection performed by a two-step biotinylated DNA–streptavidin capture assay (Casola et al., 2000). In this assay, duplex oligonucleotides were chemically synthesized containing 59 biotin on a flexible linker (Bio-Synthesis). Dithioated aptamers with the biotin linker were synthesized in house. One milligram of P388D1 cell nuclear extract was incubated at 4 uC for 30 min with 50 pmol biotinylated AP-1 or XBY-S2, in the absence or presence of a 10-fold molar excess of non-biotinylated wild-type or mutated AP-1 sites. The binding buffer contained 8 mg poly(dI : C) (as non-specific competitor) and 5 % (v/v) glycerol, 12 mM HEPES, 80 mM NaCl, 5 mM DTT, 5 mM MgCl2 and 0.5 mM EDTA. One hundred microlitres of a 50 % slurry of prewashed streptavidin–agarose beads was then added to the sample, which was incubated at 4 uC for an additional 20 min with gentle rocking. Pellets were washed twice with 500 ml binding buffer and the washed pellets were resuspended in 100 ml 16 SDSPAGE sample buffer. After SDS-PAGE separation, proteins were transferred to Immobilon P (Millipore) membranes for immunoblot analysis. Immunoblots. Samples (10–20 mg) were electrophoresed by standard SDS-PAGE (8 % gels) and transferred to Immobilon-P. Filters were blocked in 5 % non-fat dry milk–Tris-buffered saline (TBS) overnight and then incubated with a primary antibody diluted in milk–TBS plus 100 mg BSA ml21 for 1 h. Following a series of TBS– Tween (0.5 %) washes, the filters were incubated with horseradish peroxidase-conjugated secondary antibody in milk–TBS–BSA for 1 h. Following another series of washes, the filters were soaked in the Pierce SuperSignal chemiluminescent substrate (Pierce Biotechnology) and exposed to film. Cytokine assays. IL-12p40, IL-12p40/p70, IL-6 and TNF-a from mouse cells were measured using ELISA kits (Biosource International) following the instructions of the manufacturer. Guinea pig TNF-a was measured by bioassay (Flick & Gifford, 1984) and guinea pig IL-8 was measured using an ELISA kit for human IL-8 (R&D Systems). Stably transfected reporter P388D1 assays of thioaptamer action. A reporter vector containing the firefly luciferase gene with AP-1 cis-enhancer elements (pHTS-AP1) was obtained from Biomyx Technology. Control vectors had a multiple cloning site in lieu of enhancer sequences upstream of the reporter gene (designated pHTS-MCS). P388D1 cells were maintained prior to transfection in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % FBS, 100 U penicillin ml21 and 100 mg streptomycin ml21. Cells were transfected using the calcium phosphate precipitation method and were subsequently placed in medium containing 300 mg hygromycin ml21. After 3–4 weeks, colonies were screened for the presence of the reporter gene by stimulating monolayers with 20 ng recombinant human TNF-a ml21 (R&D Systems), and assaying for luciferase activity 6–24 h later using the luciferase assay system (Promega). The P388D1 cells stably transfected with the pHTS-AP1 luciferase reporter plasmid were maintained with high-glucose DMEM containing 300 mg hygromycin ml21 and 10 % FBS. For the testing of thioaptamer influence on the expression of the AP-1 driven luciferase reporter gene, 56105 P388AP1 cells were seeded into the wells of a six-well plate and grown at 37 uC overnight. XBY-S2 (10 mg per well) was added to the appropriate wells and the cultures were grown for an additional 18 h. LPS (100 ng ml21) was then added to the appropriate wells. Each control and treatment group consisted of three wells. After 6 h, the cells were rinsed twice with PBS and then lysed by the addition of 400 ml 16 lysis buffer per well (40 mM Tricine pH 7.8, 50 mM NaCl, 2 mM EDTA, 1 mM MgSO4, 5 mM DTT and 1 % Triton X-100) and incubated for 15 min at room temperature with gentle rocking. Samples were either used immediately to assay the luciferase activity or stored at 280 uC. http://vir.sgmjournals.org Virus and aptamer/liposome preparation. Pooled guinea pig spleen stock of PICV P18 virus derived from Pichinde Munchique strain (CoAn 4763) was diluted to 1000 p.f.u. ml21 in endotoxinfree PBS containing Ca2+ and Mg2+. For XBY-S2 and XBY-S1 treatment, Tfx-50 liposomes (Promega) were used as a delivery vehicle, with a constant lipid : DNA charge ratio of 1.3 : 1 (Ono et al., 1998) and prepared according to the manufacturer’s