Analysis of multimerization of the SARS coronavirus nucleocapsid protein

Abstract

Severe Acute Respiratory Syndrome (SARS), an emerging disease characterized by atypical pneumonia, has recently been attributed to a novel coronavirus. The genome of SARS Coronavirus (SARS-CoV) has recently been sequenced, and a number of genes identified, including that of the nucleocapsid protein (N). It is noted, however, that the N protein of SARS-CoV (SARS-CoV N) shares little homology with nucleocapsid proteins of other members of the coronavirus family [Science 300 (2003) 1399; Science 300 (2003) 1394]. N proteins of other coronavirus have been reported to be involved in forming the viral core and also in the packaging and transcription of the viral RNA. As data generated from some viral systems other than coronaviruses suggested that viral N-N self-interactions may be necessary for subsequent formation of the nucleocapsid and assembly of the viral particles, we decided to investigate SARS-CoV N-N interaction. By using mammalian two-hybrid system and sucrose gradient fractionations, a homotypic interaction of N, but not M, was detected by the two-hybrid analysis. The mammalian two-hybrid assay revealed an approximately 50-fold increase in SEAP activity (measurement of protein-protein interaction) in N-N interaction compared to that observed in either M-M or mock transfection. Furthermore, mutational analyses characterized that a serine/arginine-rich motif (SSRSSSRSRGNSR) between amino acids 184 and 196 is crucial for N protein oligomerization, since deletion of this region completely abolished the N protein self-multimerization. Finally, the full-length nucleocapsid protein expressed and purified from baculovirus system was found to form different levels of higher order structures as detected by Western blot analysis of the fractionated proteins. Collectively, these results may aid us in elucidating the mechanism pertaining to formation of viral nucleocapsid core, and designing molecular approaches to intervene SARS-CoV replication.

Recombinant Severe Acute Respiratory Syndrome (SARS) Coronavirus Nucleocapsid Protein Forms a Dimer through Its C-terminal Domain

“…The oligomerization states of coronavirus N proteins are reported to be dimeric (36), trimeric (16,30), and higher (37). In this report, three independent methods, analytical ultracentrifugation, SEC-LS, and chemical cross-linking, were used to study the self-association of the SARS-CoV N protein.…”

Crystal Structure of the Severe Acute Respiratory Syndrome (SARS) Coronavirus Nucleocapsid Protein Dimerization Domain Reveals Evolutionary Linkage between Corona- and Arteriviridae

“…The full-length dimeric N protein has a propensity to form tetramers and higher molecular weight oligomers in vitro (23). A serine/arginine-rich motif (residues 184 -196) was shown to be important for N protein oligomerization (31). Since constructs containing residues 211-422 or 285-422 of SARS N do not form oligomers larger than dimers (23), it is possible that the serine/arginine-rich motif located outside the dimerization domain is necessary to mediate further association of N protein dimers.…”

Severe acute respiratory syndrome coronavirus nucleocapsid protein expressed by an adenovirus vector is phosphorylated and immunogenic in mice

“…However, the N protein is distributed predominantly in the cytoplasm of SARS-CoV-infected and N gene-transfected cells (Chang et al, 2004). The SARS-CoV N protein is a highly charged, basic protein that can selfassociate to form dimers (He et al, 2004;Surjit et al, 2004). The three-dimensional structure of the N-terminal portion of the protein is similar to those of other RNA-binding proteins (Huang et al, 2004).…”