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The Journal of Immunology, 2006, 176: 451-462.
Copyright © 2006 by The American Association of Immunologists

The Viral Protein A238L Inhibits TNF-{alpha} Expression through a CBP/p300 Transcriptional Coactivators Pathway1

Aitor G. Granja*, Maria L. Nogal*, Carolina Hurtado*, Carmen del Aguila{dagger}, Angel L. Carrascosa*, María L. Salas*, Manuel Fresno* and Yolanda Revilla2,*

* Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Cientificas-Universidad Autónoma de Madrid), Universidad Autónoma de Madrid, Madrid, Spain; and {dagger} Universidad San Pablo, Centro de Enseñanza Universitaria, Madrid, Spain


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
African swine fever virus (ASFV) is able to inhibit TNF-{alpha}-induced gene expression through the synthesis of A238L protein. This was shown by the use of deletion mutants lacking the A238L gene from the Vero cell-adapted Ba71V ASFV strain and from the virulent isolate E70. To further analyze the molecular mechanism by which the viral gene controls TNF-{alpha}, we have used Jurkat cells stably transfected with the viral gene to identify the TNF-{alpha} regulatory elements involved in the induction of the gene after stimulation with PMA and calcium ionophore. We have thus identified the cAMP-responsive element and {kappa}3 sites on the TNF-{alpha} promoter as the responsible of the gene activation, and demonstrate that A238L inhibits TNF-{alpha} expression through these DNA binding sites. This inhibition was partially reverted by overexpression of the transcriptional factors NF-AT, NF-{kappa}B, and c-Jun. Furthermore, we present evidence that A238L inhibits the activation of TNF-{alpha} by modulating NF-{kappa}B, NF-AT, and c-Jun trans activation through a mechanism that involves CREB binding protein/p300 function, because overexpression of these transcriptional coactivators recovers TNF-{alpha} promoter activity. In addition, we show that A238L is a nuclear protein that binds to the cyclic AMP-responsive element/{kappa}3 complex, thus displacing the CREB binding protein/p300 coactivators. Taken together, these results establish a novel mechanism in the control of TNF-{alpha} gene expression by a viral protein that could represent an efficient strategy used by ASFV to evade the innate immune response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The TNF-{alpha} is a pleiotropic cytokine able to inhibit viral infection. TNF-{alpha} is secreted by multiple cell types such as macrophages and lymphocytes after viral infection or stimulation through cell surface receptors, and induces the synthesis of a wide array of molecules that mediate the inflammatory response and regulate immune cell function (1). TNF-{alpha} has been demonstrated to act as a potent antiviral cytokine that is directly cytotoxic to cells infected with both RNA and DNA viruses (2), and has been shown to inhibit the replication of HSV, varicella-zoster virus, CMVs, and African swine fever virus (ASFV)3 in vitro (3, 4, 5).

The promoter sequences required for induction of the human TNF-{alpha} gene by a variety of stimuli, including viruses, phorbol esters, calcium ionophore, or LPS, have been identified, and serve as the primary control point of the regulation of TNF-{alpha} production (6, 7, 8, 9). These studies have established that NF-{kappa}B, NF-AT, activating transcription factor-2 (ATF-2), Jun, Ets/Elk, and Sp-1 proteins and the CREB-binding protein (CBP) and p300 coactivator proteins are involved in the specific regulation of the human TNF-{alpha} gene, depending on the cell type and the stimuli (6, 10, 11, 12, 13).

In activated T cells, TNF-{alpha} is an immediately early gene induced by calcium fluxes through a calcineurin-dependent process. This activation requires a cAMP response element (CRE) (14), which binds ATF-2/Jun, and two NF-AT binding sites, the –76-NF-AT and {kappa}3-NF-AT sites (6). In macrophages, the basal and PMA-induced TNF-{alpha} promoter activity is very similar to that observed in T lymphocytes. Expression of c-Jun in U937 human macrophage and in MLA 144 T cell lines caused consistent augmentation of promoter activity, indicating that AP-1 plays an important role in transcriptional regulation of the TNF-{alpha} promoter (8).

ASFV, the sole member of the Asfarviridae family (15), encodes a protein, A238L, which has been described to inhibit calcineurin phosphatase activity (16). A238L also down-regulates the activation of the NF-{kappa}B and NF-AT transcription factors, both when expressed in Jurkat cells or during ASFV infection (17, 18). In a previous report, we have shown that ASFV is thus able to control the transcriptional activation of immunomodulatory genes dependent on NF-{kappa}B and NF-AT pathways, such as cyclooxygenase-2 (COX-2) (18).

In this study, we have investigated the ability of ASFV to inhibit TNF-{alpha} gene expression through the synthesis of A238L protein. To achieve this, we have used deletion mutants lacking the A238L gene from the Vero cell-adapted Ba71V ASFV strain and from the virulent isolate E70. Using these tools, we have characterized events involved in TNF-{alpha} gene induction following infection of Vero cells or porcine macrophages, the natural target of the infection. To further analyze the function of A238L on the control of TNF-{alpha}, we have used Jurkat cells stably transfected with the viral gene to identify the TNF-{alpha} promoter regulatory elements involved in the induction of the gene after stimulation with PMA plus calcium ionophore (PMA/Ion) and the modulation by A238L. We have identified the CRE and {kappa}3 sites on the TNF-{alpha} promoter as the responsible of the gene activation in our system, and demonstrate that A238L inhibits the TNF-{alpha} expression through these sites. This inhibition was reverted by overexpression of NF-AT, NF-{kappa}B, and c-Jun, but not by c-Fos. Furthermore, we present preliminary evidence that the mechanism by which A238L inhibits the activation of TNF-{alpha} involves the modulation of trans activation of NF-{kappa}B, NF-AT, and c-Jun, through the transcriptional coactivators CBP and p300. Taken together, these results establish a novel mechanism in the control of TNF-{alpha} gene expression by a viral protein that could represent an efficient strategy used by ASFV to evade the innate immune response.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell culture, viruses, and reagents

Vero (African green monkey kidney) cells and COS-7 (African green monkey kidney) cells were obtained from the American Type Culture Collection (ATCC) and grown in DMEM supplemented with 5% FBS (Invitrogen Life Technologies). Jurkat human leukemia T cell line was obtained from the ATCC and cultured in RPMI 1640 medium supplemented with 10% FBS. Porcine alveolar macrophages were obtained by bronchoalveolar lavage of pigs and cultured in DMEM supplemented with 10% homologous swine serum (19). All medium were supplemented with 2 mM L-glutamine, 100 U/ml gentamicin, and nonessential amino acids. Cells were grown at 37°C in 7% CO2 in air saturated with water vapor. Jurkat and Vero cells were stimulated by PMA (Sigma-Aldrich) at 15 ng/ml and A23187 Ion (Sigma-Aldrich) at 1 µM. Cyclosporin A (CsA, 100 ng/ml; Novartis Pharmaceuticals) was added 1 h before the addition of PMA and Ion. The Vero-adapted ASFV strain Ba71V was propagated and titrated by plaque assay on Vero cells, as described (20). The virulent ASFV strain E70 was propagated and titrated by plaque assay on swine alveolar macrophages, as described (19, 21).

ASFV A238L deletion mutants construction

The A238L-defective mutant {Delta}A238L viruses were obtained by insertion of the Escherichia coli {beta}-glucuronidase ({beta}-gus) gene into the viral A238L open reading frame. The recombinant Ba71V{Delta}A238L virus was obtained, as previously described (18). The recombinant E70{Delta}A238L was obtained using the same transfection-infection protocol, but infecting COS-7 cell monolayers with the viral strain E70.

