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

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 SIDS-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.