Regulation of Inducible Nitric Oxide Synthase ... - Journal of Virology

JOURNAL OF VIROLOGY, Nov. 2006, p. 10487–10496 0022-538X/06/$08.00⫹0 doi:10.1128/JVI.00862-06 Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Vol. 80, No. 21

Regulation of Inducible Nitric Oxide Synthase Expression by Viral A238L-Mediated Inhibition of p65/RelA Acetylation and p300 Transactivation Aitor G. Granja, Prado Sabina, Marıá L. Salas, Manuel Fresno, and Yolanda Revilla* Centro de Biologıá Molecular Severo Ochoa, Universidad Auto ńoma de Madrid, 28049 Madrid, Spain Received 27 April 2006/Accepted 31 July 2006

Uncontrolled generation of nitric oxide (NO) by inducible nitric-oxide synthase (iNOS) can cause damage to host cells and inflammation, two undesirable events for virus spreading. African swine fever virus (ASFV) infection regulates iNOS-induced gene expression through the synthesis of the A238L virus protein. We here explored the role of A238L, an NF-␬B and NFAT inhibitor, in the regulation of iNOS transcription in macrophages. NO production and iNOS mRNA and protein levels as well as iNOS promoter activity after lipopolysaccharide (LPS)-gamma interferon (IFN-␥) treatment were down-regulated both during ASFV infection and in Raw 264.7 cells stably expressing the viral protein. Overexpression of p300, but not of a histone acetyltransferase (HAT) defective mutant, reverted the A238L-mediated inhibition of both basal and LPS– IFN-␥-induced iNOS promoter activity. Following stimulation with LPS–IFN-␥, p65 and p300 interaction was abolished in Raw-A238L cells. Expression of A238L also inhibited p65/relA and p300 binding to the distal NF-␬B sequence of the iNOS promoter together with p65 acetylation. Finally, A238L abrogated p300 transactivation mediated by a GAL4-p300 construction. These results provide evidence for an unique viral mechanism involved in transcriptional regulation of iNOS gene expression. African swine fever virus (ASFV), the sole member of the Asfarviridae family (13), encodes a protein, A238L, which has been described to inhibit the activation of the NF-␬B and NFAT transcription factors both when expressed in different cells and during ASFV infection (28, 34). In previous reports, we have also shown that A238L is thus able to down-regulate the transcriptional activation of immunomodulatory genes, such as cyclooxygenase-2 (COX-2) and tumor necrosis factor alpha (TNF-␣), by a mechanism involving the control of CBP/ p300 activation (18, 19). A238L contains ankyrin repeats homologous to those found in the I␬B family and behaves as a bona fide I␬B-␣ viral homologue, since it binds p65 NF-␬B and prevents translocation and binding of p65-p50 NF-␬B dimers to their target sequence in the DNA (34). The generation of nitric oxide (NO) from oxidation of Larginine (to give citrulline and NO) is catalyzed by three distinct members of a nitric oxide synthase (NOS) family. They are either constitutively expressed in neurons and endothelial cells or induced (iNOS) by endotoxin and/or proinflammatory cytokines such as interleukin-1, TNF-␣, and gamma interferon (IFN-␥) mainly in macrophages (20, 22). The sequences of cloned iNOS promoters of all species investigated so far exhibit homologies to binding sites for numerous transcription factors known to be involved in the lipopolysaccharide (LPS)-cytokine-mediated induction of transcription (9, 24, 25, 38, 39, 41). Coactivators that are involved in iNOS promoter activation have been recently reported (11), showing the binding of p300 to the iNOS promoter region and

* Corresponding author. Mailing address: Centro de Biologıá Molecular Severo Ochoa, Universidad Auto ńoma de Madrid, Madrid 28049, Spain. Phone: 34914978486. Fax: 34914974799. E-mail: yrevilla @cbm.uam.es.

demonstrating that p300 overexpression increases LPS–IFN-␥induced iNOS promoter activity. Although much is known about the mechanisms of iNOS induction by viral infections, few transcriptional repression mechanisms developed by viruses have been described. Thus, p300 is a member of a family of transcriptional coactivator molecules with distinct functional domains that have been shown to interact with E1A and several other viral proteins, such as simian virus 40 large T antigen or herpesvirus E6 and E7. Here we have investigated the ability of ASFV to inhibit iNOS gene expression through the synthesis of A238L protein. To achieve this, we have used a deletion mutant lacking the A238L gene from the virulent isolate E70. Using this tool, we have characterized events involved in iNOS gene induction following infection of porcine macrophages, the natural target of the infection. We also describe here that A238L abrogates the stimulating effect of combined LPS–IFN-␥ on the iNOS promoter in Raw 264.7 cells stably expressing the viral protein. Our results show that A238L prevents the enhancement of iNOS promoter activity mediated by p300 overexpression as well as the LPS–IFN-␥-increased p300 interaction with promoter-bound NF-␬B–p65. NF-␬B sites seem to be essential for the inhibition induced by the viral protein, since overexpression of p65 recovered the normal levels of the iNOS promoter activity both in basal and in stimulated conditions. Finally, we also provide evidence that A238L impairs the p300 transactivation, thereby decreasing p300-mediated acetylation of the p65 subunit. Taken together, these results represent a new and sophisticated viral mechanism to regulate NO production. MATERIALS AND METHODS Cell culture, viruses, and reagents. The mouse macrophage cell line Raw 264.7 was obtained from the ATCC and cultured in RPMI 1640 medium supplemented