The lack of gene in the recombinant E70{Delta}A238L virus was assessed by Southern blot hybridization. Briefly, DNA samples obtained from purified E70 and {Delta}A238L viruses were digested with the restriction endonuclease EcoRI, subjected to electrophoresis in agarose gels, and transferred to nylon membranes following standard procedures (22). The DNA probes, specific for the {beta}-gus and A238L genes and for the SalI I' fragment of E70 genome, were labeled with a digoxigenin DNA labeling kit (Boehringer Mannheim) using manufacturer’s instructions, and hybridizations were done, as described elsewhere (23).

mRNA analysis

Total RNA was prepared from Jurkat-pcDNA, Jurkat-A238L, ASFV-infected porcine macrophages, or ASFV-infected Vero cells by the TRIzol reagent RNA protocol (Invitrogen Life Technologies). Total RNA (1 µg) was reverse transcribed into cDNA by the RevertAid First Strand cDNA synthesis kit (MBI Fermentas), and used for PCR amplification with the addition of TaqDNA polymerase (Roche) following the manufacturer’s instructions. Specific primers used in PCR were porcine TNF-{alpha} (forward, 5'-CTCTTCTGCCTACTGCACTTCGAGG-3' and reverse, 5'-CTGGGAGTAGATGAGGTACAGCCCA-3'), porcine GAPDH (forward, 5'-AGCTTGTCATCAATGGAAAGG-3' and reverse, 5'-AGAAGCAGGGATGATGTTCTG-3'), human TNF-{alpha} (forward, 5'-TCAGATCATCTTCTCGCACCC-3' and reverse, 5'-GACTCGGCAAAGTCGAGATAG-3'), human {beta}-actin (forward, 5'-GAGAAGATGACCCAGATCATG-3' and reverse, 5'-TCAGGAGGAGCAATGATCTTG-3'), viral A238L (forward, 5'-CGCGCGTCTAGATTACTTTCCATACTTGTT-3' and reverse, 5'-GCGCGCAAGCTTATGGAACACATGTTTCCA-3'), and viral p72 (forward, 5'-CGCGGATCCATGGCATCAGGAGGAG-3' and reverse, 5'-CGCGAGATCTAGCTGACCATGGGCC-3'). The PCR were performed by 30 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min. Amplified cDNAs were separated by agarose gel electrophoresis.

Western blot analysis

Cytosolic and nuclear extracts from mock-infected or ASFV-infected Vero cells, or Jurkat-pcDNA and Jurkat-A238L cells unstimulated or stimulated with 15 ng/ml PMA plus 1 µM Ion and treated or not with 100 ng/ml CsA were prepared, as described previously (18). To prepare whole cell extracts, mock-infected or ASFV-infected Vero cells and Jurkat-pcDNA and Jurkat-A238L cells were washed twice with PBS and lysed in radio immunolabeling protein assay buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, and 0.25% Na-deoxycholate, and supplemented with protease inhibitor mixture tablets (Roche). In each case, protein concentration was determined by the bicinchoninic acid spectrophotometric method (Pierce). Cell lysates (30 µg of protein) were fractionated by SDS-12% PAGE and electrophoretically transferred to an Immobilon extra membrane (Amersham Biosciences), and the separated proteins were reacted with specific primary Abs raised against A238L (generated as described in Ref.17), NF-{kappa}B-p65 (sc-109; Santa Cruz Biotechnology), c-Jun (sc-45; Santa Cruz Biotechnology), JNK1 (sc-474; Santa Cruz Biotechnology), and {beta}-actin (H-196; Santa Cruz Biotechnology). Membranes were exposed to HRP-conjugated secondary Abs (Amersham Biosciences), followed by ECL (Amersham Biosciences) detection by autoradiography.

Quantitation of TNF-{alpha} in culture supernatants

Alveolar porcine macrophages were infected with the E70 wild-type (wt) and E70{Delta}A238L viruses at a multiplicity of infection (MOI) of 5 PFU/cell, and supernatants were recovered at the indicated postinfection times. TNF-{alpha} protein levels were measured using Quantikine (R&D Systems) ELISA following the manufacturer’s instructions.

Plasmid constructs

Human TNF-{alpha} promoter constructs containing the full-length promoter sequence fused to firefly luciferase reporter gene, named pTNF(–1311)luc, or the different 5' deletion mutants, named pTNF(–751)luc, pTNF(–528)luc, pTNF(–120)luc, pTNF(–95)luc, and pTNF(–36)luc, were generated, as described (8). The full-length human NF-ATc (p1SH107c) expression plasmid (24) was a generous gift from G. Crabtree (Department of Pathology and Developmental Biology, Howard Hughes Medical Institute, Stanford University Medical School, Stanford, CA). The p65 expression plasmid pcDNA3-p65 was a gift from J. Alcamí (Universidad de Immunopatalogía, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain). The pRSV-c-Jun and the pRSV-c-Fos expression plasmids have been previously described (25). The pcDNA-A238L expression plasmid was generated by cloning the A238L open reading frame from Ba71V viral strain of ASFV into the pcDNA3.1 mammalian expression vector (Invitrogen Life Technologies). The pCDNA3-A238L-SV5 was a generous gift from L. Dixon (Institute for Animal Health, Woking, Surrey, U.K.) and generated as described (16). The GAL4-luciferase construct (pGAL4-Luc) contains five GAL4 DNA consensus binding sites derived from the yeast GAL4 gene fused to luciferase reporter gene (26). The pGAL4-hNF-AT1 construct containing the first 1–451 aa of human NF-AT1 fused to the DNA binding domain of yeast GAL4 transcription factor was originated, as described previously (27). The pGAL4-p65 construct has the yeast GAL4 DNA binding domain fused to the C-terminal trans activation domain of p65, and was generated as described (28). The pGAL4-c-Jun was generated as described (29). The CBP expression plasmid pRC/RSV-CBP-HA was a generous gift from A. Harell-Bellan (Centre National de la Recherche Scientifique-Ligue Nationale Contre le Cancer, Institut André Lwoff, Villejuif, France) and generated as described (30), and the p300 expression plasmid pCMV{beta}-p300-HA was a generous gift from J. Boyes (Institute of Cancer Research, London, U.K.) and generated as described (31).

Transfection and luciferase assays

Generation of A238L stably expressing Jurkat cells was done, as described previously (18). A238L stably expressing Vero cells were generated using the same protocol. These cellular lines were named Vero-pcDNA and Vero-A238L.

Jurkat-pcDNA and Jurkat-A238L cells, Vero cells, and porcine macrophages were transfected with 250 ng of specific reporter plasmids per 106 cells using the LipofectAMINE Plus Reagent (Invitrogen Life Technologies), according to the manufacturer’s instructions, and mixing in Opti-MEM (Invitrogen Life Technologies). In cotransfection assays, 0.05–0.5 µg of the corresponding expression plasmid per 106 cells was added. As a transfection control for luciferase assays, the Renilla luciferase control plasmid pRL-TK (Promega) was cotransfected in all of the experiments. Sixteen hours after transfection, Jurkat-pcDNA and Jurkat-A238L cells were stimulated with 15 ng/ml PMA plus 1 µM Ion during 4 h, Vero cells were infected with Ba71V or Ba71V{Delta}A238L at an MOI of 5 PFU/cell, and porcine macrophages were infected with E70 or E70{Delta}A238L at an MOI of 5 PFU/cell. At the indicated times, cells were lysed with 200 µl of Cell Culture Lysis Reagent (Promega) and microcentrifuged at full speed for 5 min at 4°C, and 20 µl of each supernatant was used to determine firefly and Renilla luciferase activity in a Monolight 2010 luminometer (Analytical Luminescence Laboratory) using Dual Luciferase Assay System. Transfections were normalized to Renilla luciferase activity, and results were expressed as the relative luminescence units after normalization of protein concentration determined by the bicinchoninic acid method, as indicated in the figure legends. Transfection experiments were performed in triplicate, and the data were presented as the mean of the relative luciferase units (RLU) (mean ± SD).

