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

Abstract

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...

The dimer interface of the SARS coronavirus nucleocapsid protein adapts a porcine respiratory and reproductive syndrome virus‐like structure

“…4). Similar observations have been reported in the literature [6]. It is evident that the tight dimer interface structure permits certain extended perturbation in the global structure without affecting the dimer structure.…”

“…It binds to the viral RNA to form a ribonucleoprotein core, which can enter the host cell and interact with cellular processes [2][3][4][5]. The free protein presumably exists as a dimer in solution, with the dimerization domain located at the C-terminus [6,7]. We have previously defined the structural domains of the SARS-CoV N protein [8].…”

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

“…In this study, we report the crystal structure of the dimerization domain of SARS-CoV N protein consisting of residues 270 -370. Consistent with biochemical studies showing that this domain mediates dimer formation of the N protein (17,23), the structure consists of a dimer with extensive interactions between subunits, suggesting that the N protein is not stable in the monomeric form and that the dimeric structure represents the functional unit of the N protein. Furthermore, the dimerization domain of SARS-CoV N protein shows a similar fold to that of the N protein of porcine reproductive and respiratory syndrome virus (PRRSV), a member of Arteriviridae.…”

Oligomerization of the carboxyl terminal domain of the human coronavirus 229E nucleocapsid protein

“…These oligomers exist predominantly as dimers [47,48]. The secondary structure alignment of the C-terminal domains from the HCoV-229E N protein with the corresponding proteins from SARS-CoV and IBV indicates that these proteins share very similar secondary structure profiles [49].…”

“…The self-association mechanism of the full-length HCoV-229E N protein has been previously reported [21,47]. To determine whether the C-terminal tail region (N351-389) plays an important role in the oligomerization of the C-terminal domain of the HCoV-229E N protein, the oligomerization of several regions of the C-terminal domain of the N protein were analyzed using analytical ultracentrifugation.…”