Calvo E, Escors D, López J A, González J M, Álvarez A, Arza E, Enjuanes L
Unidad de Proteómica, Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Sinesio Delgado 4, 28029 Madrid, Spain.
Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB, CSIC), Campus Univ. Autonoma, 3 Darwin St, Cantoblanco, 28049 Madrid, Spain.
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.
核衣壳（N）蛋白是冠状病毒传染性胃肠炎病毒（TGEV）唯一发生磷酸化的结构蛋白。通过以下多种技术相结合的方法，对感染细胞中TGEV N蛋白的磷酸化状态和细胞内分布进行了表征：（i）亚细胞分级分离，并通过二维纳米液相色谱对胰蛋白酶肽段进行分析，再与离子阱质谱联用；（ii）对经SDS-PAGE分离的N蛋白进行串联质谱分析；（iii）使用两种针对含磷酸丝氨酸基序的特异性抗血清进行蛋白质免疫印迹分析；（iv）共聚焦显微镜检查。在富含线粒体-高尔基体-内质网-高尔基体中间区室（ERGIC）的组分中，总共检测到了四种源自N蛋白的磷酸肽，包括N蛋白的磷酸丝氨酸9、156、254和256。共聚焦显微镜检查显示，在富含线粒体-高尔基体-ERGIC的组分中发现的N蛋白定位于高尔基体-ERGIC区室，而非线粒体。磷酸化的N蛋白也存在于纯化的病毒粒子中，其中至少含有磷酸丝氨酸156和256。冠状病毒N蛋白呈现出保守的二级结构元件模式，包括六条β链和四条α螺旋。虽然丝氨酸9位于一个非保守结构域中，但丝氨酸156、254和256则定位于冠状病毒N蛋白中央结构域内高度保守的二级结构元件附近。丝氨酸156高度保守，而对于其他冠状病毒N蛋白，未发现丝氨酸254和256有明显的同源位点。