Immunofluorescence and confocal microscopy

Vero cells were grown on coverslips to 2 x 105 cells/cm2. In transient overexpression experiments, the cells were transfected with 1 µg of specific expression plasmids per 106 cells using the LipofectAMINE Plus Reagent (Invitrogen Life Technologies), according to the manufacturer’s instructions, and mixing in Opti-MEM (Invitrogen Life Technologies). When the cultures were 60–70% confluence, or 24 h posttransfection in transient overexpression assays, the cells were stimulated with 15 ng/ml PMA plus 1 µM Ion during different times, as indicated in the corresponding figures. The cultures were rinsed three times with PBS and fixed with cold 99.8% methanol (Merck) for 15 min at –20°C, before rehydrating twice with PBS and blocking with 1% BSA in PBS for 10 min at room temperature. The cells were incubated during 2 h with the specific Ab against NF-ATc2 (sc-7295; Santa Cruz Biotechnology), p300 (sc-584; Santa Cruz Biotechnology), and SV5-tag (MCA1360; Serotec); rinsed extensively with PBS; and then incubated with the specific secondary Abs (Alexa; Molecular Probes) for 1 h at room temperature in the dark. Finally, the cells were rinsed successively with PBS, distilled water, and ethanol, and mounted with a drop of Mowiol on a micro slide. Visualization of stained cultures was performed under a fluorescence Axioskop2 plus (Zeiss) microscope coupled to a color charge-coupled device camera or to Confocal Microradiance (Bio-Rad) equipment. Images were digitalized, processed, and organized with Metamorph, Lasershap2000 version 4, Adobe Photoshop 7.0, Adobe Illustrator 10, and Microsoft PowerPoint SP-2 software.

Solid-phase in vitro phosphorylation kinase assay

We used 2 µg of GST-c-Jun (sc-4113; Santa Cruz Biotechnology) as the substrate for in vitro phosphorylation in which immunoprecipitated JNK1 from Jurkat-pcDNA or Jurkat-A238L were assayed. Whole cell extracts from 107 Jurkat-pcDNA or Jurkat-A238L cultured in the absence or presence of 15 ng/ml PMA plus 1 µM Ion during 30 min were prepared. The cells were lysed in radio immunolabeling protein assay buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 1% Nonidet P-40, and supplemented with phosphatase inhibitors (1 mM NaVO3, 10 mM NaF, and 10 mM Na2MoO4) and protease inhibitors (0.5 mM PMSF, 1 µg of pepstatin, 2 µg of leupeptin, and 2 µg/ml aprotinin).

Cleared extracts were incubated for 18 h with 1 µg of specific Ab against JNK1 (sc-474; Santa Cruz Biotechnology) to immunoprecipitate JNK1. Immunoprecipitates were finally resuspended in kinase buffer containing 20 mM HEPES (pH 7.6), 20 mM MgCl2, 20 mM {beta}-glycerophosphate, 20 µM ATP, and 1 µCi of [{gamma}32P]ATP (sp. act., 3000 Ci/mol) supplemented with phosphatase inhibitors and mixed with the recombinant c-Jun. After 30 min at 30°C, the kinase reaction was terminated by washing with TNT buffer containing 20 mM Trizma base (pH 7.5), 200 mM NaCl, and 1% Triton X-100, and supplemented with protease inhibitor mixture tablets (Roche). Phosphorylated proteins were separated in a SDS-12% PAGE, dried, and developed by autoradiography.

In vitro DNA-protein-binding assay

Binding of CBP, p300, or A238L proteins to CRE/{kappa}3 sequence in the TNF-{alpha} promoter DNA was analyzed by a DNA-protein-binding assay, by using streptavidin-coated beads to bind biotinylated DNA probe, which was incubated with nuclear extract proteins. Biotin-labeled double-stranded oligonucleotide probes corresponding to human CRE/{kappa}3 sequence (5'/biotin/-AGATGAGCTCATGGGTTTCTCCACC-3') or a nonrelevant DNA sequence (5'/biotin/-TTACCAACTGAGCCATCTCC-3') were synthesized by Isogen. The binding assay was performed by mixing 500 µg of nuclear extract proteins from stimulated or unstimulated Jurkat-pcDNA and Jurkat-A238L cells (obtained as described above), 5 µg of biotinylated CRE/{kappa}3 or irrelevant sequence, and 50 µl of 4% streptavidin-beaded agarose (Sigma-Aldrich) with 70% slurry. The mixture was incubated at room temperature for 1 h with shaking. Beads were then pelleted and washed three times with ice-cold PBS. The bound proteins were eluted in loading buffer and separated by 4–15% PAGE, followed by Western blot analysis probed with Abs against CBP (sc-369; Santa Cruz Biotechnology), p300 (sc-584; Santa Cruz Biotechnology), or A238L.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
A238L inhibits TNF-{alpha} promoter activity and transcription during ASFV infection in Vero cells and porcine macrophages

TNF-{alpha} transcription is regulated by several transcription factors such as NF-{kappa}B, NF-AT, ATF-2, Jun, Ets/Elk, and Sp-1 proteins. Because the viral protein A238L has been described as an inhibitor of NF-{kappa}B (17), NF-AT (16, 18), and calcineurin phosphatase (16), we have explored the possibility that this protein could inhibit TNF-{alpha} expression. To assess this, we have used the ASFV A238L deletion mutant, designated Ba71V{Delta}A238L, obtained from the Ba71V viral strain, as previously described (18). The absence of A238L protein in Ba71V{Delta}A238L-infected Vero cells was corroborated by Western blot analysis with cellular extracts from Ba71V- and Ba71V{Delta}A238L-infected cells using a specific Ab against A238L protein. Fig. 1A shows the lack of expression of the viral protein in Ba71V{Delta}A238L-infected Vero cells. In contrast, a band corresponding to the protein A238L could be detected from 6 h postinfection (hpi) in the cells infected with the parental virus. The absence of gene A238L does not affect virus replication, as shown by the similar number of lysis plaques produced by both Ba71wt and Ba71V{Delta}A238L in a plaque assay on Vero cell monolayers (Fig. 1B).



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  		FIGURE 1. TNF-{alpha} promoter activity and mRNA levels in ASFV-infected Vero cells. A, Western blot of Vero cells mock infected (M) or infected with the Vero-adapted strain Ba71V wt or Ba71V{Delta}A238L. At the indicated times postinfection (hpi), whole cell extracts were prepared, subjected to SDS-PAGE (30 µg of protein sample), and detected by immunoblotting with an A238L-specific Ab. A control of protein loading is included by {beta}-actin blotting. B, Plaque assay for Ba71Vwt and Ba71V{Delta}A238L viruses. The figure shows virus plaques developed on Vero cell monolayers by a 10–5 dilution of samples from mock-, Ba71Vwt-, or Ba71V{Delta}A238L-infected Vero cells collected at 24 hpi. C, Vero cells were transfected with the pTNF(–1311)luc plasmid (containing the full-length promoter sequence of human TNF-{alpha} gene fused to the firefly luciferase reporter gene), as described under Materials and Methods. Sixteen hours after transfection, cells were mock infected (Mock) or infected with Ba71V wt or Ba71V{Delta}A238L. Whole cell extracts were prepared at the indicated postinfection times and assayed for luciferase activity. Extracts were normalized to Renilla luciferase activity, as described in Materials and Methods. RLU/µg protein from triplicate transfections (mean ± SD). D, RT-PCR analysis of infected Vero cells. Total RNA (1 µg) from Vero cells mock infected (M) or infected with Ba71V wt or Ba71V{Delta}A238L viruses was prepared at the indicated postinfection times and analyzed by RT-PCR to measure TNF-{alpha} and A238L mRNA levels. A control using specific oligonucleotides for {beta}-actin is also included to rule out differences in PCR amplification. Amplified DNA was separated on an agarose gel and stained with ethidium bromide.

 
To investigate the role of A238L in the control of TNF-{alpha} transcription during ASFV infection, Vero cells were first transfected with the plasmid pTNF(–1311)luc, which contains the luciferase reporter gene under the control of the full-length sequence of the human TNF-{alpha} promoter. Sixteen hours after transfection, cells were infected either with the parental Ba71V or with {Delta}A238L viruses (MOI of 5 PFU/cell), and, at the indicated times after infection, luciferase activity was measured in cell extracts. As shown in Fig. 1B, a slight induction of TNF-{alpha} promoter activity was detected after 12 hpi with the wt virus. However, a much higher activity of the TNF-{alpha} promoter was observed from 6 hpi in cells infected with the deletion mutant virus. This result indicates that A238L efficiently down-regulates TNF-{alpha} promoter activation during ASFV infection in Vero cells.

We then compared the induction of TNF-{alpha} mRNA expression after infection of Vero cells with the parental Ba71V ASFV strain or with the recombinant Ba71V{Delta}A238L to determine the role of A238L in TNF-{alpha} gene expression during ASFV infection. As shown in Fig. 1C, TNF-{alpha} mRNA was identified in Vero cells after infection with the Ba71V{Delta}A238L, while no detectable TNF-{alpha}-specific mRNA was found after the infection with Ba71V wt. As expected, no specific A238L mRNA was detected in Ba71V{Delta}A238L-infected cells.