instructions. Aptamer/liposome was then diluted in endotoxin-free PBS containing Ca2+ and Mg2+ to a final concentration of 50 mg aptamer ml21. Animal XBY-S2 treatment/virus inoculation. Male Hartley outbred guinea pigs, approximately 6 weeks old, were obtained from Charles River Laboratories (colony K81). Guinea pigs were treated 2 h prior to virus infection with a 1 ml intraperitoneal (ip) injection of XBY-S2 in liposomes (50 mg thioaptamer ml21). A control group received an ip injection of endotoxin-free PBS only. Two hours after the first XBY-S2/liposome injection, 1000 p.f.u. PICV P18 was inoculated ip into guinea pigs. Two days post-virus infection, guinea pigs received a second injection of thioaptamer/liposomes or PBS at the same dose as day 0. Compiled data from two separate experiments are shown, representing a total of 18 guinea pigs per group. For XBY-S1 treatment, the injections were given 1 day before infection and on days 1 and 3 post-infection, with six animals per group. Thioaptamer treatment of primary guinea pig macrophages. Peritoneal macrophages from three male outbred Charles River guinea pigs (350 g each) were harvested by aseptic peritoneal lavage with 100 ml sterile PBS (Ca- and Mg-free). The cells were collected by centrifugation, resuspended in 2.5 ml red cell lysis buffer (0.15 M NH4Cl, 0.1 mM Na2–EDTA, 1.0 mM KHCO3, pH 7.3) and placed in a 37 uC incubator for 5 min with occasional mixing. We then added 2.5 ml 16 RPMI 1640 medium supplemented with 10 % (v/v) FBS, 100 U penicillin ml21, 100 mg streptomycin ml21 and 2 mM L-glutamine and plated cells into 96-well plates at a concentration of 16105 cells per well. Cells were then treated with XBY-S2 for 24 h. The supernatants were collected and analysed for TNF-a by bioassay (Aronson et al., 1995; Flick & Gifford, 1984) and IL-8 using a human IL-8 ELISA (R&D Systems) previously shown to be applicable for measuring guinea pig IL-8 (Kuo et al., 1997). Statistics. Where indicated, statistical significance of differences between groups was determined using Student’s t-test. LogRank analysis was performed to compare survival curves for treated and untreated animals (Sigmastat 3.0; Systat Software). RESULTS XBY-S2 influences the outcome of lethal PICV infection We have previously shown that increases in TNF-a have been associated with fatal PICV infection of guinea pigs (Aronson et al., 1995). Therefore, we hypothesized that inhibition of transcriptional factors would modulate the host immune response and reduce PICV-induced mortality in our guinea pig model. We have previously described utilization of systematic evolution of ligands by exponential enrichment (SELEX) technology to develop thioaptamers against several target proteins, including transcription factors (Yang et al., 2002a). Briefly, several double-stranded oligomers targeting transcription factors were tested for their ability to modulate an in vivo PICV infection. In this study we synthesized XBY-S2, a 14 bp DNA duplex that contains six dithioate residues (Table 1) (three on each Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 03 Aug 2017 10:24:16 983 S. M. Fennewald and others Table 1. Oligonucleotides used in this study ‘s’ denotes a dithioated linkage. Binding sites are in bold. ODN name XBY-S0 XBY-S1 XBY-S2 XBY-6 AP-1 CREB NF-kB ISRE ODN sequence CCAGGTCAGATCTG GGTCCAGTCTAGTG TsTsGCGCGC-A-AC-ATsG A-A-CGCGCGsTsTGsTA-C CC-AGTsG-ACTsC-AGTsG GGsTCA-CsTGA-GsTCA-C CC-AGG-AG-ATsTsCC-AC GGsTCCsTCsTA-A-GGsTG CGCTTGATGACTCACCGGAA GCGAACTACTGAGTGGCCTT AGAGATTGCCTGACGTCAGAGAGCTAG TCTCTAACGGACTGCAGTCTCTCGATC AGTTGAGGGGACTTTCCCAGGC TCAACTCCCCTGAAAGGGTCCG GATCGGGAAACCGAAACTGAAGCC CTAGCCCTTTGGCTTTGACTTCGG strand) that includes a consensus AP-1-binding site. Dithioate linkages contain sulfur replacements for the two non-bridging oxygens in the DNA phosphate linkage. XBYS0 and XBY-S1 are oligonucleotides containing the same base composition in each strand. XBY-S0 lacks dithioate substitutions; XBY-S1 contains the same number of dithioate linkages as XBY-S2. The other oligonucleotides used in this study are also shown in Table 1. The effectiveness of the oligonucleotides was determined in PICVinfected guinea pigs. Guinea pigs were treated with thioaptamer on days 0 and 2 relative to time of infection with a lethal dose of PICV (P18). The most effective