%0 Journal Article %A Calvo, E. %A Escors, D. %A López, J. A. %A González, J. M. %A álvarez, A. %A Arza, E. %A Enjuanes, L. %T Phosphorylation and subcellular localization of transmissible gastroenteritis virus nucleocapsid protein in infected cells %D 2005 %J Journal of General Virology, %V 86 %N 8 %P 2255-2267 %@ 1465-2099 %R https://doi.org/10.1099/vir.0.80975-0 %I Microbiology Society, %X 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 β-strands and four α-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. %U https://www.microbiologyresearch.org/content/journal/jgv/10.1099/vir.0.80975-0