Biochemical and immunological studies of nucleocapsid proteins of severe acute respiratory syndrome and 229E human coronaviruses

Abstract

Severe acute respiratory syndrome (SARS) is a serious health threat and its early diagnosis is important for infection control and potential treatment of the disease. Diagnostic tools require rapid and accurate methods, of which a capture ELISA method may be useful. Toward this goal, we have prepared and characterized soluble full-length nucleocapsid proteins (N protein) from SARS and 229E human coronaviruses. N proteins form oligomers, mostly as dimers at low concentration. These two N proteins degrade rapidly upon storage and the major degraded N protein is the C-terminal fragment of amino acid (aa) 169-422. Taken together with other data, we suggest that N protein is a two-domain protein, with the N-terminal aa 50-150 as the RNA-binding domain and the C-terminal aa 169-422 as the dimerization domain. Polyclonal antibodies against the SARS N protein have been produced and the strong binding sites of the anti-nucleocapsid protein (NP) antibodies produced were mapped to aa 1-20, aa 150-170 and aa 390-410. These sites are generally consistent with those mapped by sera obtained from SARS patients. The SARS anti-NP antibody was able to clearly detect SARS virus grown in Vero E6 cells and did not cross-react with the NP from the human coronavirus 229E. We have predicted several antigenic sites (15-20 amino acids) of S, M and N proteins and produced antibodies against those peptides, some of which could be recognized by sera obtained from SARS patients. Antibodies against the NP peptides could detect the cognate N protein clearly. Further refinement of these antibodies, particularly large-scale production of monoclonal antibodies, could lead to the development of useful diagnostic kits for diseases associated with SARS and other human coronaviruses.

SARS-CoV nucleocapsid protein antagonizes IFN-β response by targeting initial step of IFN-β induction pathway, and its C-terminal region is critical for the antagonism

“…And it as has been demonstrated that these proteins can sequester the genomic RNA in such a way that host-sensing proteins do not detect it. SARS-CoV N protein is also an RNA-binding protein [29,30]. Therefore, it may be possible that N protein employs a similar mechanism to inhibit the IFN response.…”

Oligomerization of the carboxyl terminal domain of the human coronavirus 229E nucleocapsid protein

“…The self-association mechanism of the full-length HCoV-229E N protein has been previously reported [21,47]. To determine whether the C-terminal tail region (N351-389) plays an important role in the oligomerization of the C-terminal domain of the HCoV-229E N protein, the oligomerization of several regions of the C-terminal domain of the N protein were analyzed using analytical ultracentrifugation.…”

“…The CoV N protein has also been shown to act as an RNA chaperone [26]. Moreover, the N protein is an important diagnostic marker and immunodominant antigen in host immune responses [21,27,28].…”

“…We speculate that the C-terminal tail might constitute an important molecular determinant of oligomerization for HCoV-229E NP. However, the recombinant full-length nucleocapsid N protein of the coronavirus is highly sensitive to proteolysis and aggregation that is difficult to analyze its oligomerization properties [21]. A comprehensive series of HCoV-229E N protein mutants with truncated Cterminal domains was generated based on the PrDOS prediction to clarify the role of the C-terminal tail of the HCoV-229E N protein in oligomerization (Fig.…”

“…Nucleocapsid protein, the major structural protein of CoVs, binds the viral RNA genome to form the virion core, leading to the formation of a ribonucleoprotein (RNP) complex or to a long helical nucleocapsid structure [17,18]. The formation of the RNP is important for maintaining the RNA in an ordered conformation suitable for replication and transcription of the viral genome [17,[19][20][21]. Previous studies have shown that the CoV N protein is involved in the regulation of cellular processes, such as gene transcription, actin reorganization, host cell cycle progression, and apoptosis [22][23][24][25].…”