The nucleocapsid protein of the SARS coronavirus is capable of self-association through a C-terminal 209 amino acid interaction domain

Abstract

Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) caused a severe outbreak in several regions of the world in 2003. The virus is a novel coronavirus isolated from patients exhibiting atypical pneumonia and may have originated from wild animals such as civet cats in southern China. The genome of SARS-CoV is a positive-sense, single-stranded RNA whose sequence is distantly related to all known coronaviruses that infect humans and animals. Like other known coronaviruses, SARS-CoV is an enveloped virus containing three outer structural proteins, namely the membrane (M), envelope (E), and spike (S) proteins. The nucleocapsid (N) protein together with the viral RNA genome presumably form a helical core located within the viral envelope. The SARS-CoV nucleocapsid (N) protein is a 423 amino-acid, predicted phospho-protein of 46 kDa that shares little homology with other members of the coronavirus family. A short serine-rich stretch, and a putative bipartite nuclear localization signal are unique to it, thus suggesting its involvement in many important functions during the viral life cycle. In this report we have cloned the N gene of the SARS coronavirus, and studied its property of self-association to form dimers. We expressed the N protein as a fusion protein in the yeast two-hybrid system to demonstrate self-association and confirmed dimerization of the N protein from mammalian cell lysates by coimmunoprecipitation. Furthermore, via deletion analysis, we have shown that the C-terminal 209 amino-acid region constitutes the interaction domain responsible for self-association of the N protein to form dimers.

Fig. 1

Yeast two-hybrid results showing full-length homotypic interactions of the N protein of SARS-CoV. YPD yeast peptone dextrose media (no selection), SDTrp⁻, SDLeu⁻, SDTrp⁻Leu⁻, and SDHis⁻ are restrictive growth media lacking Trp, Leu, Trp Leu, and Trp Leu His, respectively. β-Galactosidase filter assay results are also shown.

Fig. 2

Liquid β-galactosidase assay results. Single and cotransformants were analyzed for a liquid β-galactosidase assay and were compared with others. Values are given in arbitrary units. The numbers above each bar represent the mean of five independent transformants.

Fig. 3

COS1 cells were transfected with HA-N,MYC-Nor both, immunoprecipitated using respective antibodies, resolved by 10% SDS–PAGE and Western blotted using anti-HA antibody as described in Materials and methods. Lane 1 shows HA-N immunoprecipitated with the same antibody, lanes 2 and 3 show cotransfected cells immunoprecipitated using anti-myc or anti-HA antibody, respectively, lane 4 shows myc-N immunoprecipitated using anti-myc antibody.

Fig. 4

Deletion analysis for isolation of the interaction domain of the N protein of SARS-CoV. Using the yeast two-hybrid approach, each of the shown deletions was tested along with a full-length N protein and scored for marker gene activity. Histidine prototrophy was studied at 5, 10, 20, 25, and 50 mM AT concentrations on SDHis⁻ media. Liquid β-galactosidase assay was conducted on the cotransformants and mean of five samples is displayed for each deletion of the N protein. The deletions divide the N protein into four regions A, B, C, and D. Numbers above each deletion bar depict amino-acids.