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with 5% fetal bovine serum, 2 mM L-glutamine, 100 U of gentamicin per ml, and nonessential amino acids. Cells were grown at 37°C in 7% CO2 in air saturated with water vapor. Raw 264.7 cells were stimulated by LPS (Sigma-Aldrich) at 1 ␮g/ml and recombinant mouse IFN-␥ (R&D Systems) at 200 U/ml (LPS–IFN-␥). Generation of A238L stably expressing Raw 264.7 cells was done using the same protocol described in reference 19 for Jurkat T cells. These cellular lines were named Raw-pcDNA and Raw-A238L. Porcine alveolar macrophages were obtained by bronchoalveolar lavage of pigs and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% homologous swine serum, 2 mM Lglutamine, 100 U of gentamicin per ml, and nonessential amino acids, as previously described (6). The virulent ASFV strain E70 was propagated and titrated by plaque assay on swine alveolar macrophages as described previously (5, 6). The deletion mutant E70⌬A238L was generated by using a transfection-infection protocol as previously described (18). mRNA analysis. Total RNA was prepared from Raw-pcDNA or Raw-A238L or porcine alveolar macrophages and analyzed by reverse transcription (RT)PCR as previously described (18). Specific primers used in PCRs were porcine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (forward, 5⬘-AGCTTCA TCAATGGAAAGG-3⬘; reverse, 5⬘-AGAAGCAGGGATGATGTTCTG-3⬘), porcine iNOS (forward, 5⬘-TGCGTTATGCCACCAACAATG-3⬘; reverse, 5⬘-A CTCTCCAGGATGTTGTAG-3⬘), murine iNOS (forward, 5⬘-GAGAGATCCG ATTGGC-3⬘; reverse, 5⬘-GCAGATTCTGCTGGGATTTCA-3⬘), murine ␤-actin (forward, 5⬘-CTTTTGATGTCACGCACGATTTC-3⬘; reverse, 5⬘-GTGGG CCGCTCTAGGCC-3⬘), viral A238L (forward, 5⬘-CGCGCGTCTAGATTACT TTCCATACTTGTT-3⬘; reverse, 5⬘-GCGCGCAAGCTTATGGAACACATGT TTCCA-3⬘), and viral p72 (forward, 5⬘-CGGGATCCATGGCATCAGGAGGA G-3⬘; reverse, 5⬘-CGCGAGATGACCATGGGCC). Western blot analysis. Cytosolic and nuclear extracts or whole-cell extracts from Raw-pcDNA and Raw-A238L cells unstimulated or stimulated with LPS– IFN-␥, were prepared using the same protocol described previously (19) for Jurkat T cells. The specific primary antibodies used were: NF-␬B–p50 (sc-114; Santa Cruz Biotechnology), NF-␬B–p65 (sc-109; Santa Cruz Biotechnology), p300 (sc-584; Santa Cruz Biotechnology), iNOS (AB1631; Chemicon), ␤-actin (AC-15; Sigma), and acetylated lysine (Ac-K-103; Cell Signaling). Membranes were exposed to horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences), followed by chemiluminescence (ECL; Amersham Biosciences) detection by autoradiography. Densitometric analysis was performed by using TINA 2.0 software. Quantitation of nitric oxide in culture supernatants. Supernatants from RawpcDNA and Raw-A238L unstimulated or stimulated with LPS–IFN-␥ or supernatants from porcine alveolar macrophages mock infected or infected with E70wt or E70⌬A238L were recovered at the indicated poststimulation or postinfection times. The amount of NO in the culture medium was determined by using the Griess reagent system (Promega) according to the manufacturer’s instructions. Plasmid constructs. The murine iNOS promoter construct containing the full-length promoter sequence fused to the firefly luciferase reporter gene, named p(iNOS)m-luc, was generated by SalI restriction of the iNOS promoter (⫺1584/⫹161) from the pUP1 plasmid, a generous gift from Santiago Lamas (Fundacio ń Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain) and cloning in pGL3-basic plasmid (Promega). The pcDNAA238L expression plasmid was generated as described previously (19). The pRC-CMV-cRel expression plasmid, which overexpresses the NF-␬B–p50 subunit, was generated as described previously (26). The pcDNA-p65 expression plasmid was a generous gift from Jose´ Alcamı´ (Unidad de Inmunopatologıá, Centro Nacional de Microbiologıá, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain). The GAL4-luciferase construct (pGAL4-Luc) contains five GAL4 DNA consensus binding sites derived from the yeast Saccharomyces cerevisiae GAL4 gene fused to the luciferase reporter gene (27). The pGAL4-p65 construct has the yeast GAL4 DNA binding domain fused to the carboxyterminal transactivation domain of p65 and was generated as described previously (35). The GAL4-p300 construct was a generous gift from Neil Perkins (School of Life Sciences, Division of Gene Regulation and Expression, University of Dundee, Dundee, Scotland, United Kingdom) and was generated as described previously (36). The p300 wild-type expression plasmid pCl-p300 and its histone acetyltransferase (HAT) deletion mutant, pCl-p300⌬HAT, was a generous gift from Joan Boyes (Institute of Cancer Research, London, United Kingdom) and was generated as described previously (4). Transfection and luciferase assays. Raw-pcDNA and Raw-A238L or porcine alveolar macrophages were transfected with 250 ng of specific reporter plasmids per 106 cells using the Lipofectamine Plus reagent (Invitrogen) according to the manufacturer’s instructions and mixing in Opti-MEM (Invitrogen) in a six-well plate. In cotransfection assays, 0.1 to 1.6 ␮g of the corresponding expression

