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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.

SUBMITTER: Timani KA

PROVIDER: S-EPMC7114095 | biostudies-literature | 2005 Dec

REPOSITORIES: biostudies-literature

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

Timani Khalid Amine KA Liao Qingjiao Q Ye Linbai L Zeng Yingchun Y Liu Jing J Zheng Yi Y Ye Li L Yang Xiaojun X Lingbao Kong K Gao Jingrong J Zhu Ying Y

Virus research 20050629 1-2

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 analy ...[more]

PMID: 15992957

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Project description:Torovirus (ToV) has recently been classified into the new family Tobaniviridae, although historically, it belonged to the Coronavirus (CoV) family. The nucleocapsid (N) proteins of CoVs are predominantly localized in the cytoplasm, where the viruses replicate, but in some cases the proteins are partially located in the nucleolus. Many studies have investigated the subcellular localization and nucleocytoplasmic trafficking signals of the CoV N proteins, but little is known about ToV N proteins. Here, we studied the subcellular localization of the bovine ToV (BToV) N protein (BToN) and characterized its nucleocytoplasmic trafficking signals. Unlike other CoVs, BToN in infected cells was transported mainly to the nucleolus during early infection but was distributed predominantly in the nucleoplasm rather than in the nucleolus during late infection. Interestingly, a small quantity of BToN was detected in the cytoplasm during infection. Examination of a comprehensive set of substitution or deletion mutants of BToN fused with enhanced green fluorescent protein (EGFP) revealed that clusters of arginine (R) residues comprise nuclear/nucleolar localization signals (NLS/NoLS), and the C-terminal region served as a chromosomal maintenance 1 (CRM1)-independent nuclear export signal (NES). Moreover, recombinant viruses with mutations in the NLS/NoLS, but retaining nuclear accumulation, were successfully rescued and showed slightly reduced growth ability, while the virus that lost the NLS/NoLS-mediated nuclear accumulation of BToN was not rescued. These results indicate that BToN uniquely accumulates mainly in nuclear compartments during infection, regulated by an R-rich NLS/NoLS and a CRM1-independent NES, and that the BToN accumulation in the nuclear compartment driven by NLS/NoLS is important for virus growth.IMPORTANCE ToVs are diarrhea-causing pathogens detected in many species, including humans. BToV has spread worldwide, leading to economic loss, and there is currently no treatment or vaccine available. Positive-stranded RNA viruses, including ToVs, replicate in the cytoplasm, and their structural proteins generally accumulate in the cytoplasm. Interestingly, BToN accumulated predominantly in the nucleus/nucleolus during all infectious processes, with only a small fraction accumulating in the cytoplasm despite being a major structural protein. Furthermore, we identified unique nucleocytoplasmic trafficking signals and demonstrated the importance of NLS/NoLS for virus growth. This study is the first to undertake an in-depth investigation of the subcellular localization and intracellular trafficking signals of BToN. Our findings additionally suggest that the NLS/NoLS-mediated nuclear accumulation of BToN is important for virus replication. An understanding of the unique features of BToV may provide novel insights into the assembly mechanisms of not only ToVs but also other positive-stranded RNA viruses.

Dataset Information

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

Publications

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

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