Coronavirus (CoV) nucleocapsid (N) protein is a highly phosphorylated protein required for viral replication, but whether its phosphorylation and the related kinases are involved in the viral life cycle is unknown. We found the severe acute respiratory syndrome CoV N protein to be an appropriate system to address this issue. Using high resolution PAGE analysis, this protein could be separated into phosphorylated and unphosphorylated isoforms. Mass spectrometric analysis and deletion mapping showed that the major phosphorylation sites were located at the central serine-arginine (SR)-rich motif that contains several glycogen synthase kinase (GSK)-3 substrate consensus sequences. GSK-3-specific inhibitor treatment dephosphorylated the N protein, and this could be recovered by the constitutively active GSK-3 kinase. Immunoprecipitation brought down both N and GSK-3 proteins in the same complex, and the N protein could be phosphorylated directly at its SR-rich motif by GSK-3 using an in vitro kinase assay. Mutation of the two priming sites critical for GSK-3 phosphorylation in the SR-rich motif abolished N protein phosphorylation. Finally, GSK-3 inhibitor was found to reduce N phosphorylation in the severe acute respiratory syndrome CoV-infected VeroE6 cells and decrease the viral titer and cytopathic effects. The effect of GSK-3 inhibitor was reproduced in another coronavirus, the neurotropic JHM strain of mouse hepatitis virus. Our results indicate that GSK-3 is critical for CoV N protein phosphorylation and suggest that it plays a role in regulating the viral life cycle. This study, thus, provides new avenues to further investigate the specific role of N protein phosphorylation in CoV replication.The causative pathogen for the epidemic severe acute respiratory syndrome (SARS) 2 was identified as the SARS coronavirus (SCoV) in 2003 (1, 2). Its genome consists of a ϳ30-kilobase positive-sense single-stranded RNA which encodes a 3Ј co-terminal set of nine subgenomic mRNAs with a common leader sequence at their 5Ј ends (3, 4). These subgenomic RNAs encode various structural and nonstructural proteins required to produce progeny virions, including the viral nucleocapsid (N) protein.The SCoV N protein is the most abundant viral structural protein. During the viral life cycle multiple copies of the N protein interact with the viral genome to form the ribonucleoprotein complex, which is subsequently packaged by a lipid envelope during viral budding, possibly through its interaction with the viral structure membrane (M) protein (5). In addition to its structural role, the N protein is also implicated in regulating the synthesis of viral RNA and protein (4, 6, 7). Using reverse genetics, the critical role of N protein in the replication of coronaviruses has been identified in HCoV-229E, TGEV (transmissible gastroenteritis coronavirus), and IBV (infectious bronchitis virus) (8 -10). However, the molecular mechanisms in N protein participation in viral replication and the cellular gene(s) involved in regulating the process re...

BackgroundSevere acute respiratory syndrome coronavirus (SARS-CoV) caused a global panic due to its high morbidity and mortality during 2002 and 2003. Soon after the deadly disease outbreak, the angiotensin-converting enzyme 2 (ACE2) was identified as a functional cellular receptor in vitro and in vivo for SARS-CoV spike protein. However, ACE2 solely is not sufficient to allow host cells to become susceptible to SARS-CoV infection, and other host factors may be involved in SARS-CoV spike protein-ACE2 complex.ResultsA host intracellular filamentous cytoskeletal protein vimentin was identified by immunoprecipitation and LC-MS/MS analysis following chemical cross-linking on Vero E6 cells that were pre-incubated with the SARS-CoV spike protein. Moreover, flow cytometry data demonstrated an increase of the cell surface vimentin level by 16.5 % after SARS-CoV permissive Vero E6 cells were treated with SARS-CoV virus-like particles (VLPs). A direct interaction between SARS-CoV spike protein and host surface vimentin was further confirmed by far-Western blotting. In addition, antibody neutralization assay and shRNA knockdown experiments indicated a vital role of vimentin in cell binding and uptake of SARS-CoV VLPs and the viral spike protein.ConclusionsA direct interaction between vimentin and SARS-CoV spike protein during viral entry was observed. Vimentin is a putative anti-viral drug target for preventing/reducing the susceptibility to SARS-CoV infection.