J. VIROL. plasmid per 106 cells was added. The cells were incubated for 4 h, washed, incubated in serum-free medium for 24 h, and treated with or without LPS– IFN-␥. As a transfection control for luciferase assays, the Renilla luciferase control plasmid pRL-TK (Promega) was cotransfected in all of the experiments. At the indicated poststimulation times, cells were lysed with 200 ␮l of cell culture lysis reagent (Promega) and microcentrifuged at full speed for 5 min at 4°C, and 20 ␮l of each supernatant was used to determine firefly and Renilla luciferase activity in a Monolight 2010 luminometer (Analytical Luminescence Laboratory) using the Dual-Luciferase assay system (Promega). Transfections were normalized to Renilla luciferase activity, and results were expressed as relative luminescence units after normalization of the protein concentration determined by the bicinchoninic acid method, as indicated in the figure legends. Transfection experiments were performed in triplicate, and the data are presented as the means, in relative luciferase units (RLU), ⫾ standard deviations (SD). In vitro DNA-protein binding assay. Binding of the p50, p65, and p300 proteins to NF-␬B sequences in the iNOS promoter DNA was analyzed by a DNAprotein binding assay as previously described (18) for Jurkat T cells. Biotinlabeled double-stranded oligonucleotide probes corresponding to a murine distal NF-␬B sequence (5⬘-biotin-CTAGGGGGATTTTCCCTCTC-3⬘), a murine proximal NF-␬B sequence (5⬘-biotin-AACTGGGGACTCTCCCTTTG-3⬘), or a nonrelevant DNA sequence (5⬘-biotin-TTACCAACTGAGCCATCTCC-3⬘), were synthesized by Isogen. The antibodies against p50 (sc-114; Santa Cruz Biotechnology), p65 (sc-109; Santa Cruz Biotechnology), or p300 (sc-584; Santa Cruz Biotechnology) were used in the assay. Coimmunoprecipitation. Nuclear extracts were prepared from 80 to 90% confluent Raw-pcDNA and Raw-A238L cells treated with or without LPS–IFN-␥ for 6 h, and their protein concentrations were determined as described above. Nuclear extracts were incubated with a specific p65 antibody (sc-109; Santa Cruz Biotechnology) or a rabbit preimmune normal immunoglobulin G (IgG) as a negative control at a final concentration of 4 ␮g/ml. The samples were incubated at 4°C overnight. Protein A/G-Sepharose beads (Sigma) were added, incubated for 3 h at 4°C, and centrifuged. The beads were washed three times with wash buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 1 mM EDTA, and 0.5% Nonidet P-40). The immunoprecipitates were mixed with sodium dodecyl sulfate (SDS) loading buffer and analyzed by 4 to 15% SDS–polyacrylamide gel electrophoresis (PAGE) followed by Western blotting using specific antibodies against p65 and p300. Acetylation of p65. NF-␬B–p65 in nuclear extracts was immunoprecipitated with a specific antibody against p65 (sc-109; Santa Cruz Biotechnology) or a rabbit preimmune normal IgG as a negative control at a final concentration of 4 ␮g/ml, as described above. The immunoprecipitates were collected by using protein A/G-Sepharose beads (Sigma). The beads were washed three times with wash buffer, as described above. The immunoprecipitates were mixed with SDS loading buffer and analyzed by 4 to 15% SDS–PAGE. Acetylated p65 was detected by Western blotting using an antibody against acetylated lysine (Ac-K103; Cell Signaling Technology).

RESULTS A238L inhibits iNOS promoter activity and transcription during ASFV infection in porcine macrophages. iNOS transcription is regulated by several transcription factors such as NF-␬B, NF–interleukin-6, Oct-1, AP-1, C/EBP, CREB, IFN regulatory factor 1, SRF, and STAT-1␣. Since A238L has been described as an inhibitor of some of these molecules (18, 19, 28, 34), we have explored the possibility that the viral protein could inhibit iNOS activity. ASFV replicates mainly in macrophages and monocytes in vivo. This fact should play a critical role in pathogenesis, since macrophage-derived cytokines strongly determine the development of inflammatory responses against infection. Due to the fact that macrophages appear to be one of the main sources of iNOS, the role of A238L in the control of the enzyme in this cellular type is an important issue to address. To assess this point, we have generated an ASFV A238L deletion mutant (E70⌬A238L) from the virulent strain E70, which has been shown to induce a strong infection in these cells, as we previously described (18). We have used these tools to investigate the role of A238L in

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FIG. 1. Analysis of iNOS expression and NO synthesis in ASFVinfected porcine macrophages. (A) Porcine alveolar macrophages were transfected with the p(iNOS)m-luc plasmid as described in Materials and Methods and mock infected or infected with the virulent isolate E70 wt or E70⌬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 per ␮g of protein from triplicate transfections (means ⫾ SD) are shown. (B) RT-PCR analysis from infected porcine alveolar macrophages. Total RNA (1 ␮g) from macrophages mock infected (Mock) or infected with E70 wt or E70⌬A238L virus was prepared at the indicated postinfection times and analyzed by RT-PCR to measure iNOS 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 infected with the virulent isolate E70 wt or E70⌬A238L mutant, supernatants were recovered at the indicated postinfection times, and the amount of NO in the culture medium was determined by using the Griess reagent system (Promega) according to the manufacturer’s instructions, as described in Materials and Methods.

the control of iNOS transcription during ASFV infection in swine macrophages. To achieve this, macrophages were first transfected with the plasmid p(iNOS)-luc, which contains the luciferase reporter gene under the control of the full-length sequence of the murine iNOS promoter. Sixteen hours after transfection, cells were infected either with the parental E70 or with E70⌬A238L viruses (multiplicity of infection of 5 PFU/ cell), and at the indicated times after infection, luciferase activity was measured in cell extracts. As shown in Fig. 1A, a slight induction of iNOS promoter activity was detected after

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12 h postinfection (hpi) with the wild-type virus. However, a higher activity of the promoter was observed from 6 hpi in cells infected with the deletion mutant virus. This result indicates that A238L strongly down-regulates iNOS promoter activation during ASFV infection in Vero cells. We next explored by RT-PCR the expression of the A238L gene in porcine macrophages infected with the recombinant virus E70⌬A238L or with the parental virus. As shown in Fig. 1B, and as expected, no A238L-specific mRNA was detected in extracts from macrophages infected with E70⌬A238L in contrast to that obtained in E70-infected macrophages. As it is also shown in this figure, E70wt and E70⌬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 iNOS-specific mRNA, levels of the enzyme after infection of macrophages with the recombinant virus E70⌬A238L were clearly higher than those induced by the wild-type virus, demonstrating an inhibitory role of A238L in the iNOS expression during ASFV infection in macrophages (Fig. 1B). To evaluate whether the increase of iNOS promoter activity observed after E70⌬A238L infection corresponds to an increase in the iNOS product secretion, we quantified the amount of nitrite in supernatants collected from the infected macrophages. The infection with the deletion mutant virus induces an increase in NO production from 12 hpi, more clearly observed at 24 hpi. It is noteworthy that even the minimal enzyme amount observed 4 h after the infection with the wild-type virus (Fig. 1B) is able to maintain detectable levels of substrate conversion, although differences in NO can be clearly detected (Fig. 1C). Taken together, these results show the ability of A238L to control the activation of the iNOS promoter and nitrite production, which may represent an important mechanism to evade the immune response by ASFV. Overexpression of A238L down-regulates iNOS promoter activation, mRNA expression, and NO synthesis in Raw 264.7 cells. Considering the critical importance of NF-␬B in regulating iNOS transcription, we hypothesized that A238L, which has been previously described as a viral I␬B homologue which inhibits NF␬B activation (18, 34), might have a modulator role for transcriptional repression of iNOS gene expression. To test this hypothesis, we have generated Raw 264.7 cells that stably express the A238L gene by transfection with pcDNA-A238L, followed by selection using G418 as described in Materials and Methods. Raw-pcDNA or Raw-A238L cells were transfected with the plasmid p(iNOS)m-luc, which contains the luciferase reporter gene under the control of the full-length sequence of the mouse iNOS promoter. The 5⬘-flanking region of the murine iNOS gene contains two clusters of cis-acting regulatory elements that are essential for iNOS transcription, of which the proximal cluster is required for LPS-induced activation and the distal cluster is required for IFN-␥-induced activation. It is also known that LPS–IFN-␥ synergistically activates the iNOS promoter by stimulating the binding of several transcription factors, mainly NF-␬B, IFN regulatory factor 1, and C/EBP to their respective cognitive sites in these two clusters of regulatory elements (25, 39). As shown in Fig. 2A, and in parallel with that described above during ASFV infection, ectopic A238L expression strongly decreased the transcription driven

