The N Terminus of Orf Virus-Encoded Protein 002 Inhibits Acetylation of NF-κB p65 by Preventing Ser(276) Phosphorylation

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 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-κB transactivation was lost. NF-κB p65 Lys(310) acetylation and Ser(276) phosphorylation were detected in co-transfection experiments with NF-κB 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-κB p65. Further investigation indicated that ORFV002 and its C-terminal deletion mutants interfered with NF-κB p65 (Ser(276)) phosphorylation induced by mitogen- and stress-activated protein kinase-1 (MSK1) and the interaction between NF-κB p65 and MSK1. Since phosphorylated NF-κB p65 recruits transcriptional co-activators such as p300 and CBP, we concluded that the N-terminus of ORFV002 inhibits acetylation of NF-κB p65 by blocking phosphorylation of NF-κB p65 at Ser(276).