The SARS-Associated Coronavirus Nucleocapsid Protein: Molecular Mechanism And Potential Antigen For Serodiagnosis Of SARS

The sudden outbreak of the human severe acute respiratory syndrome (SARS) in 2002 prompted the establishment of a global scientific network subsuming most of the traditional rivalries in the competitive field of virology. Within months of the SARS outbreak, collaborative work revealed the identity of the disastrous pathogen as SARS-associated coronavirus (SARS-CoV). Even in a situation of no new infections, SARS remains a major health hazard, as new epidemics may arise. Therefore, further experimental and clinical research is required to understand its infectious mechanism and control the disease.Given the highly contagious and acute nature of the disease, there is an urgent need for development of diagnostic assays that can detect SARS-CoV infection, more importantly, at its early stages. One important viral protein that can serve this goal is nucleocapsid (N) due to its high antigenic nature. The N protein gene sequence of SARS-CoV Hubei isolate strain was performed and the sequence was deposited to the Genbank accession number (AY365036). The N protein gene sequence alignment with N protein of eleven isolates from different regions in China (Beijing, Guangzhu and Zhejiang, Hong Kong and Taiwan) and other countries (Canada, Singapore, Italy, Germany and USA) revealed two basses substitution in N protein gene. These nucleotide changes were located at positions 284 and 689 from (T to C) and (A to G), respectively. The changes were non-synonymous and resulted in amino aids changes, valine to alanine and arginine to glutamine, at amino acid positions 95 and 230 of the N protein, respectively.The full length of N protein is 422 amino acids (aa) with an amino-terminal hexahistidine-tag was expressed in E.coli system, BL21(DE3), and purified by Ni-NTA affinity column chromatography to homogeneity. Recombinant purified SARS-CoV N protein was employed to establish an antigen-capturing enzyme linked immunosorbent assay (ELISA) and used to measure the SARS-CoV specific IgG antibodies from 16 SARS-CoV infected patients' sera and from 131 control subjects. Specific antibody responses to the purified recombinant N protein after 10, 20, and 30 days of disease onset were observed in 13 of 16 (81.3%), 16 of 16 (100%) and L6 of 16 (100%) Comparison of detection results with a commercially available diagnostic kit coated with a mixture ofSARS-CoV viral proteins showed 9 of 16 (56.3%), 13 of 16 (81.3%), and 15 of 16 (93.7%) positive responses, respectively. None of 131 control sera subjects gave positive reaction in either assay. This data suggests that the N protein of SARS-CoV is immunodominant and this ELISA based test assay for detecting the SARS-CoV N antigen may hold a significant value for SARS diagnosis.Understanding the intracellular localization properties on N protein might shed light on its molecular mechanism during SARS infection and its possible role in SARS pathogenesis. Although coronaviruses and arteriviruses encode N protein whose principle function is to bind viral RNA, the proteins themselves are of a different size and have no discernable homology. However, an identical nucleolar-cytoplasmic localization pattern is observed. In this study, we further investigated whether this nucleolar localization pattern extends to SARS-CoV N protein. Rabbit polyclonal antibody specific for the purified N protein was generated and used to examine its intracellular localization using confocal microscopy. PSORT II program sequence analysis of the N protein identified three classical putative nuclear localization signal (NLS) motifs, labeled NLS1, NLS2 and NLS3 located at positions aa34-44, aa257-265 and aa369-390, respectively. The indirect immunofluorescence staining showed that wild type N protein localized mainly to the cytoplasm in Vero E6 infected with SARS-CoV and Vero E6, Vero and HeLa cells transfected with plasmid expressing full length N protein, and was not clear in the nucleolus.Intriguingly, the expression of C-terminal N protein was found in the cytoplasm and nucleus, concentrated in the nucleolus. This feature was confirmed to the nucleolus by using specific monoclonal antibody to nucleolar shuttling phosphorprotein B23. The results of various deletion mutants either alone or fused to enhanced green fluorescent protein (EGFP) showed that the region covering aa232-289 and overlapping NLS2 is responsible for this nucleolar localization and might considered as nucleolar localization signal (NoLS). Furthermore, deletion of leucine rich region (LRR; 220-LALLLLDRLNRL) resulted in the accumulation of N protein to the cytoplasm and nucleolus, and when fusing this peptide to EGFP localization was cytoplasmic, suggesting that the N protein may act as a shuttle protein. Leptomycin B treatment, a know inhibitor to the nuclear export signal (NES)-CRMl pathway has no effect on the localization pattern of full length N protein demonstrating that N protein maybe export via NES-CRM1 independent pathway or LRR may act as a masking signal. In addition, flow cytometric analysis and colony formation efficiency assays demonstrated that constitutive N-expressing Vero E6 cells exhibited cell growth retardation concomitantlywith prolonged Go/Gi phase and shortened S phase of the cell cycle compared with control cells. Taken together, these results demonstrated that N protein may act as a nucleolar-cytoplasmic shuttling protein and the localization to the nucleus/nucleolus implies that N protein maybe involved in the regulating of cell cycle, possibly by delaying the cell growth, to promote intracellular conditions for virus assembly and sequestering ribosomes for translation of viral proteins.Patients with SARS usually have rapid progression of pneumonitis. Approximately one-third of SARS patients developed (Acute Respiratory Distress Syndrome, ARDS), one-tenth of the patients succumbed to death and one-tenth of patients revealed pulmonary fibrosis. The mechanisms of SARS-CoV infection caused atypical pneumonia remain unclear. Nuclear factor-kB (NF-kB) is a critical regulator of the immediate early pathogen response, playing an important role in promoting inflammation and in the regulation of cell proliferation and survival; moreover, many studies have shown that NF-kB played an important role in the pathogenesis of lung disease. In this research study we further investigated the possible regulatory mechanism of SARS-CoV N protein and NF-kB by using luciferase reporter assay. Our result showed that SARS-CoV N protein can significantly activate NF-kB only in Vero E6 cells, but there is no such significant activation in Vero or HeLa cell lines compared with backbone vector, suggested that this NF-kB activation is cell type specific. Furthermore, this NF-kB activation in Vero E6 cells expressing-N protein was shown to be concentration dependent. Different deletion mutation of N protein revealed that conformation of full length N protein was crucial for the NF-kB activation. This unique function of SARS-CoV N protein from other members of coronavirus family postulated that N protein can be a good target for the development of vaccine and antiviral drugs.