title: Phosphorylation and subcellular localization of transmissible gastroenteritis virus nucleocapsid protein in infected cells.
abstract: 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.
authors: Unidad de Proteómica, Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Sinesio Delgado 4, 28029 Madrid, Spain.
authors: E
authors: E
authors: Calvo
authors: Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB, CSIC), Campus Univ. Autonoma, 3 Darwin St, Cantoblanco, 28049 Madrid, Spain.
authors: D
authors: D
authors: Escors
authors: Unidad de Proteómica, Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Sinesio Delgado 4, 28029 Madrid, Spain.
authors: J A
authors: JA
authors: López
authors: Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB, CSIC), Campus Univ. Autonoma, 3 Darwin St, Cantoblanco, 28049 Madrid, Spain.
authors: J M
authors: JM
authors: González
authors: Unidad de Citometría, Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Sinesio Delgado 4, 28029 Madrid, Spain.
authors: A
authors: A
authors: Álvarez
authors: Unidad de Citometría, Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Sinesio Delgado 4, 28029 Madrid, Spain.
authors: E
authors: E
authors: Arza
authors: Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB, CSIC), Campus Univ. Autonoma, 3 Darwin St, Cantoblanco, 28049 Madrid, Spain.
authors: L
authors: L
authors: Enjuanes
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002849:Chromatography, Gas; D019295:Computational Biology; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D013058:Mass Spectrometry; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D019590:Nucleocapsid Proteins / Q000235:genetics / Q000378:metabolism*; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D016415:Sequence Alignment; D012694:Serine; D013552:Swine; D013997:Time Factors; D005760:Transmissible gastroenteritis virus / Q000378:metabolism*
chemical_list: D019590:Nucleocapsid Proteins; D010748:Phosphopeptides; D012694:Serine