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FIG. 2. Analysis of iNOS expression in stably expressing A238L Raw cells. (A) Raw-pcDNA (gray bars) or Raw-A238L (black bars) cells were transiently transfected with the p(iNOS)m-luc murine promoter construct (250 ng/106 cells) as described in Materials and Methods. Sixteen hours after transfection, the cells were cultured in the absence or presence of 1 ␮g/ml of LPS plus 200 U/ml of IFN-␥. At the indicated times poststimulation, whole-cell extracts were prepared 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 per ␮g of protein (means ⫾ SD). Numbers above the bars represent the ratio between the RLU values obtained from Raw-pcDNA versus Raw-A238L cells at each time point. (B) Total RNA (1 ␮g) from Raw-pcDNA and Raw-A238L cells cultured in the absence (0 h) or presence of 1 ␮g/ml of LPS plus 200 U/ml of IFN-␥ was analyzed at the indicated times by RT-PCR to measure A238L and iNOS mRNA expression. A control using specific oligonucleotides for ␤-actin is also included to rule out differences in PCR amplification. (C) Western blot analysis of stably expressing A238L cells (Raw-A238L) or control cells (Raw-pcDNA) cultured in the absence (0 h) or presence of 1 ␮g/ml of LPS plus 200 U/ml of IFN-␥. At the indicated times poststimulation, whole-cell extracts were prepared, subjected to SDS-PAGE (30 ␮g of protein sample), and detected by immunoblotting with an iNOS-specific antibody. A protein loading control is included by ␤-actin blotting. (D) Nitrite accumulation in the supernatant of stably expressing A238L cells (Raw-A238L) or control cells (Raw-pcDNA) cultured in the absence or presence of 1 ␮g/ml of LPS plus 200 U/ml of IFN-␥. At the indicated times poststimulation, culture supernatants were recovered and nitrite content was measured using Griess reagent as described in Materials and Methods.

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by the p(iNOS)m-luc construction after stimulation with LPS– IFN-␥. Figure 2B shows the expression of specific mRNA for A238L in Raw 264.7 cells stably expressing A238L that is absent in Raw-pcDNA control cells. It has been previously reported that macrophage activation induced by LPS–IFN-␥ increases iNOS mRNA levels. In agreement with these data, iNOS mRNA was increased in cells transfected with the empty pcDNA vector upon treatment with LPS–IFN-␥. More interestingly, we found lower levels of iNOS transcript in cells expressing A238L after stimulation with LPS–IFN-␥ (Fig. 2B), indicating that the viral protein is able to regulate iNOS expression. To address whether iNOS mRNA inhibition was paralleled by iNOS protein decrease, we performed Western blot analysis with cellular extracts from Raw-pcDNA or Raw-A238L, using a specific antibody against iNOS protein. As expected, iNOS protein levels were clearly diminished after LPS–IFN-␥ activation in Raw-A238L cells compared to control cells (Fig. 2C), showing an inhibition similar to that obtained in mRNA analysis. NO production in the form of nitrite was also determined in culture supernatants from Raw-pcDNA or Raw-A238L after stimulation with LPS–IFN-␥. Unstimulated cells produced undetectable levels of NO2⫺, while after stimulation, the amounts of nitrite secreted in culture supernatants differed considerably depending on A238L expression. Raw-pcDNA cells secreted significant NO2⫺ amounts upon LPS–IFN-␥ treatment, whereas the amounts of nitrite detected in supernatants from Raw-A238L were lower (approximately 50%) from 12 h poststimulation, showing a parallelism with the down-regulation of iNOS mRNA and protein expression (Fig. 2D). p300 overexpression reverts the A238L-mediated inhibition of the iNOS promoter activity and iNOS protein synthesis. p300 plays a major role as a coactivator for multiple transcription factors in the induction of several proinflammatory genes such as TNF-␣, COX-2, IFN-␤, and iNOS by viruses and LPS– IFN-␥ (11, 12, 15, 37). Binding of p300 to the iNOS promoter region and increased iNOS gene transcription by p300 overexpression has been recently described (11). On the other hand, we have previously shown that CBP/p300 overexpression reverts the A238L-mediated inhibition of the pTNF(⫺120)luc promoter activity. To characterize a putative involvement of transcriptional coactivator p300 in the iNOS promoter activity down-regulation induced by the viral protein, we have cotransfected increasing amounts of expression plasmid for p300 (pClp300 wt) together with p(iNOS)m-luc into Raw-pcDNA and Raw-A238L cells. Sixteen hours after transfection, the cells were cultured in the absence or presence of LPS–IFN-␥ for 6 h and assayed for luciferase activity. As shown in Fig. 3A, a dose response induction of iNOS promoter activity was shown by overexpression of p300 in Raw-pcDNA cells after stimulation with LPS–IFN-␥. More interestingly, p300 rescued the activity of the promoter in a dose-dependent manner in Raw-A238L cells, indicating the involvement of the coactivator in the inhibition of iNOS expression by A238L. Transfection of pClp300⌬HAT, a HAT deletion mutant of p300, in Raw-pcDNA or Raw-A238L did not have costimulatory activity and did not revert the A238L inhibition, consistent with the involvement of p300 HAT in iNOS transactivation and suggesting that this domain prevents A238L down regulation (Fig. 3A). To confirm the involvement of p300 in the inhibitory mechanism induced