ASFV replicates mainly in macrophages and monocytes in vivo. This fact has been considered to play a critical role in the pathogenesis of the disease, because macrophage-derived cytokines strongly determine the development of inflammatory responses against infection. Because tissue macrophages appear to be the main source of TNF-{alpha}, and previous studies have demonstrated impairment of chemotactic activities and toxic oxygen radicals release in macrophages infected with different strains of ASFV (32), the role of A238L in the control of TNF-{alpha} in this cellular type is an important issue to address. To study this point, we have generated an ASFV A238L deletion mutant (E70{Delta}A238L) from the virulent strain E70, which has been shown to induce a strong infection in these cells (21). Recombinant virus expressing the {beta}-gus gene was purified, and genomic DNA from wt and {Delta}A238L virus was analyzed by Southern blot, using digoxigenin-labeled DNA probes. As shown in Fig. 2A, DNA fragments of predicted size were observed in both viruses when probed with the parental DNA fragment SalI I', while the {beta}-gus gene probe hybridized only with DNA from E70{Delta}A238L. As expected, the A238L gene probe failed to hybridize with DNA from E70{Delta}A238L.



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  		FIGURE 2. TNF-{alpha} promoter activity. mRNA and protein levels in ASFV-infected macrophages. A, Characterization of the E70 A238L deletion mutant by Southern blot analysis of parental E70 virus (WT) and E70{Delta}A238L ({Delta}). Purified viral DNA digested with EcoRI was electrophoresed, blotted, and hybridized with the indicated probes, as described in Material and Methods. B, RT-PCR analysis of infected porcine alveolar macrophages. Total RNA (1 µg) from macrophages mock infected (M) or infected with E70 wt or E70{Delta}A238L virus was prepared at the indicated postinfection times and analyzed by RT-PCR to measure TNF-{alpha} and A238L mRNA levels. A control of the infection by using specific oligonucleotides to detect viral p72 mRNA is also included. A control using specific oligonucleotides for porcine GAPDH is also included to rule out differences in PCR amplification. Amplified DNA was separated on an agarose gel and stained with ethidium bromide. C, Porcine alveolar macrophages were transfected with the pTNF(–1311)luc plasmid, as described in Materials and Methods, and mock infected (Mock) or infected with the virulent isolate E70 wt or E70{Delta}A238L 4 h after transfection. Whole cell extracts were prepared at the indicated postinfection times and assayed for luciferase activity. Extracts were normalized to Renilla luciferase activity, as described in Materials and Methods. RLU/µg protein from triplicate transfections (mean ± SD) are shown. D, Porcine alveolar macrophages were infected with the virulent isolate E70 wt or E70{Delta}A238L mutant, and supernatants were recovered at the indicated postinfection times and assayed in TNF-{alpha} ELISA, as described in Materials and Methods.

 
We next studied by RT-PCR the expression of A238L gene in porcine macrophages infected with the recombinant virus E70{Delta}A238L or with the wt virus. As shown in Fig. 2B, no A238L mRNA was detected in extracts from macrophages infected with E70{Delta}A238L. As also can be seen in this figure, E70 wt and E70{Delta}A238L induce similar levels of mRNA specific for the major capsid protein p72, indicating that the absence of A238L in this virus isolate does not impair virus replication in macrophages. When we used these samples to amplify the TNF-{alpha}-specific mRNA, relatively higher levels of TNF-{alpha} mRNA were detected compared with those obtained after Ba71V infection of Vero cells (Fig. 1B), either due to the different cell type or to the fact that virulent isolates of ASFV are better inducers of TNF-{alpha} (33). More importantly, levels of TNF-{alpha} mRNA after infection of macrophages with the recombinant virus E70{Delta}A238L were much higher than those induced by the parental virus, demonstrating an inhibitory role of A238L in the TNF-{alpha} expression during ASFV infection in macrophages (Fig. 2B).

The activity of the TNF-{alpha} promoter was also studied, as described above, in swine macrophages during the infection with E70 or E70{Delta}A238L, revealing a similar pattern to that obtained (Fig. 1B) during the infection with Ba71V or Ba71V{Delta}A238L in Vero cells. As shown in Fig. 2C, a higher activity could be detected after 12 hpi in cells infected with the deletion mutant, in correspondence to the levels of TNF-{alpha} mRNA obtained.

To evaluate whether the increase of TNF-{alpha} mRNA observed after E70{Delta}A238L infection corresponds to an increase in the TNF-{alpha} protein secretion, we quantified the amount of porcine TNF-{alpha} in supernatants collected from the infected macrophages. Fig. 2D shows the almost undetectable TNF-{alpha} production after the infection with the E70 wt virus. In contrast, the infection with the deletion mutant virus induces a substantial increase in TNF-{alpha} production from 12 hpi. Taken together, these results show the ability of A238L to control the activation of the TNF-{alpha} promoter and protein secretion, representing an important mechanism to evade the immune response by ASFV.

A238L regulates TNF-{alpha} transcription and promoter activity in Jurkat cells through {kappa}3 and CRE sites

To further analyze the mechanism by which A238L regulates TNF-{alpha} expression out of the context of ASFV infection, we have generated Jurkat cells that stably express the A238L gene by transfection with pcDNA-A238L expression plasmid, followed by selection using G418, as previously described (18). Stimulation with PMA/Ion increased levels of TNF-{alpha} mRNA in Jurkat-pcDNA cells, which were significantly higher than those found in Jurkat-A238L (Fig. 3A). Thus, expression of A238L strongly decreased TNF-{alpha} transcription upon PMA/Ion treatment in Jurkat cells, in concordance with the up-regulation of TNF-{alpha} mRNA levels by the absence of A238L during viral infection.



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  		FIGURE 3. Analysis of TNF-{alpha} expression in stably expressing A238L Jurkat cells. A, Total RNA (1 µg) from Jurkat-pcDNA and Jurkat-A238L cells cultured in the absence (NS) or presence of 15 ng/ml PMA plus 1 µM Ion (PMA + Ion) at the indicated times was analyzed by RT-PCR to measure TNF-{alpha} and A238L mRNA expression. A control using specific oligonucleotides for {beta}-actin is also included to rule out differences in PCR amplification. Amplified DNA was separated on an agarose gel and stained with ethidium bromide. B, Jurkat-pcDNA or Jurkat-A238L cells were transiently transfected with the pTNF(–1311)luc TNF-{alpha} human promoter construct, as described in Materials and Methods. Sixteen hours after transfection, cells were cultured in the absence ({square}) or presence of 15 ng/ml PMA plus 1 µM Ion ({cjs2108}) for 4 h and assayed for luciferase activity. Extracts were normalized to Renilla luciferase activity, as described in Materials and Methods. Results from triplicate assays are shown in RLU/µg protein (mean ± SD). Numerical data of RLUs are shown over the corresponding bars.

 
To analyze whether inhibition of TNF-{alpha} mRNA levels by A238L correlated with a decrease in the transcriptional activity mediated by the TNF-{alpha} promoter, Jurkat-pcDNA or Jurkat-A238L cells were transfected with the plasmid pTNF(–1311)luc, which contains the luciferase reporter gene under the control of the full-length sequence of the human TNF-{alpha} promoter. As shown in Fig. 3B, and in parallel with the down-regulation of TNF-{alpha} mRNA levels, ectopic A238L expression strongly decreased the transcription driven by this construction. It is noteworthy that the expression of the viral protein induces decreased levels (2.1-fold) of TNF-{alpha} reporter activity in control unstimulated cells, although inhibition is more clearly observed (3.6-fold) upon PMA/Ion stimulation.

To investigate the molecular mechanism by which A238L regulates TNF-{alpha} promoter activity, we have explored the TNF-{alpha} promoter sequences required for the transcriptional inhibition of TNF-{alpha} in Jurkat-A238L. Jurkat-pcDNA and Jurkat-A238L cells were transfected with different 5' deletions of the TNF-{alpha} promoter, pTNF(–751)luc, pTNF(–528)luc, pTNF(–120)luc, pTNF(–95)luc, and pTNF(–36)luc. Sixteen hours after transfection, cells were cultured in the absence or presence of PMA/Ion for 4 h and assayed for luciferase activity (Fig. 4).



