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. 2013 Jun;1834(6):1054-62.
doi: 10.1016/j.bbapap.2013.03.003. Epub 2013 Mar 15.

Crystal structure-based exploration of the important role of Arg106 in the RNA-binding domain of human coronavirus OC43 nucleocapsid protein

I-Jung Chen  1 , Jeu-Ming P Yuann, Yu-Ming Chang, Shing-Yen Lin, Jincun Zhao, Stanley Perlman, Yo-Yu Shen, Tai-Huang Huang, Ming-Hon Hou
Affiliations

Crystal structure-based exploration of the important role of Arg106 in the RNA-binding domain of human coronavirus OC43 nucleocapsid protein

I-Jung Chen et al. Biochim Biophys Acta. 2013 Jun.

Abstract

Human coronavirus OC43 (HCoV-OC43) is a causative agent of the common cold. The nucleocapsid (N) protein, which is a major structural protein of CoVs, binds to the viral RNA genome to form the virion core and results in the formation of the ribonucleoprotein (RNP) complex. We have solved the crystal structure of the N-terminal domain of HCoV-OC43 N protein (N-NTD) (residues 58 to 195) to a resolution of 2.0Å. The HCoV-OC43 N-NTD is a single domain protein composed of a five-stranded β-sheet core and a long extended loop, similar to that observed in the structures of N-NTDs from other coronaviruses. The positively charged loop of the HCoV-OC43 N-NTD contains a structurally well-conserved positively charged residue, R106. To assess the role of R106 in RNA binding, we undertook a series of site-directed mutagenesis experiments and docking simulations to characterize the interaction between R106 and RNA. The results show that R106 plays an important role in the interaction between the N protein and RNA. In addition, we showed that, in cells transfected with plasmids that encoded the mutant (R106A) N protein and infected with virus, the level of the matrix protein gene was decreased by 7-fold compared to cells that were transfected with the wild-type N protein. This finding suggests that R106, by enhancing binding of the N protein to viral RNA plays a critical role in the viral replication. The results also indicate that the strength of N protein/RNA interactions is critical for HCoV-OC43 replication.

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Figures

Fig. 1
Fig. 1
The structure and topology of the HCoV-OC43 N-NTD. (A) A ribbon diagram of the HCoV-OC43 N-NTD depicts the presence of five β strands, two 310 helices, and several disordered regions. (B) The topology of the HCoV-OC43 N-NTD shows the relative positions of the secondary structures of the truncated protein. (C) The surface charge distribution of the HCoV-OC43 N-NTD.
Fig. 2
Fig. 2
Amino acid sequences of N-NTDs from various coronaviruses. (A) Secondary structural alignment of the amino acids in N-NTDs from HCoV-OC43, SARS-CoV (PDB ID: 2ofz), IBV (PDB ID: 2gec), and MHV (PDB ID: 3hd4). (B) Superimposition of the HCoV-OC43 N-NTD (green) with N-NTDs from SARS-CoV (magenta), IBV (yellow), and MHV (blue). (C) Surface charge distribution of N-NTDs from HCoV-OC43, SARS-CoV, IBV, and HCoV-229E. The structure of HCoV-229E N-NTD was modeled in the Modeler program using the SARS-CoV N-NTD as a template.
Fig. 3
Fig. 3
(A) Amino acid sequence alignment, performed using T-coffee, of N-NTDs from HCoV-OC43 (NP_937954), SARS-CoV (ABI96968), HCoV-229E (AAG48597), IBV (AAB24054), and MHV (ACO72897), all of which were retrieved from GenBank. Residue 106 is indicated with an asterisk. (B) Structural superimposition of the HCoV-OC43 N-NTD (green) with N-NTDs from SARS-CoV (magenta) (PDB ID: 2ofz), IBV (yellow) (PDB ID: 2gec), and MHV (blue) (PDB ID: 3hd4). The inset depicts the location of R106.
Fig. 4
Fig. 4
(A) SPR traces from an RNA binding affinity assay assessing WT HCoV-OC43 N protein and five mutant N proteins. (B) Binding capacity of the WT and mutant N proteins at 0.1 μM for RNA.
Fig. 5
Fig. 5
Kinetic analyses of HCoV-OC43 WT and mutant N proteins binding to RNA: (A) ka and (B) kd.
Fig. 6
Fig. 6
Predicted interactions between RNA and R106 in the HCoV-OC43 N-NTDs. A single-stranded RNA, 5′-UCUAAAC-3′, was generated using the DS module in biopolymer; the subsequent docking calculations were performed using HADDOCK 1.3 software. The εN of R106 forms hydrogen bond with 5′-phosphate group of adenine residue in RNA (cyan dashed lines). Additionally, the ηN of the Arg106 guanidinium side chain also forms hydrogen bonds with ribose 2′-hydroxyl group and O3′ atom of RNA (black dashed lines). Nitrogen and oxygen atoms were represented by blue ball and red ball, respectively.
Fig. 7
Fig. 7
Virus replication by expression of wild type and R106A N proteins in trans. Cells were transfected with plasmid, pcDNA3.1, encoding (A) the WT N protein or (B) the mutant (R106A) N protein and then infected with HCoV-OC43 as described in Materials and Methods section. Samples were then analyzed for levels of Matrix protein (MP) gene transcript. (C) No transfection and (D) no infection controls are also shown. Quantitative data are shown as mean ± S.D., n = 3.
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References

    1. St-Jean J.R., Jacomy H., Desforges M., Vabret A., Freymuth F., Talbot P.J. Human respiratory coronavirus OC43: genetic stability and neuroinvasion. J. Virol. 2004;78:8824–8834. - PMC - PubMed
    1. Vabret A., Mourez T., Gouarin S., Petitjean J., Freymuth F. An outbreak of coronavirus OC43 respiratory infection in Normandy, France. Clin. Infect. Dis. 2003;36:985–989. - PMC - PubMed
    1. El-Sahly H.M., Atmar R.L., Glezen W.P., Greenberg S.B. Spectrum of clinical illness in hospitalized patients with “common cold” virus infections. Clin. Infect. Dis. 2000;31:96–100. - PMC - PubMed
    1. Gagneur A., Sizun J., Vallet S., Legr M.C., Picard B., Talbot P.J. Coronavirus-related nosocomial viral respiratory infections in a neonatal and paediatric intensive care unit: a prospective study. J. Hosp. Infect. 2002;51:59–64. - PMC - PubMed
    1. Wenzel R.P., Hendley J.O., Davies J.A., Gwaltney J.M., Jr. Coronavirus infections in military recruits. Three-year study with coronavirus strains OC43 and 229E. Am. Rev. Respir. Dis. 1974;109:621–624. - PubMed

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