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Structure of the SARS coronavirus nucleocapsid protein RNA-binding dimerization domain suggests a mechanism for helical packaging of viral RNA.

ABSTRACT: Coronavirus nucleocapsid proteins are basic proteins that encapsulate viral genomic RNA to form part of the virus structure. The nucleocapsid protein of SARS-CoV is highly antigenic and associated with several host-cell interactions. Our previous studies using nuclear magnetic resonance revealed the domain organization of the SARS-CoV nucleocapsid protein. RNA has been shown to bind to the N-terminal domain (NTD), although recently the C-terminal half of the protein has also been implicated in RNA binding. Here, we report that the C-terminal domain (CTD), spanning residues 248-365 (NP248-365), had stronger nucleic acid-binding activity than the NTD. To determine the molecular basis of this activity, we have also solved the crystal structure of the NP248-365 region. Residues 248-280 form a positively charged groove similar to that found in the infectious bronchitis virus (IBV) nucleocapsid protein. Furthermore, the positively charged surface area is larger in the SARS-CoV construct than in the IBV. Interactions between residues 248-280 and the rest of the molecule also stabilize the formation of an octamer in the asymmetric unit. Packing of the octamers in the crystal forms two parallel, basic helical grooves, which may be oligonucleotide attachment sites, and suggests a mechanism for helical RNA packaging in the virus.

SUBMITTER: Chen CY

PROVIDER: S-EPMC7094638 | biostudies-literature | 2007 May

REPOSITORIES: biostudies-literature

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Structure of the SARS coronavirus nucleocapsid protein RNA-binding dimerization domain suggests a mechanism for helical packaging of viral RNA.

Chen Chun-Yuan CY Chang Chung-Ke CK Chang Yi-Wei YW Sue Shih-Che SC Bai Hsin-I HI Riang Lilianty L Hsiao Chwan-Deng CD Huang Tai-Huang TH

Journal of molecular biology 20070302 4

Coronavirus nucleocapsid proteins are basic proteins that encapsulate viral genomic RNA to form part of the virus structure. The nucleocapsid protein of SARS-CoV is highly antigenic and associated with several host-cell interactions. Our previous studies using nuclear magnetic resonance revealed the domain organization of the SARS-CoV nucleocapsid protein. RNA has been shown to bind to the N-terminal domain (NTD), although recently the C-terminal half of the protein has also been implicated in R ...[more]

PMID: 17379242

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Project description:UnlabelledThe coronavirus nucleocapsid (N) protein forms a helical ribonucleoprotein with the viral positive-strand RNA genome and binds to the principal constituent of the virion envelope, the membrane (M) protein, to facilitate assembly and budding. Besides these structural roles, N protein associates with a component of the replicase-transcriptase complex, nonstructural protein 3, at a critical early stage of infection. N protein has also been proposed to participate in the replication and selective packaging of genomic RNA and the transcription and translation of subgenomic mRNA. Coronavirus N proteins contain two structurally distinct RNA-binding domains, an unusual characteristic among RNA viruses. To probe the functions of these domains in the N protein of the model coronavirus mouse hepatitis virus (MHV), we constructed mutants in which each RNA-binding domain was replaced by its counterpart from the N protein of severe acute respiratory syndrome coronavirus (SARS-CoV). Mapping of revertants of the resulting chimeric viruses provided evidence for extensive intramolecular interactions between the two RNA-binding domains. Through analysis of viral RNA that was packaged into virions we identified the second of the two RNA-binding domains as a principal determinant of MHV packaging signal recognition. As expected, the interaction of N protein with M protein was not affected in either of the chimeric viruses. Moreover, the SARS-CoV N substitutions did not alter the fidelity of leader-body junction formation during subgenomic mRNA synthesis. These results more clearly delineate the functions of N protein and establish a basis for further exploration of the mechanism of genomic RNA packaging.ImportanceThis work describes the interactions of the two RNA-binding domains of the nucleocapsid protein of a model coronavirus, mouse hepatitis virus. The main finding is that the second of the two domains plays an essential role in recognizing the RNA structure that allows the selective packaging of genomic RNA into assembled virions.

