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 b-strands and four a-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. INTRODUCTIONTransmissible gastroenteritis virus (TGEV) is a member of the Coronaviridae, a family of enveloped, positive-strand RNA viruses divided into three groups based on antigenic and genetic criteria (Enjuanes et al., 2000a; González et al., 2003). Group 1, to which TGEV belongs, includes members infecting human and NL63], porcine [TGEV and Porcine epidemic diarrhea virus (PEDV)], canine [Canine coronavirus (CCoV)] and feline [Feline coronavirus (FCoV)] species. All coronaviruses contain at least four structural proteins. The spike (S), membrane (M) and envelope (E) proteins are embedded in the virus envelope. The nucleocapsid (N) protein binds to the RNA genome forming the helical nucleocapsid (Sturman et al., 1980), which is arranged in TGEV virions as a spherical core by binding to the M protein (Escors et al., 2001a, b;Narayanan et al., 2000). Coronavirus N proteins are highly basic with a molecular mass ranging from 40 to 63 kDa, depending on the species and strain (Cologna & Hogue, 1998;Cologna et al., 2000;Nelson et al., 2000;Robbins et al., 1986;Stohlman et al., 1988). The N protein binds to the approximately 27-31 kb coronavirus RNA genome forming the helical nucleocapsid (Escors et al., 2001a;Kuo & Masters, 2002;Narayanan et al., 2000;Risco et al., 1998;Salanueva et al., 1999;Sturman et al., 1980) and is also involved in virus replication (Almazán et al., 2004;Bost et al., 2001;Chang & Brian, 1996; Prentice et al., 2004;Yount...

Porcine epidemic diarrhea virus N protein prolongs S-phase cell cycle, induces endoplasmic reticulum stress, and up-regulates interleukin-8 expression

“…It has been reported that the N proteins of MHV and IBV localized in the nucleolus of a small number of infected cells (Wurm et al, 2001). TGEV N protein was not observed in the nucleolus of infected ST cell lines (Calvo, 2005). In the case of our study, PEDV N protein was not observed in the nucleolus of the cells by confocal microscopy.…”

Mouse Hepatitis Coronavirus Nucleocapsid Phosphorylation

“…We did not detect any difference in phosphorylation of the protein from the infected cells and that in extracellular virions. Calvo et al (2005) suggested the possibility that phosphorylation/ dephosphorylation could play a role in TGEV assembly. The details of virus release are not fully understood, but virions are thought to transport through the constitutive secretory pathway after budding.…”

“…Representative SDS-PAGE used to isolate MHV N protein and peptide map coverage. Positions of the phosphorylated sites on MHV N, conserved resides in the group II viruses BCoV, HCoV OC43 and SARS CoV, and recently identified sites on IBV (Chen et al, 2005) and TGEV (Calvo et al, 2005) N proteins are highlighted. Boxes indicate the areas of gel excision.…”

Glycogen Synthase Kinase-3 Regulates the Phosphorylation of Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein and Viral Replication

“…The C-terminal domain contributes to its di-or multimerization assembly (13,14), and the central region contains a serine/ arginine (SR)-rich motif with unknown function but which is possibly also involved in the regulation of its multimerization (15). Notably, N proteins are highly phosphorylated in infected cells (16,17). Surjit et al (18) provided evidence that SCoV N protein undergoes phosphorylation in cells, mainly at the serine residues.…”