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The N terminus of orf virus-encoded protein 002 inhibits acetylation of NF-?B p65 by preventing Ser(276) phosphorylation.

ABSTRACT: Orf virus-encoded protein 002 (ORFV002) inhibits NF-?B signaling pathway by decreasing the acetylation of NF-?B-p65 through interference of NF-?B p65's association with NF-?B p300. However, the precise mechanism of how ORFV002 interferes with the NF-?B p65/p300 association is still unknown. Due to similarities of the amino acid sequences of ORFV002 and the adenovirus type 12 (Ad12) E1A protein (E1A-12), we hypothesized that the N-terminal 52 amino acids of ORFV002 might play an important role in this inhibition and constructed several in-frame fusions of ORFV002 to an enhanced green fluorescent protein (EGFP) reporter, including C-terminal and N-terminal deletion mutants of ORFV002. When the N-terminus of ORFV002 was absent, the localization of ORFV002 shifted mainly from the nucleus to the cytoplasm, and it's inhibition of NF-?B transactivation was lost. NF-?B p65 Lys(310) acetylation and Ser(276) phosphorylation were detected in co-transfection experiments with NF-?B p65 and ORFV002 or its mutants with, or without, the N-terminal region. The results showed that the N-terminus of ORFV002 plays a crucial role in inhibiting both the acetylation and phosphorylation of NF-?B p65. Further investigation indicated that ORFV002 and its C-terminal deletion mutants interfered with NF-?B p65 (Ser(276)) phosphorylation induced by mitogen- and stress-activated protein kinase-1 (MSK1) and the interaction between NF-?B p65 and MSK1. Since phosphorylated NF-?B p65 recruits transcriptional co-activators such as p300 and CBP, we concluded that the N-terminus of ORFV002 inhibits acetylation of NF-?B p65 by blocking phosphorylation of NF-?B p65 at Ser(276).

SUBMITTER: Ning Z

PROVIDER: S-EPMC3594181 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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The N terminus of orf virus-encoded protein 002 inhibits acetylation of NF-κB p65 by preventing Ser(276) phosphorylation.

Ning Zhangyong Z Zheng Zewei Z Hao Wenbo W Duan Chaohui C Li Wei W Wang Yuanyuan Y Li Ming M Luo Shuhong S

PloS one 20130311 3

Orf virus-encoded protein 002 (ORFV002) inhibits NF-κB signaling pathway by decreasing the acetylation of NF-κB-p65 through interference of NF-κB p65's association with NF-κB p300. However, the precise mechanism of how ORFV002 interferes with the NF-κB p65/p300 association is still unknown. Due to similarities of the amino acid sequences of ORFV002 and the adenovirus type 12 (Ad12) E1A protein (E1A-12), we hypothesized that the N-terminal 52 amino acids of ORFV002 might play an important role in ...[more]

PMID: 23536830

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Project description:Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease caused by neurotropic polyomavirus, JC virus (JCV), a virus that causes lytic infection of CNS glial cells. After primary infection, JCV is controlled by the immune system but virus persists asymptomatically. Rarely, when immune function is impaired, it can reemerge to cause PML. The mechanisms of JCV persistence and reactivation are not well understood but our earlier work implicated epigenetic control by protein acetylation since histone deacetylase inhibitors such as trichostatin A (TSA) strongly stimulate JCV transcription. Since both TNF-α and TSA activate JCV transcription via the same unique NF-κB site in the JCV control region, we investigated a role for acetylation of NF-κB in JCV regulation. A site-directed mutagenesis strategy was employed targeting the known lysine acetylation sites of NF-κB p65: K218, K221, and K310. We individually mutated each lysine to arginine, which cannot be acetylated and retains a positive charge like lysine. K218R and K221R impaired transactivation of JCV early promoter transcription either alone or combined with TSA treatment or coexpression of acetyltransferase transcriptional coactivator p300 but K310R was largely without effect. Mutation of lysine to glutamine gives mutants with a negative charge like acetyllysine. However, K218Q and K221Q showed impaired activity and only K310Q showed enhanced transactivation. NF-κB acetylation can regulate several aspects of the process of activation including complex formation with IκB, translocation to the nucleus, and DNA binding and transcriptional activation. Cell fractionation studies revealed that the mutants had no defect in translocation to the nucleus whereas gel shift studies revealed reduced binding to the JCV NF-κB site. Thus, acetylation regulates NF-κB p65 activity toward JCV at the level of p65 binding to the JCV control region and activation of JCV transcription.

Dataset Information

The N terminus of orf virus-encoded protein 002 inhibits acetylation of NF-?B p65 by preventing Ser(276) phosphorylation.

Publications

The N terminus of orf virus-encoded protein 002 inhibits acetylation of NF-κB p65 by preventing Ser(276) phosphorylation.

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