The Viral Protein A238L Inhibits Cyclooxygenase-2 Expression through a Nuclear Factor of Activated T Cell-dependent Transactivation Pathway

Abstract

Cyclooxygenase-2 is transiently induced upon cell activation or viral infections, resulting in inflammation and modulation of the immune response. Here we report that A238L, an African swine fever virus protein, efficiently inhibits cyclooxygenase-2 gene expression in Jurkat T cells and in virus-infected Vero cells. Transfection of Jurkat cells stably expressing A238L with cyclooxygenase-2 promoter-luciferase constructs containing 5 -terminal deletions or mutations in distal or proximal nuclear factor of activated T cell (NFAT) response elements revealed that these sequences are involved in the inhibition induced by A238L. Overexpression of a constitutively active version of the calciumdependent phosphatase calcineurin or NFAT reversed the inhibition mediated by A238L on cyclooxygenase-2 promoter activation, whereas overexpression of p65 NF B had no effect. A238L does not modify the nuclear localization of NFAT after phorbol 12-myristate 13-acetate/calcium ionophore stimulation. Moreover, we show that the mechanism by which the viral protein downregulates cyclooxygenase-2 activity does not involve inhibition of the binding between NFAT and its specific DNA sequences into the cyclooxygenase-2 promoter. Strikingly, A238L dramatically inhibited the transactivation mediated by a GAL4-NFAT fusion protein containing the N-terminal transactivation domain of NFAT1. Taken together, these data indicate that A238L down-regulates cyclooxygenase-2 transcription through the NFAT response elements, being NFAT-dependent transactivation implicated in this down-regulation.Viruses have been known for a long time to use a variety of strategies not only to alter the host metabolism via their signaling proteins but also to hijack cellular signaling pathways and transcription factors to control them to their own advantage. Both the nuclear factor-B (NF B) 1 and the nuclear factor of activated T cells (NFAT) pathways appear to be attractive targets for common viral pathogens, probably due to their ability to promote the expression of numerous proteins involved in adaptative and innate immunity (1, 2). Several viruses, including hepatitis C virus (3), immunodeficiency virus (4), herpes viruses (5), and African swine fever virus (ASFV) (6 -8) have been shown to modulate the activation of NFAT or NF B.NF B is a collective term referring to a class of dimeric transcription factors belonging to the rel family. In resting cells, NF B exists in the cytoplasm as an inactive complex bound to inhibitory proteins of the I B family (9, 10). In response to a variety of stimuli, I B proteins undergo phosphorylation of Ser 32 and Ser 36 (11, 12), followed by ubiquitination and degradation in the proteasome, thus unmasking the nuclear localization sequence of the transactivating heterodimers and allowing translocation of active NF B to the nucleus. Recently, there is accumulating evidence suggesting that another level of NF-〉 regulation independent on I B degradation exists. This second level of regulation relies in the activation of the transcription...

The Viral Protein A238L Inhibits TNF-α Expression through a CBP/p300 Transcriptional Coactivators Pathway

“…Because the viral protein A238L has been described as an inhibitor of NF-B (17), NF-AT (16,18), and calcineurin phosphatase (16), we have explored the possibility that this protein could inhibit TNF-␣ expression. To assess this, we have used the ASFV A238L deletion mutant, designated Ba71V⌬A238L, obtained from the Ba71V viral strain, as previously described (18). The absence of A238L protein in Ba71V⌬A238L-infected Vero cells was corroborated by Western blot analysis with cellular extracts from Ba71V-and Ba71V⌬A238L-infected cells using a specific Ab against A238L protein.…”

“…The recombinant Ba71V⌬A238L virus was obtained, as previously described (18). The recombinant ⌭70⌬A238L was obtained using the same transfection-infection protocol, but infecting COS-7 cell monolayers with the viral strain E70.…”

“…Generation of A238L stably expressing Jurkat cells was done, as described previously (18). A238L stably expressing Vero cells were generated using the same protocol.…”

“…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 M 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).…”

“…A238L also down-regulates the activation of the NF-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-B and NF-AT pathways, such as cyclooxygenase-2 (COX-2) (18).…”