sars9a

The nucleocapsid protein, sars9a, is a 46 kDa phosphoprotein mainly responsible for packaging viral RNA and forming the helical ribonucleoprotein complex during assembly. It is phosphorylated at multiple serine residues by host kinases like cyclin-dependent kinase, glycogen synthase kinase, mitogen-activated protein kinase, and casein kinase II . It is also sumoylated at Lys62, a post-translational modification that enhances the proteins tendency to homo-oligomerize, and affects typical N protein-mediated interference in host cell division. Several groups have successfully expressed soluble protein (both full length as well as partial domains) but its unusually high positive charge, tendency to oligomerize and extremely low stability have hampered structural studies. It possesses two RNA binding domains: an S/R-rich motif between residues 184 and 196, and the C-terminal 209 amino acids , . The solution structure of its N-terminal RNA binding domain shows that it adopts a novel fold with residues that mediate nucleic acid binding being localized on a beta-hairpin and a positively charged groove . The C-terminal domain of N has been implicated in self association.

The N protein interacts with the M protein. Some of the regions involved in this interaction have been delineated. Mammalian two-hybrid work suggested that residues 168–208 in the N protein are critical for interaction with M . Yeast two-hybrid and surface plasmon resonance indicates that residues 197–221 of M protein and residues 341–422 of N protein are involved in this interaction. Another study narrowed down the interaction site in M to residues 194-205. The N protein has also been shown to bind specifically, in vitro and in vivo, to heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), a protein involved in pre-mRNA splicing in the nucleus and translation regulation in the cytoplasm. The N protein fragment corresponding to residues 161-210 and the Gly-rich domain (residues 203-320) of hnRNP A1 are involved in this interaction. This suggests that these proteins may be involved in the replicase complex, playing a role in RNA synthesis and/or splicing. The N protein also binds with nanomolar affinity to human cyclophilin A, though the physiological significance of this finding is still unknown.

We have determined the crystal structure of the N-terminal domain of sars9a, which may provide details on assembly of the nucleocapsid protein on genomic RNA in the virion. The structure of the C-terminal domain has been determined. It forms a domain-swapped dimer and is the dimerization domain of the nucleocapsid. Our cryo-electron microscopy data indicates that the nucleocapsid protein adopts a spiral arrangement of monomers which are collected into a sphere on budding. Four monomers surround each S trimer.

 

Surjit M, Kumar R, Mishra RN, Reddy MK, Chow VT, Lal SK. The severe acute respiratory syndrome coronavirus nucleocapsid protein is phosphorylated and localizes in the cytoplasm by 14-3-3-mediated translocation. J Virol. 2005 Sep;79(17):11476-86.

Li FQ, Xiao H, Tam JP, Liu DX. Sumoylation of the nucleocapsid protein of severe acute respiratory syndrome coronavirus. FEBS Lett. 2005 Apr 25;579(11):2387-96.

R. He, F. Dobie, M. Ballantine, A. Leeson, Y. Li, N. Bastien, T. Cutts, A. Andonov, J. Cao, T.F. Booth, F.A. Plummer, S. Tyler, L. Baker and X. Li, Analysis of multimerization of the SARS coronavirus nucleocapsid protein, Biochem. Biophys. Res. Commun. 316 (2004), pp. 476–483.

Yu IM, Gustafson CL, Diao J, Burgner JW 2nd, Li Z, Zhang J, Chen J. Recombinant severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein forms a dimer through its C-terminal domain. J Biol Chem. 2005 Jun 17;280(24):23280-6. Epub 2005 Apr 21.

Surjit M, Liu B, Kumar P, Chow VT, Lal SK. The nucleocapsid protein of the SARS coronavirus is capable of self-association through a C-terminal 209 amino acid interaction domain. Biochem Biophys Res Commun. 2004 May 14;317(4):1030-6.

Huang Q, Yu L, Petros AM, Gunasekera A, Liu Z, Xu N, Hajduk P, Mack J, Fesik SW, Olejniczak ET. Structure of the N-terminal RNA-binding domain of the SARS CoV nucleocapsid protein. Biochemistry. 2004 May 25;43(20):6059-63.

He R, Leeson A, Ballantine M, Andonov A, Baker L, Dobie F, Li Y, Bastien N, Feldmann H, Strocher U, Theriault S, Cutts T, Cao J, Booth TF, Plummer FA, Tyler S, Li X. Characterization of protein-protein interactions between the nucleocapsid protein and membrane protein of the SARS coronavirus. Virus Res. 2004 Oct;105(2):121-5.

Luo H, Wu D, Shen C, Chen K, Shen X, Jiang H. Severe acute respiratory syndrome coronavirus membrane protein interacts with nucleocapsid protein mostly through their carboxyl termini by electrostatic attraction. Int J Biochem Cell Biol. 2006;38(4):589-99. Epub 2005 Nov 28.

Fang X, Ye L, Timani KA, Li S, Zen Y, Zhao M, Zheng H, Wu Z. Peptide domain involved in the interaction between membrane protein and nucleocapsid protein of SARS-associated coronavirus. J Biochem Mol Biol. 2005 Jul 31;38(4):381-5.

Luo H, Chen Q, Chen J, Chen K, Shen X, Jiang H. The nucleocapsid protein of SARS coronavirus has a high binding affinity to the human cellular heterogeneous nuclear ribonucleoprotein A1. FEBS Lett. 2005 May 9;579(12):2623-8. Epub 2005 Apr 9.

Luo H, Chen Q, Chen J, Chen K, Shen X, Jiang H. The nucleocapsid protein of SARS coronavirus has a high binding affinity to the human cellular heterogeneous nuclear ribonucleoprotein A1. FEBS Lett. 2005 May 9;579(12):2623-8. Epub 2005 Apr 9.

Luo C, Luo H, Zheng S, Gui C, Yue L, Yu C, Sun T, He P, Chen J, Shen J, Luo X, Li Y, Liu H, Bai D, Shen J, Yang Y, Li F, Zuo J, Hilgenfeld R, Pei G, Chen K, Shen X, Jiang H. Nucleocapsid protein of SARS coronavirus tightly binds to human cyclophilin A. Biochem Biophys Res Commun. 2004 Aug 27;321(3):557-65.