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1 Unidad de Proteómica, Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Sinesio Delgado 4, 28029 Madrid, Spain
2 Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB, CSIC), Campus Univ. Autonoma, 3 Darwin St, Cantoblanco, 28049 Madrid, Spain
3 Unidad de Citometría, Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Sinesio Delgado 4, 28029 Madrid, Spain
Correspondence
L. Enjuanes
L.Enjuanes{at}cnb.uam.es
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 
-strands and four 
-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.
These authors contributed equally to this work.
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