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FIG. 3. Effect of p300 overexpression in iNOS promoter activation and iNOS protein levels. (A) Raw-pcDNA (open bars) or Raw-A238L (shaded bars) cells were transiently transfected with the p(iNOS)m-luc promoter reporter plasmid and with the indicated doses (from 0 to 1.6 ␮g of DNA/106 cells) of pCl-p300 wt or p300 HAT deletion mutant (pCl-p300⌬HAT) expression plasmids. Sixteen hours after transfection, the cells were cultured in the absence (plain bars) or presence of 1 ␮g/ml of LPS plus 200 U/ml of IFN-␥ (striped bars) for 6 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 per ␮g of protein (means ⫾ SD). (B) Effect of p300 wt and p300 HAT mutated in iNOS protein levels. The figure shows p300 and iNOS protein levels determined by Western blotting in Raw-pcDNA and Raw-A238L cells transfected with 1.6 ␮g of DNA/106 cells of pCl-p300wt, pCl-p300⌬HAT, or empty pCl plasmid as a control. Sixteen hours after transfection, the cells were treated (⫹) or not (⫺) with 1 ␮g/ml of LPS plus 200 U/ml of IFN-␥ for 12 h, and whole-cell extracts were prepared, subjected to 4 to 15% SDS– PAGE, and detected by immunoblotting with p300- and iNOS-specific antibodies. A protein loading control is included by ␤-actin blotting. The result is a representative assay from two separate experiments. The Western blot shown has been performed using the same membrane and exposition. The densitometric analysis shows the amount of iNOS in each assay from two independent experiments (means ⫾ SD).

by the viral protein, we performed Western blot analysis with cellular extracts from Raw-pcDNA or Raw-A238L, previously transfected with pCl-p300 or pCl-p300⌬HAT (Fig. 3B). Using a specific antibody against iNOS, a recovery of iNOS protein levels after overexpression of wild-type p300 could be detected, whereas no effect was observed after expression of p300⌬HAT. Figure 3B also shows that the levels of p300 protein, which could be detected in both Raw-pcDNA and Raw-A238L resting cells, were not increased after treatment with LPS–IFN-␥. Taken together, these results indicate that A238L-mediated


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iNOS inhibition is accomplished by modulation of p300 transcriptional coactivator. A238L displaces p65 and p300 from the distal NF-␬B-specific DNA sequence in the iNOS promoter. To further investigate the mechanism by which A238L controls the activity of the iNOS promoter, we have carried out DNA-protein binding experiments using biotinylated distal (d-NF-␬B) and proximal (p-NF-␬B) probes. To achieve this, the biotinylated DNA probes were incubated with nuclear extracts from Raw-pcDNA or Raw-A238L cells treated or not with LPS–IFN-␥, and the complex was pulled down with streptavidin-agarose beads, as described in Materials and Methods. Finally, the proteins in the complex were analyzed by Western blotting using antibodies against p300, NF-␬B–p65, and NF-␬B–p50. As shown in Fig. 4A, p300, p65, and p50 were present to similar extents in whole nuclear extracts from either Raw-pcDNA or RawA238L and its binding in control cells increased after LPS– IFN-␥ stimulation. This increase was evident for p50, p65, and p300 in both d-NF-␬B and p-NF-␬B sites. Interestingly, the results of the pull-down assay showed that, while p300, p65, and p50 were bound to the d-NF-␬B in control cells, the levels of both p300 and p65 bound to this site were significantly lower in the case of cells expressing A238L, indicating that the viral protein partially displaces or prevents the binding of p300 and p65 from this specific site in the iNOS promoter. On the other hand, these results also suggest that A238L inhibits iNOS promoter activity by displacing p300 and p65 from the d-NF-␬Bspecific site, whereas the second NF-␬B site in the iNOS promoter, the p-NF-␬B, would be involved in a lesser, if any, degree of control of iNOS activity induced by the viral product. It is interesting that p50 remains bound to d-NF-␬B in RawA238L cells, revealing that binding of this subunit to the promoter seems not to be affected by the presence of the viral protein, suggesting that p50 is not involved in the mechanism of inhibition induced by A238L to control iNOS transcription. To confirm this last hypothesis, and to further demonstrate the involvement of p65 in the inhibition induced by A238L, RawpcDNA or Raw-A238L cells were transiently transfected with the p(iNOS)m-luc reporter plasmid together with increasing doses (from 0 to 1.6 ␮g of DNA/106 cells) of the expression plasmid pRC-CMV-cRel to overexpress the p50 subunit of NF-␬B transcription factor or pCMV-p65 to overexpress the p65 subunit. Sixteen hours after transfection, the cells were cultured in the absence or presence of LPS–IFN-␥ for 6 h and assayed for luciferase activity. The results clearly showed that p65 overexpression recovered the inhibition of iNOS promoter induced by A238L, whereas the A238L-mediated inhibition of the promoter was not affected by the expression of similar amounts of p50. A238L interferes with the interaction of p65 and p300. Since A238L was shown to interact with p65 during ASFV infection (34), and considering that the p65 subunit of NF-␬B recruits p300 to its transcriptional activation complex through the C/H1 domain (42), we investigated whether the mechanism whereby A238L suppresses the transcriptional activation of the iNOS/ NF-␬B signal transduction pathway involved direct competition for binding to p300. To achieve this, we prepared nuclear extracts from resting and LPS–IFN-␥-stimulated Raw-pcDNA and Raw-A238L cells, and the interaction between p65 and endogenous p300 was examined by immunoprecipitation with

