Mechanism of inactivation of NF-kappa B by a viral homologue of I kappa b alpha. Signal-induced release of i kappa b alpha results in binding of the viral homologue to NF-kappa B.
Activation of the nuclear factor kappa B plays a key role in viral pathogenesis, resulting in inflammation and modulation of the immune response. We have previously shown that A238L, an open reading frame from African swine fever virus (ASFV), encoding a protein with 40% homology to porcine I kappa B alpha exerts a potent anti-inflammatory effect in host macrophages, where it down-regulates NF-kappa B-dependent gene transcription and proinflammatory cytokine production. This paper reveals the mechanism of suppression of NF-kappa B activity by A238Lp. A238Lp is synthesized throughout infection as two molecular mass forms of 28 and 32 kDa, and vaccinia-mediated expression of A238L demonstrated that both proteins are produced from a single gene. Significantly, the higher 32-kDa form of A238L, but not the 28-kDa form, interacts directly with RelA, the 65-kDa subunit of NF-kappa B, indicating that the binding is dependent on a post-translational modification. Immunoprecipitation analysis shows the NF-kappa B p65-A238L p32 heterodimer is a separate complex from NF-kappa B-I kappa B alpha, and it resides in the cytoplasm. Moreover, we show that ASFV infection stimulates the NF kappa B signal transduction pathway, which results in the rapid degradation of endogenous I kappa B alpha, although both forms of A238Lp are resistant to stimulus-induced degradation. Using the proteasome inhibitor MG132, we show that when degradation of I kappa B alpha is inhibited, A238Lp binding to NF-kappa B p65 is reduced. The results suggest that the virus exploits its activation of the NF-kappa B pathway to enable its own I kappa B homologue to bind to NF-kappa B p65. Last, we show that synthesis of I kappa B alpha is increased during ASFV infection, indicating RelA-independent transcription of the I kappa B alpha gene.
Department of Immunology and Pathology, BBSRC Institute for Animal Health, Ash Road, Pirbright, Surrey, GU24 ONF, United Kingdom.
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Tait
E B
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Reid
D R
DR
Greaves
T E
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Wileman
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Powell
D000358:African Swine Fever Virus / Q000235:genetics / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D020672:I-kappa B Proteins; D008969:Molecular Sequence Data; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors*; D016366:Open Reading Frames; D011233:Precipitin Tests; D011499:Protein Processing, Post-Translational; D015398:Signal Transduction; D013552:Swine; D014158:Transcription, Genetic; D014709:Vero Cells
D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D016328:NF-kappa B; D000072000:NF-KappaB Inhibitor alpha