African Swine Fever Virus Protein A238L Interacts with the Cellular Phosphatase Calcineurin via a Binding Domain Similar to That of NFAT

Abstract

The African swine fever virus protein A238L inhibits activation of NFAT transcription factor by binding calcineurin and inhibiting its phosphatase activity. NFAT controls the expression of many immunomodulatory proteins. Here we describe a 14-amino-acid region of A238L that is needed and sufficient for binding to calcineurin. By introducing mutations within this region, we have identified a motif (PxIxITxC/S) required for A238L binding to calcineurin; a similar motif is found in NFAT proteins. Peptides corresponding to this domain of A238L bind calcineurin but do not inhibit its phosphatase activity. Binding of A238L to calcineurin stabilizes the A238L protein in cells. Although A238L-mediated suppression of NF-B-dependent gene expression occurs by a different mechanism, the A238L-calcineurin interaction may be required to stabilize A238L.Many large DNA viruses encode proteins that help the virus to evade the host immune response (1, 9, 24). African swine fever virus (ASFV), the prototypic member of the African swine fever-like virus genus (10), encodes a potent immunosuppressive protein, A238L. A238L inhibits activation of the NF-B transcription factor (25,28,32) and the activity of the calcium/calmodulin-regulated phosphatase calcineurin (CaN) (23). The virus, therefore, has the potential to inhibit transcriptional activation of immunomodulatory genes dependent on these pathways in infected macrophages.NF-B is retained in the cytoplasm in a complex with the inhibitor protein, IB, in resting cells. Following activation, IB is phosphorylated at two key Ser residues (Ser32 and Ser36 in IB-␣) and is then ubiquitinated and targeted for degradation by the 26S proteasome (for a review, see reference 16). This process exposes nuclear localization signals within nuclear factor B (NF-B) which subsequently translocates to the nucleus and binds to promoters with the appropriate DNA recognition sequence. A238L coprecipitates with the p65 subunit of NF-B (32) and therefore might function as an IB mimic which does not respond to signal-induced degradation.The second function of A238L, inhibition of CaN phosphatase activity, is mediated by direct binding to the catalytic subunit of CaN [CaN(A)] (23). CaN is activated following calcium release and binding of calmodulin, which results in displacement of the autoinhibitory (AI) domain from the enzyme active site. CaN regulates a number of different pathways, including activation of the NFAT family of transcription factors, which are present in a phosphorylated form in the cytoplasm in resting cells. Activation of CaN results in dephosphorylation and nuclear translocation of NFAT factors which, in cooperation with other transcription factors, play an essential role in transcriptional activation of cytokine and other immunomodulatory genes (8,29,30). The immunosuppressive drugs cyclosporin A (CsA) and FK506 bind to CaN in a complex with the immunophilin proteins cyclophilin A (CypA) and FKBP12, respectively (34); this drug-immunophilin complex

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

“…In this regard, it is worth mentioning that, although interaction of calcineurin with A238L has been clearly demonstrated (11), no direct inhibition of the phosphatase activity has been reported. Rather, the reported results were obtained using cell extracts and no inhibition of calcineurin by A238L binding peptide was observed (60). Thus, it is likely that A238L, by binding to calcineurin, may inhibit its activity in some settings but not in others.…”

“…These apparent differences can be ascribed to the different cellular systems used, mainly to the fact that Matsuda et al overexpressed both proteins NFAT and A238L in BHK cells, whereas we have employed both A238L stably expressing T cells or ASFV-infected Vero cells to investigate endogenous NFAT activation, thus representing, in our opinion, a more physiological system. In addition, a motif similar to the calcineurin docking motif of NFAT protein has been found in A238L (60), suggesting that the two proteins bind calcineurin at the same site. However, our results show that modulation of NFAT activity by A238L does not involve either the translocation to the nucleus or DNA binding of this factor to its DNA recognition sequences.…”

Calcineurin A–Binding Protein, a Novel Modulator of the Calcineurin-Nuclear Factor of Activated T-Cell Signaling Pathway, Is Overexpressed in Wilms' Tumors and Promotes Cell Migration

“…Alternatively, it is possible that CnABP is a substrate for calcineurin with its own NFAT-independent cellular activity, as is the case for the K + channel TRESK (54). In this respect, it is remarkable that the best-conserved domain of CnABP contains a perfect PxIxIT consensus sequence (where x represents any amino acid), which is a calcineurin docking site found in NFATs and other calcineurin substrates (54,55), as well as in calcineurin inhibitor proteins such as Cain/Cabin1 and A238L (56)(57)(58). This sequence was shown to be sufficient for binding to calcineurin (59).…”

The CD2v protein of African swine fever virus interacts with the actin-binding adaptor protein SH3P7

“…The genome is approximately 170 kbp long and is predicted to encode at least 150 proteins, including proteins involved in virus-host interactions important for virus survival and transmission (Dixon et al, 1994;Yanez et al, 1995;Yozawa et al, 1994). For example, proteins related to IAP and Bcl2 inhibit apoptosis (Brun et al, 1998;Nogal et al, 2001;Revilla et al, 1997) and the A238L protein inhibits activation of NFkBdependent gene transcription (Powell et al, 1996;Revilla et al, 1998) and also inhibits calcineurin phosphatase activity and hence NFAT transcription factor activation (Miskin et al, 1998(Miskin et al, , 2000.…”

“…African swine fever is an important haemorrhagic disease of domestic pigs, caused by a large double-stranded DNA virus, African swine fever virus (ASFV), the only member of the family Asfarviridae (Dixon et al, 2000). The genome is approximately 170 kbp long and is predicted to encode at least 150 proteins, including proteins involved in virus-host interactions important for virus survival and transmission (Dixon et al, 1994;Yanez et al, 1995;Yozawa et al, 1994).…”