ABSTRACT: 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.