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  		FIGURE 4. Effect of A238L upon the transcriptional activation of the TNF-{alpha} promoter. Jurkat-pcDNA ({square}) or Jurkat-A238L ({cjs2100}) cells were transiently transfected with the indicated TNF-{alpha} promoter constructs, as described in Materials and Methods. Sixteen hours after transfection, cells were cultured in the absence or presence of 15 ng/ml PMA plus 1 µM Ion for 4 h and assayed for luciferase activity. Extracts were normalized to Renilla luciferase activity, as described in Materials and Methods. Results from triplicate assays are shown as bars in RLU/µg protein (mean ± SD), corresponding to the series of 5' truncations ranging from –1311 to –36 represented at the left side of the figure. The cis-acting consensus sequences are represented by boxes. The extent of the 5' truncations are shown with numbers indicating their length relative to the transcription start site. pTNF(–36)luc is also included to show the absence of luciferase activity after stimulation under our experimental conditions. Numbers in the table represent the ratio between the RLU values obtained from Jurkat-pcDNA vs Jurkat-A238L cells in basal (unstimulated) or inducible (PMA/Ion-stimulated) conditions.

 
The human TNF-{alpha} gene promoter contains three NF-{kappa}B consensus sequences, named {kappa}1, {kappa}2, and {kappa}3. Consistent with studies in other cell types and with other inducers, deletion of {kappa}1 and {kappa}2 does not affect the inducibility of the gene, and 120 nt upstream of the TNF-{alpha} transcription start site are sufficient for maximal inducibility of the gene. The {kappa}3 site, but not the {kappa}1 or {kappa}2 sites, is included in the pTNF(–120)luc reporter construct. The {kappa}3 site, in contrast to the {kappa}1 and {kappa}2 sites, has been implicated in the regulation of the gene in a variety of cell types (10). It has been described that the {kappa}3 site binds NF-AT and NF-{kappa}B, and that an immediately adjacent CRE site binds ATF-2 and c-Jun proteins. The results presented in Fig. 4 show that A238L expression produced a 1.9- to 2.4-fold decrease, under unstimulated conditions (basal activity), and a 3.4- to 3.8-fold decrease, after PMA/Ion stimulation (inducible activity), in the transcription driven by –751, –528, and –120 TNF-{alpha} promoter constructs. Deletion up to –95 nt significantly reduced activation of TNF-{alpha} promoter activity by PMA/Ion, although deletion of the –95 to –36 region of the TNF-{alpha} completely abrogated TNF-{alpha} inducibility by PMA/Ion. These results indicate not only that the composite {kappa}3/CRE site seems to be essential for full transcriptional activation of the TNF-{alpha} human gene in our system, but also that the ectopic expression of A238L results in >50% reduction of the activity of the TNF-{alpha} promoter mediated by this region.

NF-AT, p65, and c-Jun, but not c-Fos, participate in the transcriptional activation of the TNF-{alpha} gene and are regulated by A238L

The above results show that TNF-{alpha} promoter induction was strongly inhibited by the expression of the A238L viral gene in Jurkat cells through {kappa}3 and CRE sites. To characterize the transcription factors that are able to bind to this region and that could be involved in the down-regulation induced by the viral protein, we have cotransfected expression plasmids for NF-AT/c2, NF-{kappa}B (p65), c-Jun, and c-Fos, together with the pTNF(–120)luc, into Jurkat-pcDNA or Jurkat-A238L cells. As shown in Fig. 5A, overexpression of NF-AT/c2 increased the activity of the promoter after stimulation with PMA/Ion in a dose-dependent manner, corroborating the involvement of NF-AT in the modulation of TNF-{alpha} expression by the viral protein. Similarly, increased doses of p65-NF-{kappa}B strongly cooperated with PMA/Ion to activate the TNF-{alpha} promoter in Jurkat-A238L cells (Fig. 5B), demonstrating the involvement of the NF-{kappa}B pathway in the mechanism of TNF-{alpha} inhibition by the viral protein. Next, we cotransfected the pTNF(–120)luc construct along with increasing amounts of c-Jun expression plasmid, a member of the AP-1 protein family. As shown in Fig. 5C, cotransfection of pRSV-c-Jun also counteracted the inhibition of the TNF-{alpha} promoter activity mediated by A238L. However, increasing doses of c-Fos, an AP-1 family member that is not required for the inducibility of the TNF-{alpha} promoter in PMA/Ion-activated T cells, were unable to recover the inhibition mediated by A238L, corroborating the specific involvement of NF-AT, NF-{kappa}B (p65), and c-Jun in this process. It is interesting to note that the ratio between Jurkat-pcDNA vs Jurkat-A238L RLU values was higher in inducible (PMA/Ion-stimulated) than in basal (nonstimulated) conditions, as described in Figs. 3 and 4, but showed equivalent numbers when the activities were reverted by increasing expression of NF-AT, p65, and c-Jun (data not shown).



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  		FIGURE 5. NF-AT, p65, and c-Jun, but not c-Fos, participate in the transcriptional activation of the TNF-{alpha} gene promoter modulated by A238L. Jurkat-pcDNA ({square}) or Jurkat-A238L ({cjs2100}) cells were transiently transfected with the pTNF(–120)luc reporter plasmid (a 5' deletion construct that preserves the CRE (–107 to –99 nt) and {kappa}3 (–97 to –88 nt) sites) and with the indicated doses (from 0 to 500 ng of DNA per million cells) of four different expression plasmids: A, p1SH107c to overexpress the NF-AT transcription factor; B, pCMV-p65 to overexpress the NF-{kappa}B transcription factor; C, pRSV-c-Jun, to overexpress the AP-1 family member c-Jun transcription factor; and D, pRSV-c-Fos to overexpress the AP-1 family member c-Fos transcription factor. Sixteen hours after transfection, the cells were cultured in the absence ({square}) or presence of 15 ng/ml PMA plus 1 µM Ion ({cjs2108}) during 4 h and assayed for luciferase activity. Extracts were normalized to Renilla luciferase activity, as described in Materials and Methods. Results from triplicate assays are shown in RLU/µg protein (mean ± SD).

 
The inhibition of TNF-{alpha} by A238L does not involve NF-AT or NF-{kappa}B nuclear translocation or c-Jun phosphorylation

As we described above, the overexpression of NF-AT, NF-{kappa}B, and c-Jun restored partially the TNF-{alpha} expression inhibited by A238L, suggesting that the mechanism by which A238L mediates this inhibition in Jurkat cells involves, at least, the activity of these transcription factors.

We have previously described that the mechanism responsible for the A238L-mediated inhibition of NF-AT does not imply dephosphorylation or translocation of this transcription factor to the nucleus upon treatment of Jurkat-pcDNA or Jurkat-A238L with PMA/Ion or during ASFV infection (18). To confirm these results in a different cellular system, we have analyzed the nuclear shuttling of NF-ATc2 both in Vero-pcDNA and Vero-A238L cells by confocal microscopy using a specific anti-NF-AT Ab. As shown in Fig. 6A, nuclear translocation of NF-AT increased after 30 min of stimulation with PMA/Ion both in the absence and in the presence of A238L expression. This translocation was not observed in cells pretreated with CsA, which prevents NF-AT nuclear activation through the inhibition of calcineurin phosphatase. Taken together, these findings suggest that the inhibition of NF-AT by A238L is not likely the result of preventing the NF-AT translocation to the nucleus in Vero cells.