oligonucleotide at modulating PICV infection in guinea pigs was XBY-S2. Treatment with XBY-S2 increased and prolonged the survival of PICV-infected animals (Fig. 1a). The majority (61 %) of the animals treated survived virus infection, which was a significant improvement over the 22 % survival rate for the untreated animals (P=0.013, LogRank survival analysis). In contrast, thioaptamer XBYS1 was tested with doses administered at 1 day before infection, and 1 and 3 days post-infection. It not only failed to modulate the virus infection but also decreased survival, though it was not statistically significant (Fig. 1b). Additional experimentation is under way to determine the optimal dosage and time of administration to protect animals from virulent arenavirus infection. EMSA analysis of P388D1 and 70Z/3 cells Since XBY-S2 proved to be effective against PICV infection in guinea pigs, we sought to investigate the mechanism of action and binding specificity of XBY-S2. An examination of the XBY-S2 sequence revealed an AP-1 consensus binding site TGA(C/G)TCA that resembled the related CRE-binding 984 Fig. 1. XBY-S2 prolongs and increases survival of PICVinfected guinea pigs. (a) Guinea pigs were treated with XBYS2 (solid line) at 2 h prior to infection with a lethal dose of PICV variant P18. A control group received an injection of endotoxin-free PBS only (dashed line). Mortality was monitored for 40 days post-infection. (b) XBY-S1 (solid line) decreases survival; treatment was at ”1, 1 and 3 days post-infection. site, TGACgTCAG. The binding specificity of the XBY-S2 oligonucleotide was determined using lysates from mouse P388D1 macrophages and 70Z/3 mouse pre-B cells. 32Plabelled XBY-S2 was found to bind specifically with proteins in the extracts, forming one discrete band (Fig. 2a). Fully phosphorothioate-substituted oligonucleotides are unable to show binding specificity in this assay due to very high levels of non-specific binding. Only a subset of the partially dithioate-substituted oligonucleotides show binding, depending on the sequence specificity of their binding sites. The XBY-S2 binding was successfully competed by unlabelled XBY-S2, indicating specific binding, and by AP-1 and CREB oligonucleotides, indicating that the binding was to proteins in the AP-1 family. Neither an oligonucleotide with an NF-kB nor an interferon-stimulated response element (ISRE)-binding site was capable of competing for binding, indicating that these immune-associated transcription factors are not bound by XBY-S2 (Fig. 2a). In the reciprocal binding assays, binding of AP-1 proteins by their consensus oligonucleotide was successfully competed by XBY-S2, confirming that XBY-S2 was binding to the same proteins as the AP-1 consensus oligonucleotide (Fig. 2b). XBY-S2 was also specifically bound by human recombinant AP-1 and c-Jun protein dimers (data not shown). Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 03 Aug 2017 10:24:16 Journal of General Virology 88 AP-1 inhibition and arenavirus infection Identification of the AP-1 complexes binding to the AP-1 and XBY-S2 oligonucleotides Fig. 2. XBY-S2 binds to AP-1 proteins in both macrophage and B cell lines. EMSA analysis to determine the complexes present in mouse 70Z/3 pre-B cells (activated with LPS) and P388D1 mouse macrophage-like cells [activated with poly(I : C)] using either a consensus AP-1 binding site (a) or XBY-S2 thioaptamer (b). Competition with 50-fold molar excess unlabelled transcription factor-binding site oligonucleotides (AP-1, CREB, XBY-S2, NFkB and ISRE) in the EMSA reaction was performed as indicated. There are more than 10 different members of the AP-1 family of proteins, which dimerize in various combinations. To determine which of the AP-1 proteins were binding to the XBY-S2 oligonucleotide, we used a combination of immunoblots, supershift EMSAs and MAP. Immunoblots revealed that P388D1 cells contain JunB, c-Jun, Fra-2, CREB, CREM and possibly JunD proteins (Fig. 3a). High background in immunoblots using anti-Fra-2(A) and antic-Fos(A) precluded their use and inclusion in the selection shown in Fig. 3(a). Subsequently, supershift EMSA reactions were used to determine which of the AP-1 proteins were binding to XBY-S2 on the EMSA gels (Fig. 3b). Where possible, we used more than one antibody from different sources, or specific to