Phosphorylation and subcellular localization of transmissible gastroenteritis virus nucleocapsid protein in infected cells

J Gen Virol. 2005 Aug;86(Pt 8):2255-2267. doi: 10.1099/vir.0.80975-0.


The nucleocapsid (N) protein is the only phosphorylated structural protein of the coronavirus Transmissible gastroenteritis virus (TGEV). The phosphorylation state and intracellular distribution of TGEV N protein in infected cells were characterized by a combination of techniques including: (i) subcellular fractionation and analysis of tryptic peptides by two-dimensional nano-liquid chromatography, coupled to ion-trap mass spectrometry; (ii) tandem mass-spectrometry analysis of N protein resolved by SDS-PAGE; (iii) Western blotting using two specific antisera for phosphoserine-containing motifs; and (iv) confocal microscopy. A total of four N protein-derived phosphopeptides were detected in mitochondria-Golgi-endoplasmic reticulum-Golgi intermediate compartment (ERGIC)-enriched fractions, including N-protein phosphoserines 9, 156, 254 and 256. Confocal microscopy showed that the N protein found in mitochondria-Golgi-ERGIC fractions localized within the Golgi-ERGIC compartments and not with mitochondria. Phosphorylated N protein was also present in purified virions, containing at least phosphoserines 156 and 256. Coronavirus N proteins showed a conserved pattern of secondary structural elements, including six beta-strands and four alpha-helices. Whilst serine 9 was present in a non-conserved domain, serines 156, 254 and 256 were localized close to highly conserved secondary structural elements within the central domain of coronavirus N proteins. Serine 156 was highly conserved, whereas no clear homologous sites were found for serines 254 and 256 for other coronavirus N proteins.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Blotting, Western
  • Chromatography, Gas
  • Computational Biology
  • Electrophoresis, Polyacrylamide Gel
  • Endoplasmic Reticulum / metabolism
  • Golgi Apparatus / metabolism
  • Mass Spectrometry
  • Microscopy, Confocal
  • Mitochondria / metabolism
  • Molecular Sequence Data
  • Nucleocapsid Proteins / genetics
  • Nucleocapsid Proteins / metabolism*
  • Phosphopeptides / chemistry
  • Phosphopeptides / metabolism
  • Phosphorylation
  • Sequence Alignment
  • Serine
  • Swine
  • Time Factors
  • Transmissible gastroenteritis virus / metabolism*


  • Nucleocapsid Proteins
  • Phosphopeptides
  • Serine