SR-Rich Motif Plays a Pivotal Role in Recombinant SARS Coronavirus Nucleocapsid Protein Multimerization

Luo, Hai‐Bin; Ye, Fei; Chen, Kaixian; Shen, Xu; Jiang, Hualiang

doi:10.1021/bi051122c

Cited by 52 publications

(71 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The oligomerization of the full-length SARS-CoV N protein has been studied widely (20)(21)(22)(23)(24), and its carboxyl terminal was proved to be necessary for its self-association (20,23). It was discovered previously that the C-terminal domain is able to form multimers (25), but it was not clear how high the formed oligomers could be, and the primary sequence for the oligomerization remained unclear. In this work, to investigate the oligomeric properties of the C-terminal domain (residues 283-422, N 283-422 ), the gel filtration (Figure 1), chemical cross-linking, and native gel electrophoresis technology based assays ( Figure 2) were applied.…”

Section: Methodsmentioning

confidence: 99%

“…The self-association domain in SARS-CoV N was first mapped out as the C-terminal 209 amino acids using the yeast twohybrid (23). Later this domain was shortened to the Cterminal 138 (residues 285-422) (20) and 140 (residues 283-422) amino acids (25) by two independent groups. Yu et al reported that residues 285-422 of SARS-CoV N formed only dimers (20), while our previous work revealed that residues 283-422 formed not only dimers but also multimers (25).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Carboxyl Terminus of Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein: Self-Association Analysis and Nucleic Acid Binding Characterization

et al. 2006

Self Cite

View full text Add to dashboard Cite

Coronavirus nucleocapsid (N) protein envelops the genomic RNA to form long helical nucleocapsid during virion assembly. Since N protein oligomerization is usually a crucial step in this process, characterization of such an oligomerization will help in the understanding of the possible mechanisms for nucleocapsid formation. The N protein of severe acute respiratory syndrome coronavirus (SARS-CoV) was recently discovered to self-associate by its carboxyl terminus. In this study, to further address the detailed understanding of the association feature of this C-terminus, its oligomerization was systematically investigated by size exclusion chromatography and chemical cross-linking assays. Our results clearly indicated that the C-terminal domain of SARS-CoV N protein could form not only dimers but also trimers, tetramers, and hexamers. Further analyses against six deletion mutants showed that residues 343-402 were necessary and sufficient for this C-terminus oligomerization. Although this segment contains many charged residues, differences in ionic strength have no effects on its oligomerization, indicating the absence of electrostatic force in SARS-CoV N protein C-terminus self-association. Gel shift assay results revealed that the SARS-CoV N protein C-terminus is also able to associate with nucleic acids and residues 363-382 are the responsible interaction partner, demonstrating that this fragment might involve genomic RNA binding sites. The fact that nucleic acid binding could promote the SARS-CoV N protein C-terminus to form high-order oligomers implies that the oligomeric SARS-CoV N protein probably combines with the viral genomic RNA in triggering long nucleocapsid formation.

show abstract

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Carboxyl Terminus of Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein: Self-Association Analysis and Nucleic Acid Binding Characterization

et al. 2006

Self Cite

View full text Add to dashboard Cite

show abstract

“…The N-terminal domain (ϳ130 residues) folds similarly to the U1A RNA-binding protein and is suggested to bind RNA (11,12). 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).…”

mentioning

confidence: 99%

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

Wu¹,

Yeh

Tsay

et al. 2009

Journal of Biological Chemistry

189

306

View full text Add to dashboard Cite

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...

show abstract

“…Nevertheless, Surjit et al [56] demonstrated that the C-terminal 209 amino-acid region constitutes the interaction domain responsible for self-association of SARS-CoV N protein to form dimers. While a recent work [57] has provided an incongruent result, which demonstrates that the C-terminal domain of SARSCoV N protein (residues 283-422) has multimeric ability, although the full-length protein tends to form dimers. Further study revealed that the multimeric ability of the Cterminal domain could by weakened by the SR-rich motif interaction with the central region (residues 211-290), and suggested that the SR-rich motif might play an important role in the transformation of SARS-CoV N protein between the dimer and multimer for self-association or dissociation.…”

Section: Nucleocapsid (N) Proteinmentioning

confidence: 98%

An Overall Picture of SARS Coronavirus (SARS-CoV) Genome-Encoded Major Proteins: Structures, Functions and Drug Development

Chen¹,

Luo²,

Chen³

et al. 2006

CPD

Self Cite

View full text Add to dashboard Cite

A severe atypical pneumonia designated as severe acute respiratory syndrome (SARS) by The World Health Organization broke out in China and menaced to more than other 30 countries between the end of the year 2002 and June of the year 2003. A novel coronavirus called severe acute respiratory syndrome coronavirus (SARS-CoV) has been recently identified as the etiological agent responsible for the infectious SARS disease. Based on extensively scientific cooperation and almost two-year's studies, remarkable achievements have been made in the understanding of the phylogenetic property and the genome organization of SARS-CoV, as well as the detailed characters of the major proteins involved in SARS-CoV life cycle. In this review, we would like to summarize the substantial scientific progress that has been made towards the structural and functional aspects of SARS-CoV associated key proteins. The progress focused on the corresponding key proteins' structure-based drug and vaccine developments has been also highlighted. The concerted and cooperative response for the treatment of the SARS disease has been proved to be a triumph of global public health and provides a new paradigm for the detection and control of future emerging infectious disease threats.

show abstract

SR-Rich Motif Plays a Pivotal Role in Recombinant SARS Coronavirus Nucleocapsid Protein Multimerization

Cited by 52 publications

References 27 publications

Carboxyl Terminus of Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein: Self-Association Analysis and Nucleic Acid Binding Characterization

Carboxyl Terminus of Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein: Self-Association Analysis and Nucleic Acid Binding Characterization

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

An Overall Picture of SARS Coronavirus (SARS-CoV) Genome-Encoded Major Proteins: Structures, Functions and Drug Development

Contact Info

Product

Resources

About