different portions of an AP-1 protein, to confirm these results. Supershift analysis revealed that AP-1 complexes in stimulated P388D1 cells contained detectable levels of Fra-2, c-Fos, JunB and JunD, as well as c-Jun and phosphorylated c-Jun (Fig. 3b). We were unable to detect the presence of active Fra-1, FosB, ATF2, CREM and CREB using supershift assays. Most of the AP-1 complexes contain Fra-2 and/or JunB since the combination of antibodies can supershift the majority of the complexes (data not shown). The same experiment was performed to identify the AP-1 proteins that bind to XBY-S2 (Fig. 3b). Fra-2, c-Fos, JunB and possibly c-Jun were found in complexes that bound XBY-S2. Fig. 3. Determination of AP-1 proteins capable of binding to the XBY-S2 thioaptamer decoy. Abbreviations: pc-Jun, phosphorylated c-Jun; pATF-2, phosphorylated ATF-2; pCREB, phosphorylated CREB. (a) Immunoblot analysis of P388D1 nuclear extracts with the indicated antibodies for AP-1/ CREB family members. Two separate antibodies for Fra-2, c-Jun and c-Fos were used and are distinguished by the A and B designation. (b) Supershift analysis of P388D1 nuclear protein complexes binding to either the AP-1 consensus oligonucleotide or the XBY-S2 thioaptamer. Antibodies are the same as those used for immunoblots and were added to each of the EMSA reactions to achieve a supershift as indicated. Supershifted bands are indicated by a dot. (c) MAP of proteins in P388D1 nuclei which bind to the XBY-S2 aptamer was followed by immunoblot analysis of the proteins that were bound. http://vir.sgmjournals.org Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 03 Aug 2017 10:24:16 985 S. M. Fennewald and others Due to the limited availability of antibodies that are effective in supershift analysis, we also used MAP in conjunction with immunoblotting to confirm the results of negative supershift data. MAP is a more sensitive assay than either immunoblotting or EMSA. XBY-S2 was end-labelled with biotin and streptavidin beads were used to purify any proteins that were bound to it. MAP results established that XBY-S2 was recognized by AP-1 dimers that also included CREM, but there was still no detectable ATF-2 or FosB (Fig. 3c). Either the complexes containing CREM are not detectable by EMSA, or the CREM antibody is not effective when used in the EMSA supershift protocol, but is effective in immunoblot analysis. Treatment of P388D1 cells with XBY-S2 eliminates AP-1 DNA-binding activity XBY-S2 includes a consensus AP-1-binding site [59-TGA(G/ C)TCA-39] and, when used as a decoy aptamer, it would be expected to influence the expression of cytokines from macrophages by acting to sequester the AP-1 proteins in cells. In order to demonstrate that XBY-S2 functions as a decoy, P388D1 macrophage cultures were treated in triplicate with liposomes, either with or without XBY-S2, for 2 h prior to stimulation with LPS; liposomes were used to deliver the thioaptamer into cells more efficiently. XBY-S0 was used as a control oligonucleotide since it contains the same base composition as the XBY-S2 oligonucleotide, though it does not contain any dithioate modifications. Nuclear extracts were harvested at 16 h post-stimulation with LPS and analysed by EMSA. The EMSA gels were quantified, to allow statistical analysis, and the results are depicted graphically in Fig. 4(a). Treatment of cells with XBY-S2 significantly reduced the AP-1-binding activity. Treatment with the control oligonucleotide XBY-S0 led to a non-significant reduction of AP-1-binding activity and was significantly higher than the levels of AP-1 in the XBY-S2treated cells. Therefore, the XBY-S2 thioaptamer appears to be an efficient and specific inhibitor of AP-1 transcription factor DNA-binding activity. Reporter assays confirm that XBY-S2 treatment of P388 cells inhibits AP-1 transcription factors activity In order to confirm that the XBY-S2 thioaptamer altered AP-1 regulated transcription, we established a P388D1derived cell line (P388AP1) stably transformed with an AP-1 driven luciferase reporter plasmid, pHTS-AP1 (Biomyx Technology). This plasmid contains a tandem repeat of six copies of an AP-1 site (TGACTAA) linked to the luciferase gene. Fig. 4(b) illustrates that the P388AP1 reporter cell line responds to LPS stimulation with a twofold increase in the expression of