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  		FIGURE 6. A238L does not inhibit either NF-AT, NF-{kappa}B-p65 nuclear translocation, or c-Jun activation. A, Subcellular localization of NF-AT in the presence or absence of A238L. Stably expressing A238L Vero cells (Vero-A238L) or control cells (Vero-pcDNA) were cultured in the absence (control) or presence of 15 ng/ml PMA plus 1 µM Ion during 15, 30, or 90 min, or preincubated for 1 h with CsA and then stimulated with 15 ng/ml PMA plus 1 µM Ion during 90 min (CsA + 90 min). Then the cells were labeled with an anti-NF-AT Ab (green), and examined by confocal microscopy. The figure shows images corresponding to one of three independent experiments performed. B, Cytosolic and nuclear extracts from Jurkat-pcDNA and Jurkat-A238L cells, nonstimulated (NS) and stimulated with 15 ng/ml PMA plus 1 µM Ion (PMA + Ion) during 30 min (30') or 90 min (90'), were prepared, subjected to SDS-PAGE (30 µg of cytosolic extract and the corresponding fraction of nuclear extract), and detected by immunoblotting with a NF-{kappa}B-p65-specific Ab. A control of cellular fractions and protein loading is included by {beta}-actin blotting. C, Whole cell extracts from Jurkat-pcDNA and Jurkat-A238L cells, nonstimulated (NS) and stimulated with 15 ng/ml PMA plus 1 µM Ion (PMA + Ion) during 15 min (15'), 30 min (30'), or 90 min (90'), were prepared, subjected to SDS-PAGE (30 µg of protein), and detected by immunoblotting with a c-Jun-specific Ab. D, Whole cell extracts from 107 stably transfected Jurkat-pcDNA and Jurkat-A238L cells cultured in the absence or presence of 15 ng/ml PMA plus 1 µM Ion during 30 min (30') were incubated and immunoprecipitated with 1 µg of JNK1-specific Ab. In the left panel, immunoprecipitates were separated by SDS-PAGE, transferred to an Immobilon membrane, and revealed with the same JNK1 Ab. In the right panel, the immunoprecipitates were used in an in vitro kinase assay, as described in Materials and Methods, using purified GST-c-Jun as the substrate. Proteins were separated by SDS-12% PAGE and developed by autoradiography.

 
In contrast, it is widely accepted that the nuclear translocation is a hallmark of the transcriptional activation of NF-{kappa}B and that the intracellular localization of this transcription factor is governed by I{kappa}Bs. We have previously described that A238L inhibits NF-{kappa}B activation (17). In this study, we have analyzed the presence of p65 in subcellular fractions from Jurkat-pcDNA and Jurkat-A238L after PMA/Ion stimulation to determine whether the control of NF-{kappa}B activation is due to the inhibition of p65 translocation to the nucleus or to an alternative mechanism. As shown in Fig. 6B, the level of p65 detected in the cytoplasm from both Jurkat-pcDNA and Jurkat-A238L was similar from 0 to 90 min after PMA/Ion stimulation. Interestingly, the expression of A238L did not impair the translocation of p65 to the nucleus of the stimulated cells.

As mentioned above, the region involved in the regulation of TNF-{alpha} transcription contains a CRE binding site, which binds ATF-2/Jun heterodimer and forms a composite element with the {kappa}3 site (6, 34, 35). ATF-2/Jun proteins become transcriptionally active upon phosphorylation by the p38 and JNK1, members of the MAPK family (36). JNK1, whose activity can be augmented by increasing levels of intracellular calcium, binds to the N-terminal region of c-Jun and phosphorylates it at Ser63/73 (37). We next evaluated the effect of A238L on c-Jun expression and JNK1 activity by analysis of extracts from Jurkat-pcDNA or Jurkat-A238L. Fig. 6C shows the c-Jun expression detected by Western blot with specific antiserum against human c-Jun. Because c-Jun activity depends mainly on its phosphorylation, the effect of A238L on the level of phosphorylated c-Jun was examined through a solid-phase in vitro phosphorylation kinase assay (Fig. 6D), in which immunoprecipitated JNK1 from both Jurkat-pcDNA and Jurkat-A238L nonstimulated or stimulated with PMA/Ion was incubated with purified GST-c-Jun as substrate. These results show that the presence of A238L does not inhibit c-Jun expression or JNK1 activity, suggesting that these are not the mechanisms used by the viral protein to down-regulate the TNF-{alpha} transcription.

A238L down-regulates the trans activation function of NF-AT, p65, and c-Jun

The above results indicate that A238L is acting on the NF-AT and NF-{kappa}B activity without altering their nuclear translocation. Besides, A238L does not affect either c-Jun expression or JNK-dependent activation. Recent evidence indicates that activation of NF-AT does not only involve its nuclear translocation, but also the intrinsic function of the trans activation domain that is located at the N terminus (38). To study the regulation of the trans-activating function of NF-AT by A238L, Jurkat-pcDNA or Jurkat-A238L were transfected with a GAL4-luc reporter plasmid along with a construct (GAL4-NFATc2 (1–415)) encoding the N-terminal region of the NF-ATc2 (aa 1–415), which contains the strong acidic trans activation domain and the whole regulatory domain fused to the GAL4 DNA binding domain. As expected from our previous results (18), expression of A238L strongly inhibits the function of NF-AT trans activation domain (Fig. 7A). Reporter activity was not induced either in Jurkat-pcDNA or Jurkat-A238L by stimulation with PMA/Ion when the control GAL4-DNA binding domain was transfected (data not shown).



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  		FIGURE 7. A238L inhibits trans activation mediated by NF-AT, p65, and c-Jun. A, Jurkat-pcDNA and Jurkat-A238L cells were cotransfected with GAL4-NF-AT (50 ng of DNA/106 cells) and GAL4-luc (150 ng of DNA/106 cells) and cultured with 15 ng/ml PMA plus 1 µM Ion. B, Jurkat-pcDNA and Jurkat-A238L cells were cotransfected with GAL4-p65 (150 ng of DNA/106 cells) and GAL4-luc (150 ng of DNA/106 cells) and cultured with 15 ng/ml PMA plus 1 µM Ion. C, Jurkat-pcDNA and Jurkat-A238L cells were cotransfected with GAL4-c-Jun (150 ng of DNA/106 cells) and GAL4-luc (150 ng of DNA/106 cells) and cultured with 15 ng/ml PMA plus 1 µM Ion. In each case, whole cell extracts were prepared at the indicated poststimulation times and luciferase activity was assayed. Extracts were normalized to Renilla luciferase activity, as described in Materials and Methods. RLU/µg protein from triplicate transfections (mean ± SD) are shown.

 
In the case of NF-{kappa}B, and although the induced nuclear translocation of NF-{kappa}B has been generally considered as the principal way to activate NF-{kappa}B-dependent gene expression, an alternative mechanism of NF-{kappa}B activation is emerging that involves the phosphorylation of the RelA/p65 trans activation subunit. To address this question, we used an approach similar to that described above for NF-AT. Thus, a plasmid encoding the GAL4-p65 fusion protein, in which the DNA binding domain of GAL4 has been joined to the trans activation domain of RelA/p65 (28), was cotransfected with a GAL4-Luc reporter, allowing us to determine whether the viral protein down-regulates TNF-{alpha} gene expression by specifically targeting the trans activation domain of the RelA/p65 subunit of NF-{kappa}B. Fig. 7B shows that in the presence of the A238L inhibitor, the ability of PMA/Ion to activate GAL4-p65 was strongly abrogated from 1 to 6 h after stimulation.

As we mentioned before, overexpression of c-Jun by cotransfection of pRSV-c-Jun counteracted the inhibition of the TNF-{alpha} promoter activity observed in Jurkat-A238L. To assess whether c-Jun trans activation is modified in cells expressing the viral protein, we cotransfected the plasmid encoding the GAL4-c-Jun fusion protein together with the GAL4-Luc plasmid in Jurkat-pcDNA and Jurkat-A238L. As shown in Fig. 7C, GAL4-c-Jun was poorly stimulated in Jurkat-A238L in comparison with the stimulation observed in Jurkat-pcDNA, suggesting that A238L targets also the trans activation domain of c-Jun.

A238L localizes in the nucleus in infected Vero cells and colocalizes with p300 in the nucleus of cotransfected Vero cells

The results presented before suggested that protein A238L might localize in the nucleus to exert its inhibitory action. To address this issue, we performed subcellular fractionation of ASFV-infected cells, followed by Western blot analysis to detect the viral protein. As can be seen in Fig. 8A, A238L was present both in the cytoplasm and in the nuclear fractions at 12 and 24 hpi, showing a higher amount of the protein in the insoluble nuclear fraction (N2) at 24 hpi. The absence of a corresponding band in the subcellular fractions of Vero cells infected with the recombinant Ba71V{Delta}A238L clearly demonstrates that the band detected in cells infected with wt virus is the A238L protein.



