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

Abstract

African swine fever virus (ASFV) is able to inhibit TNF-α-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-α, we have used Jurkat cells stably transfected with the viral gene to identify the TNF-α 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 κ3 sites on the TNF-α promoter as the responsible of the gene activation, and demonstrate that A238L inhibits TNF-α expression through these DNA binding sites. This inhibition was partially reverted by overexpression of the transcriptional factors NF-AT, NF-κB, and c-Jun. Furthermore, we present evidence that A238L inhibits the activation of TNF-α by modulating NF-κB, 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-α promoter activity. In addition, we show that A238L is a nuclear protein that binds to the cyclic AMP-responsive element/κ3 complex, thus displacing the CREB binding protein/p300 coactivators. Taken together, these results establish a novel mechanism in the control of TNF-α gene expression by a viral protein that could represent an efficient strategy used by ASFV to evade the innate immune response.

Regulation of Inducible Nitric Oxide Synthase Expression by Viral A238L-Mediated Inhibition of p65/RelA Acetylation and p300 Transactivation

“…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.…”

“…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, 5Ј-AGCTTCA TCAATGGAAAGG-3Ј; reverse, 5Ј-AGAAGCAGGGATGATGTTCTG-3Ј), porcine iNOS (forward, 5Ј-TGCGTTATGCCACCAACAATG-3Ј; reverse, 5Ј-A CTCTCCAGGATGTTGTAG-3Ј), murine iNOS (forward, 5Ј-GAGAGATCCG ATTGGC-3Ј; reverse, 5Ј-GCAGATTCTGCTGGGATTTCA-3Ј), murine ␤-actin (forward, 5Ј-CTTTTGATGTCACGCACGATTTC-3Ј; reverse, 5Ј-GTGGG CCGCTCTAGGCC-3Ј), viral A238L (forward, 5Ј-CGCGCGTCTAGATTACT TTCCATACTTGTT-3Ј; reverse, 5Ј-GCGCGCAAGCTTATGGAACACATGT TTCCA-3Ј), and viral p72 (forward, 5Ј-CGGGATCCATGGCATCAGGAGGA G-3Ј; reverse, 5Ј-CGCGAGATGACCATGGGCC).…”

“…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 IB family and behaves as a bona fide IB-␣ 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).…”

Manipulation of the nuclear factor-κB pathway and the innate immune response by viruses

“…Thus, while ASFV infection initially induces NF-kB, the degraded cellular IkBa is replaced by the viral IkB A238L, which stably inhibits the NF-kB complex (Tait et al, 2000). Of note, A238L also inhibits calcineurin, a protein phosphatase that is required for the induction of the NFAT-dependent immune response genes (Miskin et al, 1998), and the coactivator complex CBP/p300 (Granja et al, 2006). The viral IkB homolog A238L, which is expressed early in ASFV infection, appears to block NF-kB-and NFAT-dependent immune and inflammatory responses.…”

The Study of Rs1800629 (G-308а) Polymorphism of the TNF Gene in Patients With Myocardial Infarction With Elevated St Segment

“…Одним из наиболее изученных полиморфных вариантов в этом гене является rs1800629 (G-308A), располагающийся в промоторе гена. В экспериментальных исследованиях in vitro была показана функциональная значимость этого однонуклеотидного полиморфизма: данный аллель-ный вариант влияет на уровень экспрессии гена и концентрации ФНО-a [6].…”