Nuclear/nucleolar localization properties of C-terminal nucleocapsid protein of SARS coronavirus

Abstract

A novel coronavirus (CoV) has recently been identified as the aetiological agent of severe acute respiratory syndrome (SARS). Nucleocapsid (N) proteins of the Coronaviridae family have no discernable homology, but they share a common nucleolar-cytoplasmic distribution pattern. There are three putative nuclear localization signal (NLS) motifs present in the N. To determine the role of these putative NLSs in the intracellular localization of the SARS-CoV N, we performed a confocal microscopy analysis using rabbit anti-N antisera. In this report, we show that the wild type N was distributed mainly in the cytoplasm. The N-terminal of the N, which contains the NLS1 (aa38-44), was localized to the nucleus. The C-terminus of the N, which contains both NLS2 (aa257-265) and NLS3 (aa369-390) was localized to the cytoplasm and the nucleolus. Results derived from analysis of various deletion mutations show that the region containing amino acids 226-289 is able to mediate nucleolar localization. The deletion of two hydrophobic regions that flanked the NLS3 recovered its activity and localized to the nucleus. Furthermore, deletion of leucine rich region (220-LALLLLDRLNRL) resulted in the accumulation of N to the cytoplasm and nucleolus, and when fusing this peptide to EGFP localization was cytoplasmic, suggesting that the N may act as a shuttle protein. Differences in nuclear/nucleolar localization properties of N from other members of coronavirus family suggest a unique function for N, which may play an important role in the pathogenesis of SARS.

Severe Acute Respiratory Syndrome-associated Coronavirus Nucleocapsid Protein Interacts with Smad3 and Modulates Transforming Growth Factor-β Signaling

“…Several nuclear localization signals have been identified in both the N-terminal and C-terminal domains of N protein, whereas only one putative nuclear export signal is located in the middle (amino acids 220 -231) (30,31). We further mapped the region of N protein interacting with Smad3.…”

Antigenic and cellular localisation analysis of the severe acute respiratory syndrome coronavirus nucleocapsid protein using monoclonal antibodies

“…In the virion, nucleoprotein molecules associate with viral RNA forming the helical nucleocapsid (Davies et al, 1981;Nelson et al, 2000). The protein contains both nuclear localisation and export signals (Rowland et al, 2005;Timani et al, 2005;You et al, 2005) suggesting shuttling of the protein between the nuclear and the cytoplasmic compartment in virus-infected cells. Electron microscopy, conventional immunofluorescence and confocal laser scanning microscopic analyses demonstrate that the N protein is located in the cytoplasm of infected cells.…”

Characterization of the Nuclear Export Signal in the Coronavirus Infectious Bronchitis Virus Nucleocapsid Protein

“…However, in the case of severe acute respiratory syndrome coronavirus there are differing reports of subcellular localization in both virus-infected cells and cells expressing the N protein (21,31,38,40,46). Likewise, several different N proteins of arterivirus (which, together with the coronaviruses, forms part of the nidoviruses) have been shown to localize to the nucleus/nucleolus (32,39).…”