luciferase. Treatment with XBY-S2 alone stimulated nearly the same increase in luciferase expression as LPS stimulation. LPS in addition to XBY-S2 resulted in an additive increase of luciferase expression. Treatment with a thioaptamer without an AP-1 site had no effect on AP-1driven expression of luciferase (data not shown). Thus, 986 Fig. 4. XBY-S2 eliminates AP-1 DNA-binding activities in macrophages and alters AP-1 reporter gene expression in P388D1 cells. (a) P388D1 cells were incubated with liposomes with or without the indicated thioaptamers for 24 h prior to stimulation with LPS. Binding to an AP-1 consensus binding site in nuclear extracts was analysed by EMSA. InstantImager quantification of the gel is depicted in (a). Statistical analysis used Student’s t-test. (b) P388D1 cells stably transfected (P388AP1) with an AP-1-driven luciferase reporter plasmid, pHTS-AP1, were stimulated with 100 ng LPS ml”1 for 6 h with and without pretreatment overnight with 10 mg XBY-S2 per well and then assayed for luciferase activity. XBY-S2 appears to function by increasing AP-1-driven transcription, possibly by blocking Fra-2, the repressive AP-1 subunit that is present in large amounts in these cells. XBY-S2 perturbation of cytokine expression in P388D1 cells in response to poly(I : C) Poly(I : C) is a potent inducer of cytokine expression through Toll-like receptors (Alexopoulou et al., 2001). To determine whether XBY-S2 can influence cytokine gene expression, cytokines elaborated from macrophages following poly(I : C) stimulation were measured in combination with prior treatment with the XBY-S2 thioaptamer. As above, liposomes were used to deliver the aptamer more efficiently. A 2 h pre-treatment with XBY-S2 increased the expression of IL-12p40+p70 (sixfold), IL-6 (3.5-fold), and TNF-a (1.7-fold) over the levels expressed in cells stimulated with poly(I : C) alone (Fig. 5). IL12p70 was only detectable in those cells treated with XBY-S2 and poly(I : C). Interestingly, liposome treatment without thioaptamer appeared to Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 03 Aug 2017 10:24:16 Journal of General Virology 88 AP-1 inhibition and arenavirus infection cytokine production from primary peritoneal macrophages was examined following treatment with the XBY-S2 thioaptamer. Due to the limited availability of anti-guinea pig-based reagents, we are limited as to the cytokines that can be measured in the guinea pig. The supernatants of cultured primary guinea pig peritoneal macrophages were assayed for TNF-a by standard bioassay and IL-8 was measured by ELISA based on human IL-8. These two cytokines were chosen because they are among the few guinea pig macrophage-derived cytokines for which available standardized assays exist, and because AP-1 is known to be important in regulation of their transcription (Hoffmann et al., 2002). XBY-S2 increased the expression of IL-8 in a dose-dependent fashion over the levels expressed in untreated macrophages (Fig. 6). At doses below 20 mg per 105 cells, XBY-S2 appeared to increase basal level of TNF-a expression, but there was no clear dose dependence at higher doses. Therefore, these data, in part, confirm the results seen in the P388D1 cell line, which indicate that XBY-S2 either directly targets AP-1 proteins that repress the transcription of these cytokines, or AP-1 regulates the expression of another protein that serves as a repressor. XBY-S2 does not block the activities of transcription factors that activate the expression of these cytokines. However, XBY-S2 increases the basal level of expression of pro-inflammatory cytokines that could influence innate immunity induced in response to viral challenge. DISCUSSION PICV infection of guinea pigs causes a syndrome that is indicative of immune system malfunction (Aronson et al., 1995; Peters et al., 1989). Infection of guinea pigs with the virulent P18 variant is characterized by dysregulated Fig. 5. Pretreatment of P388D1 cells with XBY-S2 increases the levels of cytokine expression in response to poly(I : C) stimulation. P388D1 cells were treated with XBY-S2 liposome preparations or liposomes alone 1 h prior to stimulation with poly(I : C) (25 mg ml”1). Cell culture supernatants were collected 24 h after stimulation and the levels of the cytokines measured by ELISA. suppress cytokine gene