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  		FIGURE 8. Subcellular localization of A238L and colocalization with p300. A, At the indicated times postinfection (hpi), cytosolic (C) and soluble (N1) or insoluble (N2) fractions of nuclear extracts from mock-infected and Ba71Vwt- or Ba71V{Delta}A238L-infected Vero cells were prepared. The soluble nuclear fraction (N1) was obtained from the supernatant of the nuclear extract, and the insoluble nuclear fraction (N2) was prepared from the pellet remaining from N1 fraction by a sonication step. The fractions were subjected to SDS-PAGE (30 µg of cytosolic extract and the corresponding fraction of nuclear extracts) and detected by immunoblotting with A238L-specific Ab. B, Vero cells were transiently transfected with the pcDNA-A238L-SV5 expression plasmid, as described in Materials and Methods. Twenty-four hours after transfection, cells were cultured in the absence (Unstimulated) or presence of 15 ng/ml PMA plus 1 µM Ion during 15 or 30 min. Then the cells were labeled with an anti-SV5 Ab and examined by confocal microscopy. The figure shows images corresponding to one of three independent experiments performed. C, Cultures of Vero cells were transiently transfected with pcDNA-A238L-SV5 and pCMV{beta}-p300-HA expression plasmids, as described in Materials and Methods. Twenty-four hours after transfection, cells were incubated in the absence (control) or presence of 15 ng/ml PMA plus 1 µM Ion during 15, 30, or 60 min. Then the cells were labeled with anti-SV5 (green) and with anti-p300 (red) Abs and examined by confocal microscopy. The figure shows images corresponding to one of three independent experiments performed.

 
We next investigated the localization of A238L in Vero cells transfected with plasmid pcDNA3-A238L-SV5 by confocal microscopy using an anti-SV5 Ab. The results showed that A238L localized in the cytoplasm of unstimulated cells and that after treatment with PMA/Ion the protein was translocated to the nucleus (Fig. 8B). Interestingly, when the cells were cotransfected with pcDNA3-A238L-SV5 and pCMV{beta}-p300-HA, which expresses the coactivator protein p300 fused to a hemagglutinin tag, a colocalization of both proteins in the nucleus was observed upon stimulation with PMA/Ion (Fig. 8C). This finding may be related to the role of coactivators in the inhibition of TNF-{alpha} promoter activity, as described below.

CBP/p300 overexpression reverts the A238L-mediated inhibition of the pTNF(–120)luc promoter activity

CBP/p300 proteins play a critical role in the induction of TNF-{alpha} transcription by virus, TCR ligands, and LPS (6, 39, 40). CBP/p300 proteins function as coactivators for multiple transcription factors, including NF-{kappa}B, NF-AT, and c-Jun. In contrast, previous studies have shown that the composite TNF-{alpha} CRE/{kappa}3 site, which binds ATF-2/Jun and NF-AT proteins and is required for induction of the TNF-{alpha} gene by TCR engagement, virus infection, and calcium influx, is a CBP/p300-dependent element (12).

To characterize the involvement of transcriptional coactivators CBP/p300 in the TNF-{alpha} promoter activity down-regulation induced by the viral protein, we have cotransfected expression plasmids for CBP (pCMV-CBP), p300 (pRSV-p300), or both, together with the pTNF(–120)luc, into Jurkat-pcDNA or Jurkat-A238L cells. Sixteen hours after transfection, the cells were cultured in the absence or presence of PMA/Ion during 4 h and assayed for luciferase activity. As shown in Figs. 8 and 9, overexpression of CBP in A238L cells rescued the activity of the promoter after stimulation with PMA/Ion in a dose-dependent manner, indicating the involvement of CBP in the modulation of TNF-{alpha} expression by the viral protein. We have performed a similar experiment using the pRSV-p300 construct that drives the expression of p300. As shown in Figs. 8 and 9, a high recovery of TNF-{alpha} promoter activity could be found in Jurkat-A238L in the presence of increasing doses of pRSV-p300, thus demonstrating the involvement of p300 in the mechanism of TNF-{alpha} inhibition by the viral protein. Next, we cotransfected the pTNF(–120)luc construct along with increasing amounts of pCMV-CBP and pRSV-p300 expression plasmids together. As shown in Figs. 8 and 9, a complete reversion of the inhibition of TNF-{alpha} promoter activity was obtained. These results suggest that A238L-mediated TNF-{alpha} inhibition is accomplished by modulation of CBP/p300 transcriptional coactivators representing an accurate viral mechanism to evade the inflammatory immune response.



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  		FIGURE 9. CBP and p300 expression counteracts the inhibition of TNF-{alpha} promoter activity induced by A238L. Jurkat-pcDNA ({square}) or Jurkat-A238L ({cjs2113}) cells were transiently transfected with the pTNF(–120)luc reporter plasmid (a 5' deletion construct that preserves the CRE (–107 to –99 nt) and {kappa}3 (–97 to –88 nt) sites) and with the indicated doses (from 0 to 500 ng of DNA per 106 cells) of CBP and p300 expression plasmids: A, pRC/RSV-CBP-HA to overexpress the CBP transcriptional coactivator; B, pCMV{beta}-p300-HA to overexpress the p300 transcriptional coactivator; C, pCMV{beta}-p300-HA and pRC/RSV-CBP-HA to overexpress both coactivators. Sixteen hours after transfection, the cells were cultured in the absence ({square}) or presence of 15 ng/ml PMA plus 1 µM Ion ({cjs2108}) during 4 h and assayed for luciferase activity. Results from triplicate assays are shown in RLU/µg protein (mean ± SD). Extracts were normalized to Renilla luciferase activity, as described in Materials and Methods.

 
A238L displaces CBP/p300 from the CRE/{kappa}3 complex

To further investigate the mechanism by which A238l and CBP/p300 coactivators control the activity of the TNF-{alpha} promoter, we have performed pull-down experiments using a biotinylated CRE/{kappa}3 probe. For this, the biotinylated probe was incubated with nuclear extracts from Jurkat-pcDNA or Jurkat-A238L cells treated or not with PMA/Ion, and the complex was pulled down with streptavidin-agarose beads. Finally, the proteins in the complex were analyzed by Western blotting using Abs against p300, CBP, or A238L. As shown in Fig. 10, p300 and CBP were present in whole nuclear extracts from both Jurkat-pcDNA and Jurkat A238L, while A238L was detected only in the nuclear extracts from Jurkat-A238L, as expected. The results of the pull down assay showed that, while both p300 and CBP were present in the CRE/{kappa}3 complex in the absence of A238L expression, the coactivators were not detected in the case of cells expressing A238L, indicating that the viral protein displaces CBP/p300 from the complex (Fig. 10). It is interesting to note that A238L remains bound to the CRE/{kappa}3 complex, revealing a direct or indirect interaction with the CRE/{kappa}3 site. These results suggest that A238L inhibits TNF-{alpha} promoter activity by binding to the CRE/{kappa}3 complex and in this way displacing the CBP/p300 coactivators.



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  		FIGURE 10. Binding of p300 and CBP coactivators to a biotinylated CRE/{kappa}3 probe. Nuclear extracts from Jurkat-pcDNA and Jurkat-A238L cells treated or not with PMA/Ion were incubated with the indicated biotinylated probe, and the complex was pulled down with streptavidin-agarose beads. After extensive washing, proteins in the complex were analyzed by Western blotting using Abs against p300, CBP, or A238L. Control probe is a biotinylated 22-bp nonrelevant DNA sequence. Nuclear extracts were also included to demonstrate the presence of the analyzed proteins in Jurkat cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In the current study, we show that A238L specifically inhibits the TNF-{alpha} transcription by a mechanism that involves CBP/p300. The important role of A238L in controlling TNF-{alpha} synthesis was shown by the effect of virus deletion mutant both in infected Vero cells and porcine macrophages.

