Carboxyl terminus of severe acute respiratory syndrome coronavirus nucleocapsid protein: self-association analysis and nucleic acid binding characterization.
Coronavirus nucleocapsid (N) protein envelops the genomic RNA to form long helical nucleocapsid during virion assembly. Since N protein oligomerization is usually a crucial step in this process, characterization of such an oligomerization will help in the understanding of the possible mechanisms for nucleocapsid formation. The N protein of severe acute respiratory syndrome coronavirus (SARS-CoV) was recently discovered to self-associate by its carboxyl terminus. In this study, to further address the detailed understanding of the association feature of this C-terminus, its oligomerization was systematically investigated by size exclusion chromatography and chemical cross-linking assays. Our results clearly indicated that the C-terminal domain of SARS-CoV N protein could form not only dimers but also trimers, tetramers, and hexamers. Further analyses against six deletion mutants showed that residues 343-402 were necessary and sufficient for this C-terminus oligomerization. Although this segment contains many charged residues, differences in ionic strength have no effects on its oligomerization, indicating the absence of electrostatic force in SARS-CoV N protein C-terminus self-association. Gel shift assay results revealed that the SARS-CoV N protein C-terminus is also able to associate with nucleic acids and residues 363-382 are the responsible interaction partner, demonstrating that this fragment might involve genomic RNA binding sites. The fact that nucleic acid binding could promote the SARS-CoV N protein C-terminus to form high-order oligomers implies that the oligomeric SARS-CoV N protein probably combines with the viral genomic RNA in triggering long nucleocapsid formation.
Luo
Haibin
H
Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Chen
Jing
J
Chen
Kaixian
K
Shen
Xu
X
Jiang
Hualiang
H
D000595:Amino Acid Sequence / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002850:Chromatography, Gel; D000086462:Coronavirus Nucleocapsid Proteins; D019281:Dimerization; D016679:Genome, Viral / Q000502:physiology; D019251:Nucleocapsid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019590:Nucleocapsid Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D045473:Severe acute respiratory syndrome-related coronavirus / Q000737:chemistry / Q000502:physiology*; D017384:Sequence Deletion; D055672:Static Electricity; D019065:Virus Assembly / Q000502:physiology*
D000086462:Coronavirus Nucleocapsid Proteins; D019590:Nucleocapsid Proteins; D012367:RNA, Viral