expression under these conditions. These data suggest that either binding of AP-1 proteins to the XBY-S2 decoy aptamer eliminates transcriptional repressors of these cytokines, or a specific set of AP-1 proteins regulates the expression of another protein that serves as a repressor. These data also suggest that XBY-S2 modulation of AP-1 proteins alters the expression of proinflammatory cytokines that could influence innate immunity induced in response to viral challenge. XBY-S2 perturbation of cytokine expression in primary guinea pig macrophages To determine whether XBY-S2 can also influence cytokine gene expression in primary cells from guinea pigs, the http://vir.sgmjournals.org Fig. 6. XBY-S2 increases cytokine expression in primary guinea pig macrophages. Primary peritoneal macrophages were harvested by lavage from guinea pigs and treated with varying amounts of XBY-S2 for 48 h. TNF-a and IL-8 were measured in culture supernatants. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 03 Aug 2017 10:24:16 987 S. M. Fennewald and others pro-inflammatory cytokine production (Aronson et al., 1995) with profound terminal shock. The macrophage tropism of PICV is a characteristic of many haemorrhagic fever viruses (reviewed by Peters et al., 1989) and may be an important contributor to either the ability of the virus to evade the immune system and/or the eventual fatal shock syndrome. Virus infection of macrophages presents an opportunity for a virus to evade the immune system by modulating the immune response of the infected cell. Inhibition of macrophage activation would favour viral spread, as activation of macrophages is known to increase the microbicidal action of these phagocytes (Oswald et al., 1992). Therefore, treatment with an immunomodulating agent such as the thioaptamer XBY-S2 may alter the expression of a number of cytokines that are important in generating an effective early innate response, in the development of the protective Th-1 type antiviral response critical in determining the outcome of infection, and ultimately the development of the adaptive immune response. In previous studies, macrophages explanted from PICV-infected guinea pigs and stimulated ex vivo with LPS showed some suppression of TNF-a induction (Aronson et al., 1994; Fennewald et al., 2002). We previously described that the attenuated PICV P2 variant is associated with the appearance of activated forms of transcription factors NF-kB in macrophages, while the lethal PICV P18 variant is associated with the ‘non-activated’ state of these transcription factors that appears to correlate with a failure to mount an effective immune response (Fennewald et al., 2002). Here we demonstrate that modulation of the DNA-binding activity of another transcription factor, AP-1, appears to eliminate the repression of cytokine gene expression, elevating the basal and, in some circumstances, the induced cytokines. Modulation of AP-1 is capable of protecting guinea pigs from lethal PICV infection and we hypothesize that this is the result of changes in host cell gene expression that counteract inhibitions by pathogenic PICV. Though we were somewhat surprised to find that our most active aptamer was binding to AP-1, it is known that AP-1 is important in the immune response (Adcock, 1997). Although there are over 50 members of the AP-1 family of proteins and potentially over 1000 different homo- and heterodimeric forms (Newman & Keating, 2003), the majority of the detectable AP-1 DNA-binding activity in mouse macrophages consists of dimers that include Fra-2 and JunB. As Fra-2 can also negatively regulate c-Jun activity (Suzuki et al., 1991; Sonobe et al., 1995) and can form heterodimers with JunB that act as transcriptional repressors in keratinocytes (Rutberg et al., 1997), it is conceivable that the negative influence of c-Jun and JunB on basal cytokine expression in macrophages may arise from formation of repressive Jun/Fra-2 heterodimers. The treatment of macrophages with the thioaptamer XBY-S2 can completely abrogate AP-1 DNA binding activity and hence could be acting as a decoy to effectively outcompete the repressive Jun/Fra-2 dimers. By modulating the activity of AP-1 we 988 have shown that we can increase the expression of several key cytokines including TNF-a, IL-8, IL-6 and IL-12 and increase the survival of