Structure of the N-Terminal RNA-Binding Domain of the SARS CoV Nucleocapsid Protein

Huang, Qiulong; Yu, Liping; Petros, Andrew M.; Gunasekera, Angelo; Liu, Zhihong; Xu, Nan; Hajduk, Philip J.; Mack, J.; Fesik, Stephen W.; Olejniczak, Edward T.

doi:10.1021/bi036155b

Cited by 230 publications

(282 citation statements)

References 26 publications

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“…Extensive hydrogen-bonding and hydrophobic interactions are observed between the two subunits within the dimer found in the crystal, suggesting that the functional unit of the N protein is dimeric. Strong protein-protein interactions at the dimerization region may be essential to hold the putatively monomeric, highly charged RNA-binding domains in close proximity (16), thereby facilitating the formation of a large helical nucleocapsid core. Association of the N protein dimers is necessary for further assembly of the core.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Crystal Structure of the Severe Acute Respiratory Syndrome (SARS) Coronavirus Nucleocapsid Protein Dimerization Domain Reveals Evolutionary Linkage between Corona- and Arteriviridae

Oldham

Zhang

et al. 2006

Journal of Biological Chemistry

127

156

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The causative agent of severe acute respiratory syndrome (SARS) is the SARS-associated coronavirus, SARS-CoV. The nucleocapsid (N) protein plays an essential role in SARS-CoV genome packaging and virion assembly. We have previously shown that SARS-CoV N protein forms a dimer in solution through its C-terminal domain. In this study, the crystal structure of the dimerization domain, consisting of residues 270 -370, is determined to 1.75 Å resolution. The structure shows a dimer with extensive interactions between the two subunits, suggesting that the dimeric form of the N protein is the functional unit in vivo. Although lacking significant sequence similarity, the dimerization domain of SARS-CoV N protein has a fold similar to that of the nucleocapsid protein of the porcine reproductive and respiratory syndrome virus. This finding provides structural evidence of the evolutionary link between Coronaviridae and Arteriviridae, suggesting that the N proteins of both viruses have a common origin.Coronaviruses are enveloped, single-stranded, positive-sense RNA viruses that infect a variety of mammals and birds. Although previously identified human coronaviruses cause only mild respiratory infections, in the 2003 outbreak of severe acute respiratory syndrome (SARS), 3 a disease caused by a new type of coronavirus (SARS-CoV), there were more than 8,000 cases resulting in 774 deaths (ϳ10% mortality). Phylogenetic analysis suggests that SARSCoV diverged early from group 2 coronaviruses and has evolved independently for a long period of time (1).The coronavirus genome, containing ϳ30,000 bases, is the largest among positive-sense RNA viruses (2, 3). It encodes non-structural proteins including the RNA polymerase and helicase, as well as the spike (S), envelope (E), membrane (M), and nucleocapsid (N) structural proteins. The coronavirus virion is about 120 nm in diameter and consists of a lipid envelope containing three or four anchored glycoproteins and a helical ribonucleoprotein core (4). The surface projections forming the crown-like structure observed via electron microscopy are made up of the S protein, which is responsible for receptor recognition and membrane fusion (5-9). The integral membrane proteins M and E are essential for virus budding. When co-expressed in animal cells, the M and E proteins are sufficient to form virus-like particles (10). The N protein interacts with the viral genome to form the ribonucleoprotein core and has been shown to be involved in viral RNA synthesis, transcriptional regulation of genomic RNA, translation of viral proteins, and budding (2,11,12).Coronaviruses are related to arteriviruses by their similar genome organizations and viral replication mechanisms (3, 13). Recently, Coronaviridae and Arteriviridae were united to form the new order Nidovirales. The name of the order comes from a property common to both viruses, a nested set of subgenomic mRNAs for structural protein expression (Latin nidus, meaning nest). The replicase genes of arteriviruses and coronaviruses are thought to ha...

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Section: Discussionmentioning

confidence: 99%

“…High resolution structures of the N-terminal domain (ϳ130 residues) were determined by NMR (16) and crystallography (17). This region folds similarly to the U1A RNA-binding protein and is suggested to bind RNA (16,17). The central region of the N protein has also been shown by several laboratories to be an RNA-binding domain (18 -21).…”

mentioning

confidence: 99%

Crystal Structure of the Severe Acute Respiratory Syndrome (SARS) Coronavirus Nucleocapsid Protein Dimerization Domain Reveals Evolutionary Linkage between Corona- and Arteriviridae

Oldham

Zhang

et al. 2006

Journal of Biological Chemistry

127

156

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“…Although lacking sequence homology to previously described RNA-binding motifs, the central domain of N protein has been shown to be the RNA-binding region by several laboratories (18 -21). Recently, the structure of the Nterminal region consisting of residues 49 -178 of SARS-CoV N protein has been determined by NMR (22). Although the structure reveals a fold similar to the U1A RNA-binding protein (22), whether this N-terminal domain indeed binds RNA remains to be determined.…”

mentioning

confidence: 99%

“…Recently, the structure of the Nterminal region consisting of residues 49 -178 of SARS-CoV N protein has been determined by NMR (22). Although the structure reveals a fold similar to the U1A RNA-binding protein (22), whether this N-terminal domain indeed binds RNA remains to be determined.…”

mentioning

confidence: 99%

Recombinant Severe Acute Respiratory Syndrome (SARS) Coronavirus Nucleocapsid Protein Forms a Dimer through Its C-terminal Domain