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FIG. 4. Effect of A238L on the binding of p50/p65 and p300 to NF-␬B sites in the iNOS promoter. (A) Nuclear extracts from Raw-pcDNA and Raw-A238L cells treated (⫹) or not (⫺) with 1 ␮g/ml of LPS plus 200 U/ml of IFN-␥ for 6 h were incubated with the indicated biotinylated probe, and the complex was pulled down with streptavidin-agarose beads, as described in Materials and Methods. After extensive washing, proteins in the complex were analyzed by Western blotting using antibodies against p300, p65, or p50. The control probe is a biotinylated 22-bp nonrelevant DNA sequence of the murine iNOS promoter. Inputs were also included to show the presence of the analyzed proteins in nuclear protein extracts of Raw cells. (B) Raw-pcDNA (open bars) or Raw-A238L (shaded bars) cells were transiently transfected with the p(iNOS)m-luc reporter plasmid and with the indicated doses (from 0 to 1.6 ␮g of DNA/106 cells) of two different expression plasmids: pRC-CMV-cRel to overexpress the p50 subunit of NF-␬B or pCMV-p65 to overexpress the p65 subunit of NF-␬B transcription factor. Sixteen hours after transfection, the cells were cultured in the absence (plain bars) or presence of 1 ␮g/ml of LPS plus 200 U/ml of IFN-␥ (striped bars) for 6 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 per ␮g of protein (means ⫾ SD).

specific antibodies for p65 or control serum. The presence of p300 and p65 in the immunoprecipitate was analyzed by Western blotting. The results shown in Fig. 5 indicate that p300 complexed with NF-␬B–p65 was increased by LPS–IFN-␥ treatment as previously described (11), but more importantly, the presence of A238L prevented this interaction. A control rabbit preimmune IgG did not precipitate a p300-containing complex. Thus, A238L abrogates the binding of p300 to p65 in the nucleus both at basal and LPS–IFN-␥-stimulated cellular states, suggesting that iNOS transcriptional inhibition by the viral protein is dependent on its ability to compete with p65 for binding to the p300 coactivator, although a direct interaction between A238L and p300 has not yet been demonstrated. Inhibition of p65 acetylation by A238L. We have shown above that the increase of the iNOS promoter activity by overexpression of wild-type (wt) p300 in Raw-pcDNA was inhibited in Raw-A238L cells, and in parallel, that p65 and p300 binding to the iNOS promoter distal NF␬B probe was interfered with by A238L in resting and LPS–IFN-␥-treated cells. Activation of the iNOS promoter has been described to be regulated by

p300-mediated acetylation of NF-␬B–p50 (11). To determine whether the mechanism by which A238L inhibits the iNOS promoter activity might be regulated by acetylation, we prepared nuclear extracts from resting and LPS–IFN-␥-treated Raw-pcDNA and Raw-A238L cells and immunoprecipitated the nuclear extract proteins with an antibody against p65, and acetylation in the immunoprecipitate was detected by Western blotting with an anti-acetyl lysine antibody. Acetylated p65 was detectable at the basal cell state and increased by about 3-fold in Raw-pcDNA LPS–IFN-␥-treated cells, whereas the expression of the viral protein resulted in strong reduction of acetylated p65 both at the basal state and in LPS–IFN-␥-stimulated Raw-A238L cells (Fig. 6). These results indicate a relationship between p65 acetylation and A238L regulation of the iNOS promoter. A238L down-regulates the transactivation function of p65 and p300. Regulation of NF-␬B following stimulation with LPS–IFN-␥ occurs via activation of at least two pathways. The best characterized of these regulates the release of NF-␬B from I␬B␣ and the subsequent translocation of NF-␬B to the nucleus. However, this is not sufficient to activate NF-␬B-

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FIG. 5. A238L inhibits the direct interaction between NF-␬B–p65 and p300. Nuclear extracts from 107 stably transfected Raw-pcDNA and Raw-A238L cells treated (⫹) or not (⫺) with 1 ␮g/ml of LPS plus 200 U/ml of IFN-␥ for 6 h were incubated and immunoprecipitated (IP) with 4 ␮g of rabbit polyclonal NF-␬B–p65 specific antibody (p65) or rabbit preimmune normal IgG (control IgG) as a negative control of coimmunoprecipitation, as described in Materials and Methods. Immunoprecipitates were separated by 4 to 15% SDS–PAGE, electrophoretically transferred to an Immobilon membrane, and detected by immunoblotting with the same NF-␬B–p65 (␣p65) antibody to determinate levels of p65 in the precipitate, or with p300 (␣p300)-specific antibody to detect the interaction between p65 and p300. The densitometric analysis shows the ratio between coimmunoprecipitated p300 amount and p65 precipitated amount from three independent experiments (mean ⫾ SD). WB, Western blot.

dependent gene transcription. The second pathway, which involves posttranslational modifications, regulates the transactivating ability of the p65 subunit of NF-␬B. We have previously described that A238L, a viral I␬B␣ homologue, does not con-

FIG. 6. A238L inhibits the acetylation of p65 in stimulated Raw cells. Nuclear extracts from 107 stably transfected Raw-pcDNA and Raw-A238L cells, treated (⫹) or not (⫺) with 1 ␮g/ml of LPS plus 200 U/ml of IFN-␥ for 6 h, were incubated and immunoprecipitated (IP) with 4 ␮g of rabbit polyclonal NF-␬B–p65-specific antibody (␣p65), as described in Materials and Methods. Immunoprecipitates were separated by 8% SDS–PAGE, electrophoretically transferred to an Immobilon membrane, and detected by immunoblotting with the same NF␬B–p65 (␣p65) antibody to detect the levels of p65 in the precipitate and with an anti-acetylated-lysine-specific antibody to detect the levels of acetylated p65 (␣Ac.p65) in the precipitate. The densitometric analysis shows the ratio between the amounts of acetylated p65 and total p65 immunoprecipitated from three independent experiments (means ⫾ SD). WB, Western blot.