guinea pigs to lethal PICV infection. The increased expression of IL-12 may be particularly important. It is consistent with the inhibitory action of some AP-1 dimers and the work of Roy et al. (1999), who demonstrated that a deficiency in c-Fos increased macrophage IL-12 production. Previous research has demonstrated that IL-12 administration at low doses can be efficacious in mice infected with LCMV, resulting in inhibition of virus replication and enhanced CD-8 responses (Orange et al., 1995). Similarly, low doses of IL-12 are also effective in protecting mice against Encephalomyelocarditis virus, Murine hepatitis virus and herpes simplex virus, and promotes clearance of vesicular stomatitis virus while having other positive effects on the outcome of a number of other virus infections (Komastu et al., 1998). Though we are unable to confirm the observations in vivo in PICVinfected guinea pigs due to the lack of antibody reagents or a bioassay, the increase in IL-12 production following XBY-S2 treatment could contribute to the efficacy in the animal. As with all treatments, it is not entirely proven that the in vivo efficacy is due to the measured in vitro activity – that the ability of XBY-S2 to prolong survival is due to its in vitro ability to inhibit AP-1 proteins. While phosphorothioate oligonucleotides are known to give strong non-specific effects, our modestly modified aptamers continue to show specific binding in assays and only the XBY-S2 aptamer showed significant protection in the animal model. Still, we cannot rule out the possibility that additional activities, including Toll-like receptor responses, are important in the in vivo efficacy. The XBY-S2 activity increases the interest in the AP-1 proteins and their significance in both positive and negative regulation of the immune response. Inhibition of AP-1 with XBY-S2 treatment increases cytokine gene expression and protects against virus infection. It is of interest to note that Lassa virus infection of macrophages leads to the release of virus particles, but not to an increase in TNF-a, IL-1b, IL-12p35 and p40, IL-10, IL-6, TGF-b, IFN-c or CD25 synthesis (Baize et al., 2004). Interestingly, these authors also demonstrated that Lassa virus infection of dendritic cells led to the expression of elevated levels of IL-8 but no other chemokines. Therefore, our data using virulent PICV correlate with results from studies with Lassa virus that indicate that there is viral suppression of the normal response of macrophages and dendritic cells to dsRNA or viral infection. ACKNOWLEDGEMENTS We wish to express our deep regrets at the death of Dr Robert E. Shope on 19 January 2004 in Galveston and to acknowledge his leadership and participation in this research. Dr Shope had a world-class career of more than 40 years of identifying and isolating infectious diseases worldwide. His contributions to the scientific arena are extensive, but more importantly, he consistently exemplified and fostered an attitude Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 03 Aug 2017 10:24:16 Journal of General Virology 88 AP-1 inhibition and arenavirus infection of service and collegiality. Dr Shope was admired and beloved by students and scientific colleagues alike, and his contributions to science and society will long be remembered. We wish to thank Barry Elsom for his excellent technical assistance and Dr Debra Kallick for her helpful discussions. This work was supported by grants from DARPA (DAAD19011037), DTRA (DAAD17-01-D0001), NIH (N01-HV28184, U01 AI054827, R01 A127744) and by a grant from NIAID to Dr Herzog through the Western Regional Center of Excellence for Biodefence and Emerging Infectious Disease Research, NIH grant number U54 AI057156. Falvo, J. V., Uglialoro, A. M., Brinkman, B. M., Merika, M., Parekh, B. S., Tsai, E. Y., King, H. 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Shaulian, E. & Karin, M. (2001). AP-1 in cell proliferation and survival. Oncogene 20, 2390–2400. 990 Yang, X., Hodge, R. P., Luxon, B. A., Shope, R. & Gorenstein, D. G. (2002b). Separation of synthetic oligonucleotide dithioates from monothiophosphate impurities by anion-exchange chromatography on a mono-q column. Anal Biochem 306, 92–99. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 03 Aug 2017 10:24:16 Journal of General Virology 88