Yu¹,

Gustafson²,

Diao³

et al. 2005

Journal of Biological Chemistry

104

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The causative agent of severe acute respiratory syndrome (SARS) is the SARS-associated coronavirus, SARS-CoV. The viral nucleocapsid (N) protein plays an essential role in viral RNA packaging. In this study, recombinant SARS-CoV N protein was shown to be dimeric by analytical ultracentrifugation, size exclusion chromatography coupled with light scattering, and chemical cross-linking. Dimeric N proteins selfassociate into tetramers and higher molecular weight oligomers at high concentrations. The dimerization domain of N was mapped through studies of the oligomeric states of several truncated mutants. Although mutants consisting of residues 1-210 and 1-284 fold as monomers, constructs consisting of residues 211-422 and 285-422 efficiently form dimers. When in excess, the truncated construct 285-422 inhibits the homodimerization of full-length N protein by forming a heterodimer with the full-length N protein. These results suggest that the N protein oligomerization involves the C-terminal residues 285-422, and this region is a good target for mutagenic studies to disrupt N protein self-association and virion assembly.Coronaviruses are responsible for ϳ30% of human upper respiratory infections each year. In November 2002, a new coronavirus, known as the severe acute respiratory syndrome (SARS) 1 -associated coronavirus, SARS-CoV, emerged in China and caused more than 8000 cases of SARS worldwide. Approximately 10% of these cases were fatal. Similar to other coronaviruses, SARS-CoV is an enveloped, single-stranded (ss) RNA virus. The SARS-CoV genome contains ϳ29,700 nucleotides (1, 2), encoding the RNA-dependent RNA polymerase and four structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N). In addition, intergenic regions encode several open reading frames for nonstructural proteins of unknown function (1, 2). The S protein is a surface glycoprotein that mediates viral entry by binding to the cellular receptor angiotensin-converting enzyme 2 (ACE2) (3) and inducing membrane fusion. The receptorbinding domain has been mapped to amino acids 318 -510 (4, 5), and structures of the heptad repeat region of S protein (6, 7) indicate that it is a class I membrane fusion protein. The M protein of coronavirus is the most abundant protein component of the envelope. This protein plays a predominant role in the formation and release of the virion envelope. When co-expressed with the E protein, virus-like particles with sizes and shapes similar to those of virions are assembled (8, 9). Recently, virus-like particles of SARS-CoV were obtained by recombinant expression of S, E, and M proteins in insect cells (10). Inside the envelope, the N protein associates with the genomic RNA to form a long, flexible, helical ribonucleoprotein. The N protein is typically 350 -450 amino acids in length, highly basic, and serine-phosphorylated, but the extent and physiological relevance of phosphorylation is unclear (11, 12). In addition to its structural role, several additional functions are postulated for the N protei...

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“…The order/disorder state of HCoV-NL63 N was different to that of other coronavirus N-proteins, with HCoV-NL63 N having only one disordered region (residues 109 to 248) compared to the 2 to 3 disordered regions within other coronavirus N-proteins . Interestingly, many of the motifs and/or regions important for the interaction of SARS-CoV N with other proteins fall within these disordered regions (He et al, 2004;Huang et al, 2004;Luo et al, 2004;Surjit et al, 2004b). For this reason, residues 109 to 248 of HCoV-NL63 N could be crucial for interactions between HCoV-NL63 and other proteins.…”

Section: Coronavirusmentioning

confidence: 99%

Characterisation of human coronavirus-NL63 nucleocapsid protein

Berry¹

2012

Afr. J. Biotechnol.

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Coronavirus N is a multifunctional protein that plays an essential role in enhancing the efficiency of virus transcription and assembly. This manuscript reports the analysis of HCoV-NL63 N protein by comparing the amino acid sequences of coronavirus N-homologues. A ~50 kDa protein was expressed in both a mammalian cell and bacterial cell system that is similar in size to the predicted ~42.6 kDa HCoV-NL63 N protein. PSORTII identified two putative nuclear localisations signals and PONDR identified one disordered region in HCoV-NL63 N. The reported protein analysis serves as a prelude to laboratory analysis to understand the processing of HCoV-NL63 N.

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Structure of the N-Terminal RNA-Binding Domain of the SARS CoV Nucleocapsid Protein

Cited by 230 publications

References 26 publications

Crystal Structure of the Severe Acute Respiratory Syndrome (SARS) Coronavirus Nucleocapsid Protein Dimerization Domain Reveals Evolutionary Linkage between Corona- and Arteriviridae

Crystal Structure of the Severe Acute Respiratory Syndrome (SARS) Coronavirus Nucleocapsid Protein Dimerization Domain Reveals Evolutionary Linkage between Corona- and Arteriviridae

Recombinant Severe Acute Respiratory Syndrome (SARS) Coronavirus Nucleocapsid Protein Forms a Dimer through Its C-terminal Domain

Characterisation of human coronavirus-NL63 nucleocapsid protein

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