BackgroundHead and neck squamous cell carcinoma (HNSCC) is a highly lethal cancer that contains cellular and functional heterogeneity. Previously, we enriched a subpopulation of highly tumorigenic head and neck cancer initiating cells (HN-CICs) from HNSCC. However, the molecular mechanisms by which to govern the characteristics of HN-CICs remain unclear. GRP78, a stress-inducible endoplasmic reticulum chaperone, has been reported to play a crucial role in the maintenance of embryonic stem cells, but the role of GRP78 in CICs has not been elucidated.ResultsInitially, we recognized GRP78 as a putative candidate on mediating the stemness and tumorigenic properties of HN-CICs by differential systemic analyses. Subsequently, cells with GRP78 anchored at the plasma membrane (memGRP78+) exerted cancer stemness properties of self-renewal, differentiation and radioresistance. Of note, xenotransplantation assay indicated merely 100 memGRP78+ HNSCCs resulted in tumor growth. Moreover, knockdown of GRP78 significantly reduced the self-renewal ability, side population cells and expression of stemness genes, but inversely promoted cell differentiation and apoptosis in HN-CICs. Targeting GRP78 also lessened tumorigenicity of HN-CICs both in vitro and in vivo. Clinically, co-expression of GRP78 and Nanog predicted the worse survival prognosis of HNSCC patients by immunohistochemical analyses. Finally, depletion of GRP78 in HN-CICs induced the expression of Bax, Caspase 3, and PTEN.ConclusionsIn summary, memGRP78 should be a novel surface marker for isolation of HN-CICs, and targeting GRP78 signaling might be a potential therapeutic strategy for HNSCC through eliminating HN-CICs.

The terminal proteins of linear Streptomyces chromosomes and plasmids: a novel class of replication priming proteins tides are similar in length (184-185 amino acids) and sequences, which include a putative helix domain that is homologous to part of the DNA-binding 'thumb' domain of HIV reverse transcriptase, and a putative amphiphilic beta-sheet that may be involved in the observed self-aggregation of the TP and/or the proposed membrane binding. IntroductionSoil bacteria of the genus Streptomyces are unusual among bacteria in having protein-capped linear chromosomes . The terminal sequences of these chromosomes consist of inverted repeats (TIR) of variable lengths ranging from 20 to 550 kb. The nucleotide sequences of these TIR are generally not conserved except for the first ª 200 bp, which are packed with palindromic sequences (Huang et al., 1998a). Streptomyces species also harbour many linear plasmids of a wide range of sizes (tens to hundreds of kilobases). Like the Streptomyces chromosomes, these linear plasmids contain TIR and covalently bound terminal proteins (TPs).Both linear chromosomes and linear plasmids of Streptomyces are replicated from internal origins (Chang and Cohen, 1994;Musialowski et al., 1994). This would leave single-stranded gaps at the 3¢ ends of the replicons (Chang and Cohen, 1994), which are patched by an as yet undefined mechanism presumably involving the TPs (Chen, 1996).The presence of the covalently linked TPs at the telomeres has been indicated by various lines of experimental evidence, including immobilization or retardation of the telomere DNA during electrophoresis in the absence of proteolytic or denaturing treatments (Ito et al., 1978), protection against exonuclease digestion and binding to silica (Coombs and Pearson, 1978;Thomas et al., 1979). The TPs may be readily removed when subjected to mild alkaline conditions, suggesting that the DNA-protein phosphodiester linkage is at a serine residue on the TP. That the TP is linked to the 5¢ end of the DNA has been suggested by protection of the terminal DNA from 5¢ exonucleases but not 3¢ exonucleases, although in at least one case, both 5¢ and 3¢ ends are protected .Linear DNA molecules with 5¢ covalently bound TPs were discovered previously in many eukaryotic and prokaryotic viruses and plasmids, the best studied of which are adenoviruses and bacillus phage f29. These viral genomes have relatively very short (as short as 6 bp in adenoviruses) TIR and are replicated from end to end using the TP as the primer. The TP-primed replication initiation of these viruses contrasts with the proposed TPprimed patching at the Streptomyces telomeres, in that the substrate for the former is double stranded, whereas that for the latter is single stranded. This difference may explain the need for extensive palindromes in the latter, which supposedly fold into elaborate secondary structures containing many hairpin loops and 'bulges' closed by purine:purine sheared pairs resembling those found in the genomes of autonomous parvoviruses (Huang...