Nucleocapsid protein of SARS-CoV activates the expression of cyclooxygenase-2 by binding directly to regulatory elements for nuclear factor-kappa B and CCAAT/enhancer binding protein

Int J Biochem Cell Biol. 2006;38(8):1417-28. doi: 10.1016/j.biocel.2006.02.003. Epub 2006 Mar 3.

Abstract

SARS-associated coronavirus (SARS-CoV) causes inflammation and damage to the lungs resulting in severe acute respiratory syndrome. To evaluate the molecular mechanisms behind this event, we investigated the roles of SARS-CoV proteins in regulation of the proinflammatory factor, cyclooxygenase-2 (COX-2). Individual viral proteins were tested for their abilities to regulate COX-2 gene expression. Results showed that the COX-2 promoter was activated by the nucleocapsid (N) protein in a concentration-dependent manner. Western blot analysis indicated that N protein was sufficient to stimulate the production of COX-2 protein in mammalian cells. COX-2 promoter mutations suggested that activation of COX-2 transcription depended on two regulatory elements, a nuclear factor-kappa B (NF-kappaB) binding site, and a CCAAT/enhancer binding protein (C/EBP) binding site. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) demonstrated that SARS-CoV N protein bound directly to these regulatory sequences. Protein mutation analysis revealed that a Lys-rich motif of N protein acted as a nuclear localization signal and was essential for the activation of COX-2. In addition, a Leu-rich motif was found to be required for the N protein function. A sequence of 68 residuals was identified as a potential DNA-binding domain essential for activating COX-2 expression. We propose that SARS-CoV N protein causes inflammation of the lungs by activating COX-2 gene expression by binding directly to the promoter resulting in inflammation through multiple COX-2 signaling cascades.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Binding Sites / genetics
  • CCAAT-Enhancer-Binding Protein-beta / metabolism*
  • Cell Line
  • Chromatin Immunoprecipitation
  • Cyclooxygenase 2 / genetics
  • Cyclooxygenase 2 / metabolism*
  • Electrophoretic Mobility Shift Assay
  • Gene Expression Regulation, Enzymologic
  • Humans
  • Mutagenesis, Site-Directed / methods
  • Mutation
  • NF-kappa B / metabolism*
  • Nucleocapsid Proteins / genetics
  • Nucleocapsid Proteins / metabolism
  • Nucleocapsid Proteins / physiology*
  • Plasmids / genetics
  • Promoter Regions, Genetic / genetics
  • Protein Binding
  • SARS Virus / genetics
  • SARS Virus / metabolism*
  • Transcriptional Activation

Substances

  • CCAAT-Enhancer-Binding Protein-beta
  • NF-kappa B
  • Nucleocapsid Proteins
  • Cyclooxygenase 2