Despite the relevance of TNF-{alpha} in the control of viral infections, the role that this cytokine could play during ASFV infection in vivo (41, 42) or in vitro (33, 43), and the molecular mechanisms involved in the potential control of TNF-{alpha} by ASFV remained largely unknown. Previous reports have shown an up-regulation of TNF-{alpha} gene expression after infection with ASFV E75-infected macrophages (33). However, in stimulated macrophages, an inhibition of TNF-{alpha} production was observed from early times postinfection (43). These data could be expected if A238L, a protein synthesized from 6 hpi during the viral cycle, would inhibit NF-{kappa}B/NF-AT-dependent gene activation, although a direct effect of A238L on TNF-{alpha} gene expression was needed to explain these controversial results. Indeed, we and others have previously shown that A238L regulates the activity of NF-AT and NF-{kappa}B (16, 17). In a recent work, we have demonstrated that A238L down-regulates COX-2 transcription in an NF-AT-dependent, but NF-{kappa}B-independent manner. The NF-{kappa}B site was not required for A238L inhibition and p65 NF-{kappa}B did not revert this inhibition, identifying NF-AT as the target of A238L-mediated down-regulation of COX-2 promoter (18).

In this study, we provide compelling evidence that establishes that A238L efficiently controls TNF-{alpha} production during the ASFV infection of Vero cells or macrophages, because A238L deletion viruses induced an exacerbated TNF-{alpha} secretion. Moreover, ectopic expression of the viral protein also inhibits the TNF-{alpha} mRNA expression after PMA/Ion stimulation in Jurkat cells.

The molecular mechanism by which A238L inhibits TNF-{alpha} has been investigated by examining the regulation of the TNF-{alpha} promoter activity by the viral protein. The region involved in the regulation of TNF-{alpha} transcription contains a CRE binding site, which binds ATF-2/Jun heterodimer and forms a composite element with the {kappa}3 site (6, 34, 39). By using serial deletion constructs of TNF-{alpha} promoter, we found that the pTNF(–120)luc reporter plasmid, a deletion mutant of the promoter that only contains the composite CRE/{kappa}3 site, was inhibited by A238L. In T cells activated by PMA/Ion treatment, a specific set of transcription factors is involved in the activation of the TNF-{alpha} enhancer. ATF-2/Jun is constitutively bound to this enhancer and NF-AT binds to multiple sites in the promoter in response to ionophore stimulation (34). NF-AT DNA-binding components can form Rel/NF-{kappa}B-like dimers on certain types of NF-AT-binding DNA elements (14, 44, 45), such as the {kappa}3 element of the TNF-{alpha} promoter that binds NF-AT dimers (14) as well as certain Rel-containing dimers (46).

In line with these results, we have found that the overexpression of NF-AT, p65, and c-Jun activates TNF-{alpha} transcription and counteracts the inhibition of the TNF-{alpha} promoter activity mediated by A238L. This result not only corroborates that these transcription factors are involved in the control of TNF-{alpha} in Jurkat cells after PMA/Ion treatment, but also, and more importantly, that A238L interferes with all of them in the activation of TNF-{alpha}. In parallel with the nuclear accumulation of NF-{kappa}B and NF-AT, activation of JNK1 by PMA/Ion in T cells results in the rapid phosphorylation of the constitutively bound c-Jun and ATF-2 heterodimer in the TNF-{alpha} promoter (47). In this regard, it is worth mentioning that neither down-regulation of c-Jun expression nor direct inhibition of the kinase activity of JNK1 could be found in Jurkat-A238L after PMA/Ion stimulation, indicating that A238L is interfering with c-Jun-dependent TNF-{alpha} gene expression in a step downstream phosphorylation by JNK1.

There are several mechanisms by which A238L could act to inhibit NF-{kappa}B activation. The similarity between ankyrin repeats in A238L and I{kappa}B suggests that, like I{kappa}B, A238L may bind directly to NF-{kappa}B. We have previously shown that p65 is coprecipitated with A238L from cell extracts infected with ASFV, suggesting that A238L is present in a complex with NF-{kappa}B (17, 48) during the viral infection. Purified recombinant A238L protein added to nuclear extracts from stimulated cells inhibited binding of NF-{kappa}B to target DNA sequences and displaced preformed NF-{kappa}B complexes from DNA. Furthermore, we also showed in these experiments that recombinant A238L protein inhibited the formation of complexes containing p65/p50 heterodimers, rather than those containing p50 homodimers, as was expected, because p50 homodimers act to suppress NF-{kappa}B-dependent transcription, whereas p50/p65 heterodimers act as transcriptional trans activators.

In the present work, we demonstrate that the expression of A238L in Jurkat cells does not inhibit the translocation of p65 to the nucleus after PMA/Ion stimulation, further supporting the hypothesis that A238L-mediated NF-{kappa}B inhibition does not involve hijacking of p65 in the cytoplasm. In relation to this, it is known that, although a major step in NF-{kappa}B activation is the removal of I{kappa}B from the NF-{kappa}B/I{kappa}B complex, allowing its nuclear translocation, a second level of regulation of NF-{kappa}B exists that depends on phosphorylation of trans activation domain enhancing its transcriptional activity. Thus, p65 phosphorylation at its trans activation domain is thought to enhance transcriptional competency by recruiting coactivator proteins such as CBP/p300 to NF-{kappa}B.

p300 is a member of a family of transcriptional coadaptor molecules with distinct functional domains that have been shown to interact with several viral proteins such as the adenovirus protein E1A, SV40 large T Ag (49), and herpes virus E6 and E7 (50). The consequence of this interaction on the biological effects on p300 function differs depending on the specific viral proteins and, although both adenovirus E1A and SV40 large T Ag interact with p300 in overlapping locations, large T Ag inhibits, whereas E1A enhances, the phosphorylation of p300 (51).

Although the A238L induced inhibition of the stimulus-induced trans activation of NF-AT, p65, and c-Jun, or the effect on the recovery of the inhibition of the TNF (–120)luc promoter construct activity by overexpression of CBP/p300 described in this work could explain how A238L down-regulates TNF-{alpha} promoter activity, the molecular basis for the A238L-induced inhibition of TNF-{alpha} gene expression is still not clearly defined. CBP and p300 are essential for the optimal transcriptional activity of TNF-{alpha} and COX-2 (12, 52), two genes inhibited by A238L. It is interesting to speculate that a viral gene such as A238L, which inhibits the trans activation of NF-AT, NF-{kappa}B, and c-Jun in response to PMA/Ion, may have evolved this level of flexibility to accomplish novel patterns of gene regulation to evade the host response. Our results indicate that A238L, which localizes in the nucleus of infected cells and after PMA/Ion stimulation of transfected cells, binds to the CRE/{kappa}3 complex and displaces the coactivators CBP/p300, thus inhibiting the trans activation of factors that associate with them as NF-AT, NF-{kappa}B, and c-Jun.

Thus, the full understanding of TNF-{alpha} gene regulation in ASFV-infected cells and in A238L stably expressing human T cells could potentially lead to novel therapeutic manipulations that interrupt the signaling cascade, resulting in TNF-{alpha} gene transcription and subsequent protein production in conditions characterized by TNF-{alpha}-mediated immunopathologic effects. A detailed understanding of the mechanism by which A238L inhibits TNF-{alpha} gene transcription in different cells also offers potential novel insights regarding the role of TNF-{alpha} in several inflammatory pathologies.

The analysis presented in this work indicates for the first time that A238L is a novel viral protein with a potent regulatory function on TNF-{alpha} that might be mediated by the control of CBP/p300 activity.


   Acknowledgments
 
We are grateful to Dr. Miguel Iñiguez and Dr. José Salas for critical reading of the manuscript, and Maria José Bustos for excellent technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from Ministerio de Educación y Ciencia (BFU2004-00298/BMC), the Wellcome Trust (075813/C/04/z), and by an institutional grant from the Fundación Ramón Areces. C.H. was a fellow from Fundación Ramón Areces. Back

2 Address correspondence and reprint requests to Dr. Yolanda Revilla, Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, Madrid 28049, Spain. E-mail address: yrevilla{at}cbm.uam.es Back

3 Abbreviations used in this paper: ASFV, African swine fever virus; ATF, activating transcription factor; {beta}-gus, {beta}-glucuronidase; CBP, CREB binding protein; COX, cyclooxygenase; CRE, cAMP-responsive element; CsA, cyclosporine; hpi, hours postinfection; Ion, calcium ionophore; MOI, multiplicity of infection; RLU, relative luciferase unit; wt, wild type. Back

Received for publication February 17, 2005. Accepted for publication October 17, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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