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FIG. 7. A238L inhibits NF-␬B–p65 and p300 transactivation. RawpcDNA (gray bars) and Raw-A238L (black bars) cells were cotransfected with GAL4-luc (150 ng DNA/106 cells) and GAL4-p65 (A) or GAL4-p300 (B), at a final concentration of 50 ng DNA/106 cells, and cultured with 1 ␮g/ml of LPS plus 200 U/ml of IFN-␥. At the indicated poststimulation times, whole-cell extracts were prepared and luciferase activity was assayed. Extracts were normalized to Renilla luciferase activity as described in Materials and Methods. RLU per ␮g of protein from triplicate transfections (means ⫾ SD) are shown.

trol the nuclear translocation of NF-␬B but regulates the transactivation of this transcription factor in Jurkat cells (18). Due to the fact that A238L interferes with the interaction of p65 and p300 and inhibits p65 acetylation when overexpressed in Raw 264.7 cells, it is plausible that the viral protein might inhibit p65 transactivation as a part of the mechanism used in the down-regulation of the iNOS promoter in mouse macrophages. To address this question, we used a plasmid encoding the GAL4-p65 fusion protein, where the DNA-binding domain of GAL4 has been joined to the transactivation domain of RelA/p65. Raw-pcDNA cells or Raw-A238L cells were cotransfected with GAL4-p65 and a GAL4-Luc reporter, allowing us to determine whether the viral protein down-regulates iNOS gene expression by specifically targeting the transactivation domain of the RelA/p65 subunit of NF-␬B. Figure 7A shows that, in the presence of the A238L protein, the ability of LPS–IFN-␥ to activate GAL4-p65 was strongly inhibited in stimulated Raw 264.7 cells. The p300/CBP acetyltransferases regulate different functions of NF-␬B, including transcriptional activation, by targeting distinct lysine residues of RelA/p65 (14, 17, 21, 33). Previously, we have shown that the ability of p300 to coactivate NF-␬B is strongly down-regulated by expression of A238L and that overexpression of p65 could recover the inhibition of TNF-␣ promoter (18). However, the need to recruit p300 to the promoter through NF-␬B, which itself is a regulated event

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(42), made it difficult to conclude that the effect of A238L was directly on p300 itself. To enable the analysis of this possibility, we have used the plasmid GAL4-p300. This construction contains the complete p300 sequence fused to the GAL4 DNA binding domain (36). By using this approach, we also overcome any side effects from endogenous p300 because the iNOS promoter is targeted through GAL4. GAL4-p300 was then cotransfected with the GAL4-luc reporter plasmid into RawpcDNA and Raw-A238L cells. As expected, GAL4-p300 stimulated luciferase activity after LPS–IFN-␥ in Raw-pcDNA cells (Fig. 7B). Interestingly, the presence of A238L resulted in a strong down-regulation of p300 transactivation, while GAL4 alone was unaffected (data not shown). These results confirmed that the transcriptional activity of p300 is specifically inhibited by A238L. DISCUSSION This is the first report describing the modulation of iNOS promoter activation and NO production in macrophages following ASFV infection. Macrophages, the natural target of ASFV infection, represent an essential part of innate immunity, and the infection of macrophages results in the release of multiple proinflammatory mediators, such as NO. NO, a free radical species that is predominantly produced by activated macrophages, exhibits an inhibitory effect on virus replication as well as cytotoxicity to the infected cells (29). Thus, the virus-induced production of NO in macrophages can be considered one of the important factors in innate immunity, especially in controlling the early stage of viral replication at the peripheral sites of virus infection. Despite the relevance of NO in the control of viral infections and the role that this product could play during ASFV infection in vivo or in vitro, no information existed on the potential control of NO by ASFV. We show here that iNOS promoter activity is induced after ASFV infection and that NF-␬B is involved in this activation. On the other hand, we have also found that the viral protein A238L down-regulates iNOS levels and NO production both during infection or when ectopically expressed in transfected murine macrophages. The sustained high output of NO accounts for its antimicrobial effects on a variety of pathogens, including viruses (10, 23, 30). Thus, the regulation of the iNOS promoter activity by A238L could be an important checkpoint in the virus cycle. However, as described here and as reported before (31), A238L is nonessential for virus growth in vitro. Also, it has been shown that deletion of the ASFV A238L gene from the highly virulent Malawi Lil-20/1 strain does not affect the virulence phenotype in domestic pigs (31). These findings may be surprising, since the proposed role for this gene is to interfere not only with iNOS transcription, as shown here, but also with the expression of other immunomodulatory molecules, such as TNF-␣ or COX-2 (19, 20). Immunomodulation by the viral protein could be subtler and play a significant role not in acute ASFV infections, which cause a fulminating death of the animal, but rather in subacute and chronic infections of domestic pigs. The complex regulation of iNOS gene transcription involves the interaction of both basally expressed and inducible transcription factors with the coactivators CBP/p300, which could

