Multiple Nucleic Acid Binding Sites and Intrinsic Disorder of Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein: Implications for Ribonucleocapsid Protein Packaging

Abstract

The nucleocapsid protein (N) of the severe acute respiratory syndrome coronavirus (SARS-CoV) packages the viral genomic RNA and is crucial for viability. However, the RNA-binding mechanism is poorly understood. We have shown previously that the N protein contains two structural domains-the N-terminal domain (NTD; residues 45 to 181) and the C-terminal dimerization domain (CTD; residues 248 to 365)-flanked by long stretches of disordered regions accounting for almost half of the entire sequence. Small-angle X-ray scattering data show that the protein is in an extended conformation and that the two structural domains of the SARS-CoV N protein are far apart. Both the NTD and the CTD have been shown to bind RNA. Here we show that all disordered regions are also capable of binding to RNA. Constructs containing multiple RNA-binding regions showed Hill coefficients greater than 1, suggesting that the N protein binds to RNA cooperatively. The effect can be explained by the "coupled-allostery" model, devised to explain the allosteric effect in a multidomain regulatory system. Although the N proteins of different coronaviruses share very low sequence homology, the physicochemical features described above may be conserved across different groups of Coronaviridae. The current results underscore the important roles of multisite nucleic acid binding and intrinsic disorder in N protein function and RNP packaging.Severe acute respiratory syndrome (SARS) is the first pandemic of the 21st century that spread to multiple nations, with a fatality rate of ca. 8%. The disease is caused by a novel SARS-associated coronavirus (SARS-CoV) closely related to the group II coronaviruses, which include the human coronavirus OC43 and murine hepatitis virus (6, 18). Traditional antiviral treatments have had little success against SARS during the outbreak, and vaccines have yet to be developed (35).Coronaviruses are positive-sense single-stranded RNA (ssRNA) viruses. The coronavirus genomic RNA is encapsidated into a helical capsid by the nucleocapsid (N) protein, which is one of the most abundant coronavirus proteins (19). The N protein has nonspecific binding activity toward nucleic acids, including ssRNA, single-stranded DNA, and double-stranded DNA (33). It can also act as an RNA chaperone (39). However, the mechanism of binding of the N protein to nucleic acids is poorly understood.The SARS-CoV N protein is a homodimer composed of 422 amino acids (aa) in each chain. The N protein can be divided into two structural domains interspersed with disordered (unstructured) regions (Fig. 1A) (2). The N-terminal domain (NTD; also called RBD) serves as a putative RNA-binding domain, while the C-terminal domain (CTD; also called DD) is a dimerization domain (13,36). Both the NTD and the CTD bind to nucleic acids through electropositive regions on their surfaces (3, 13, 32). All coronaviruses share similar domain architectures at both the sequence and structure levels. No structure of N protein or any of its domains in complex with nucleic acids is ava...

SARS-CoV nucleocapsid protein antagonizes IFN-β response by targeting initial step of IFN-β induction pathway, and its C-terminal region is critical for the antagonism

“…And it as has been demonstrated that these proteins can sequester the genomic RNA in such a way that host-sensing proteins do not detect it. SARS-CoV N protein is also an RNA-binding protein [29,30]. Therefore, it may be possible that N protein employs a similar mechanism to inhibit the IFN response.…”

“…A deletion mutant of CTD, which includes aa 281-365, totally lost the ability to bind to nucleic acid and retains dimerization activity [23]. Besides, it has been demonstrated that all three disordered regions of SARS-CoV N protein are involved in RNA-binding [30]. N truncation assays showed that SenV-induced IFN-b production was significantly suppressed by expression of CTD-containing constructs, while the C-CTD (aa 281-365) almost lost the ability to reduce IFN-b promoter activity (Fig.…”

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

“…Despite their low sequence homology, CoV N proteins from different strains can show a high level of conservation in some motifs [30]. Chang et al reported results from an order-disorder prediction and secondary structure prediction coupled with sequence alignment, which suggested that all CoV N proteins share the same modular organization [31]. Self-association of the N protein is an important step in virus particle assembly for many CoVs [32].…”

Characterisation of human coronavirus-NL63 nucleocapsid protein

“…Since interactions between coronavirus RNA and N are assumed to predominantly take place at the nucleocapsid N-terminal domain, the possible role of this conserved motif in RNA-N interaction will be studied. However, several other binding sites within N have also previously been identified, indicating that both N-and C-terminal domains are probably involved in RNA binding (Chang et al, 2009).…”