Project description:Alphaviruses are a group of small, enveloped viruses which are widely distributed on all continents. In infected cells, alphaviruses display remarkable specificity in RNA packaging by encapsidating only their genomic RNA while avoiding packaging of the more abundant viral subgenomic (SG), cellular messenger and transfer RNAs into released virions. In this work, we demonstrate that in spite of evolution in geographically isolated areas and accumulation of considerable diversity in the nonstructural and structural genes, many alphaviruses belonging to different serocomplexes harbor RNA packaging signals (PSs) which contain the same structural and functional elements. Their characteristic features are as follows. (i) Sindbis, eastern, western, and Venezuelan equine encephalitis and most likely many other alphaviruses, except those belonging to the Semliki Forest virus (SFV) clade, have PSs which can be recognized by the capsid proteins of heterologous alphaviruses. (ii) The PS consists of 4 to 6 stem-loop RNA structures bearing conserved GGG sequences located at the base of the loop. These short motifs are integral elements of the PS and can function even in the artificially designed PS. (iii) Mutagenesis of the entire PS or simply the GGG sequences has strong negative effects on viral genome packaging and leads to release of viral particles containing mostly SG RNAs. (iv) Packaging of RNA appears to be determined to some extent by the number of GGG-containing stem-loops, and more than one stem-loop is required for efficient RNA encapsidation. (v) Viruses of the SFV clade are the exception to the general rule. They contain PSs in the nsP2 gene, but their capsid protein retains the ability to use the nsP1-specific PS of other alphaviruses. These new discoveries regarding alphavirus PS structure and function provide an opportunity for the development of virus variants, which are irreversibly attenuated in terms of production of infectious virus but release high levels of genome-free virions.

Project description:The severe acute respiratory syndrome coronavirus (SARS-CoV) was recently identified as the etiology of SARS. The virus particle consists of four structural proteins: spike (S), small envelope (E), membrane (M), and nucleocapsid (N). Recognition of a specific sequence, termed the packaging signal (PS), by a virus N protein is often the first step in the assembly of viral RNA, but the molecular mechanisms involved in the assembly of SARS-CoV RNA are not clear. In this study, Vero E6 cells were cotransfected with plasmids encoding the four structural proteins of SARS-CoV. This generated virus-like particles (VLPs) of SARS-CoV that can be partially purified on a discontinuous sucrose gradient from the culture medium. The VLPs bearing all four of the structural proteins have a density of about 1.132 g/cm(3). Western blot analysis of the culture medium from transfection experiments revealed that both E and M expressed alone could be released in sedimentable particles and that E and M proteins are likely to form VLPs when they are coexpressed. To examine the assembly of the viral genomic RNA, a plasmid representing the GFP-PS580 cDNA fragment encompassing the viral genomic RNA from nucleotides 19715 to 20294 inserted into the 3' noncoding region of the green fluorescent protein (GFP) gene was constructed and applied to the cotransfection experiments with the four structural proteins. The SARS-CoV VLPs thus produced were designated VLP(GFP-PS580). Expression of GFP was detected in Vero E6 cells infected with the VLP(GFP-PS580), indicating that GFP-PS580 RNA can be assembled into the VLPs. Nevertheless, when Vero E6 cells were infected with VLPs produced in the absence of the viral N protein, no green fluorescence was visualized. These results indicate that N protein has an essential role in the packaging of SARS-CoV RNA. A filter binding assay and competition analysis further demonstrated that the N-terminal and C-terminal regions of the SARS-CoV N protein each contain a binding activity specific to the viral RNA. Deletions that presumably disrupt the structure of the N-terminal domain diminished its RNA-binding activity. The GFP-PS-containing SARS-CoV VLPs are powerful tools for investigating the tissue tropism and pathogenesis of SARS-CoV.

Dataset Information

Structure of the SARS coronavirus nucleocapsid protein RNA-binding dimerization domain suggests a mechanism for helical packaging of viral RNA.

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Structure of the SARS coronavirus nucleocapsid protein RNA-binding dimerization domain suggests a mechanism for helical packaging of viral RNA.

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