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be targeted by A238L to modulate NO production during ASFV infection of macrophages. This notion is supported by our finding that the A238L-mediated inhibition of iNOS promoter activity and iNOS levels was reverted by overexpression of p300 in the murine macrophage cell line Raw 264.7. Both p300 and CBP contain a histone acetyltransferase (HAT) enzymatic activity that regulates gene expression through acetylation of the N-terminal tails of histones (32). In addition to modifying histones, p300/CBP also directly acetylates several transcription factors, including p65 and p50 (2, 3, 7, 8). Acetylation of these factors is a critical step in transcriptional regulation, leading to changes in their biological activity, such as alterations in DNA binding affinity, transcriptional activity, and interaction with other proteins (2, 3, 7, 16). It is noteworthy that the p300⌬HAT deletion mutant construct is unable to restore the iNOS protein levels inhibited by the viral protein, in agreement with the general concept that the core HAT domain is required for p300-mediated iNOS promoter activity and suggesting that iNOS inhibition by A238L might be related to the acetylase activity of p300. In keeping with this, we show here that expression of the protein A238L inhibits the acetylation of p65. The p300 binding and interaction with DNA-bound NF-␬B subunits p65 and p50 at the iNOS promoter has been shown to be enhanced by LPS–IFN-␥ stimulation (11, 14). Our results corroborate this and, more interestingly, also indicate that p300 and p65 are strongly displaced from the complex in cells expressing the A238L protein. In agreement with this, increasing doses of p65 as well as of p300, as mentioned above, not only induced iNOS promoter transcription but, more importantly, reverted the inhibition of iNOS promoter induced by A238L, supporting the involvement of these proteins in the control of iNOS by the viral protein. The p65 subunit of NF-␬B interacts with p300 to recruit this coactivator to the transcriptional activation complex (42). As described here, the presence of A238L impairs this interaction, suggesting that the viral protein may suppress the transcriptional activation of the iNOS/p65 signal transduction pathway by competing with p65 for binding to p300. This interference could be due to the previously described interaction of A238L with p65, which might block the binding sites in the NF-␬B subunit, but the possible formation of a complex of the viral protein with p300 should also be taken into account. Furthermore, it cannot be discarded that the effects of A238L could be due to interactions in an upstream step of this pathway. The mechanism by which p300/CBP enhances NF-␬B transcriptional activity is likely multifactorial. In connection with this, we present here a clear correlation between p65 acetylation, p65 binding to DNA, and p300 recruitment to the distal NF-␬B sequence in the iNOS promoter in macrophages treated with LPS–IFN-␥. Furthermore, expression of the viral A238L protein induces a concordant decrease in p65/d-NF-␬B binding, p300-mediated acetylation, and p300-p65 interaction. The transcriptional activities of p300 and CBP are themselves directly regulated, providing an additional complexity to the control of promoter activity. A number of signaling pathways, including p300 and/or CBP phosphorylation, have been demonstrated during cell differentiation, cell cycle progression, and cell signaling via the protein kinase C and cyclin E-CdK2 pathways (1, 40). Our previous results showed that A238L,

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a nuclear viral protein that colocalizes with p300, binds to the CRE/␬3 complex in the TNF-␣ promoter and displaces the coactivators CBP/p300 to inhibit the transactivation of the associated factors NFAT, NF-␬B, and c-Jun (18). Herein, we demonstrate that the viral protein also inhibits the transactivation of p300. Further experiments are needed to explore whether the mechanism used by A238L to inhibit the transactivation of p300 involves regulation of this coactivator phosphorylation. In conclusion, the data presented here establish a new viral mechanism of p300 transcription coactivator activity downregulation to modulate iNOS activation. However, future work is required to address the precise strategy used by this viral protein to achieve this effect on p300 transactivation. A detailed understanding of the mechanism by which iNOS gene transcription is controlled by A238L in macrophages offers potential novel insights regarding the role of iNOS in several inflammatory pathologies. ACKNOWLEDGMENTS This work was supported by grants from the Ministerio de Educacio ń y Ciencia (BFU2004-00298/BMC), the Ministerio de Ciencia y Tecnologıá (AGL2002-10220-E), the European Commission (QLRT2000-02216), and the Wellcome Trust (no. 075813/C/04/Z) and by an institutional grant from the Fundacio ń Ramo ń Areces. We thank Neil Perkins for the generous gift of the GAL4-p300 plasmid, Carmen del Aguila for the generation of Raw-A238L cells, Virginia Vila-del Sol for the generous gift of the p(iNOS)m-luc plasmid, and Maria L. Nogal for excellent and continuous technical assistance. The helpful advice of Angel L. Carrascosa is also very much appreciated. REFERENCES 1. Ait-Si-Ali, S., S. Ramirez, F. X. Barre, F. Dkhissi, L. Magnaghi-Jaulin, J. A. Girault, P. Robin, M. Knibiehler, L. L. Pritchard, B. Ducommun, D. Trouche, and A. Harel-Bellan. 1998. Histone acetyltransferase activity of CBP is controlled by cycle-dependent kinases and oncoprotein E1A. Nature 396:184–186. 2. Bannister, A. J., and E. A. Miska. 2000. Regulation of gene expression by transcription factor acetylation. Cell. Mol. Life Sci. 57:1184–1192. 3. Berger, S. L. 1999. Gene activation by histone and factor acetyltransferases. Curr. Opin. Cell Biol. 11:336–341. 4. Boyes, J., P. Byfield, Y. Nakatani, and V. Ogryzko. 1998. Regulation of activity of the transcription factor GATA-1 by acetylation. Nature 396:594– 598. 5. Bustos, M. J., M. L. Nogal, Y. Revilla, and A. L. Carrascosa. 2002. Plaque assay for African swine fever virus on swine macrophages. Arch. Virol. 147:1453–1459. 6. Carrascosa, A. L., J. F. Santaren, and E. Vinuela. 1982. Production and titration of African swine fever virus in porcine alveolar macrophages. J. Virol. Methods 3:303–310. 7. Chen, H., M. Tini, and R. M. Evans. 2001. HATs on and beyond chromatin. Curr. Opin. Cell Biol. 13:218–224. 8. Chen, L. F., Y. Mu, and W. C. Greene. 2002. Acetylation of RelA at discrete sites regulates distinct nuclear functions of NF-kappaB. EMBO J. 21:6539– 6548. 9. Chu, S. C., J. Marks-Konczalik, H. P. Wu, T. C. Banks, and J. Moss. 1998. Analysis of the cytokine-stimulated human inducible nitric oxide synthase (iNOS) gene: characterization of differences between human and mouse iNOS promoters. Biochem. Biophys. Res. Commun. 248:871–878. 10. Croen, K. D. 1993. Evidence for antiviral effect of nitric oxide. Inhibition of herpes simplex virus type 1 replication. J. Clin. Investig. 91:2446–2452. 11. Deng, W. G., and K. K. Wu. 2003. Regulation of inducible nitric oxide synthase expression by p300 and p50 acetylation. J. Immunol. 171:6581– 6588. 12. Deng, W. G., Y. Zhu, and K. K. Wu. 2003. Up-regulation of p300 binding and p50 acetylation in tumor necrosis factor-alpha-induced cyclooxygenase-2 promoter activation. J. Biol. Chem. 278:4770–4777. 13. Dixon, L. K., C. C. Abrams, G. Bowick, L. C. Goatley, P. C. Kay-Jackson, D. Chapman, E. Liverani, R. Nix, R. Silk, and F. Zhang. 2004. African swine fever virus proteins involved in evading host defence systems. Vet. Immunol. Immunopathol. 100:117–134.

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