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.

Figures

Fig. 1

Characterization of the rabbit anti-N polyclonal antibody. (A) Total cell lysates from SARS–CoV-infected Vero E6 (lane 1) and Vero E6 cells only as a control (lane 2) were resolved by 15% SDS-PAGE. (B) Total cell lystaes from E. coli expressing the recombinant N protein after IPTG induction (described elsewhere; (Timani et al., 2004)) (lane 1), N protein constitutively expressing Vero E6 cells (lane 2) and neor control (lane 3) were resolved by 10% SDS-PAGE. Both (A) and (B) were analyzed by Western blot. The transferred PVDF membranes were probed with rabbit anti-N polyclonal antibody. The molecular mass of N protein in the blot was verified as ∼47 kDa. Approximate mobilities of protein markers are indicated on the left of (A).

Fig. 2

Detection of SARS–CoV N protein by indirect immunofluorescence assay in infected cells. Vero E6 cells were infected with SARS–CoV WHU strain. After 24 h cells were fixed and analyzed by indirect immunofluorescence using rabbit anti-N polyclonal antibody (green) ((A) and (B)) or pre-immune rabbit serum ((A) inset panel), followed by staining with PI (red) ((A) and (C)) to visualize the nuclear DNA. Differentially fluorescing images were gathered separately ((A) and (D)) using confocal microscope. Magnification 20 and 40× for (A) and (B)–(D), respectively.

Fig. 3

(A) The diagram identifies the location of putative NLSs and LRR domains within SARS–CoV N protein. Amino acid sequence analysis software program, PSORT II (Nakai and Kanehisa, 1992) was used to locate the classical NLS motifs. The numbers identified the amino acid position covered by each domain. The NLS was classified into “pat4”, “pat7” and “bipartite” motifs. For details, see the text. (B) Schematic representation of a summary of different deletion mutants of SARS–CoV N and their respective intracellular localization. Dash lines and numbers indicate the deleted amino acids and amino acids positions, respectively while bars represent translated amino acids. N: nucleus; C: cytoplasm. (+) and (−) designate for presence and absence of protein expression, respectively.

Fig. 4

Intracellular localization of various deletion mutants of SARS–CoV N protein in the transfected cells. Vero E6 cells were transfected with different deleted mutants. After 24 h, the cells were fixed and analyzed by indirect immunofluorescence assay using rabbit anti-N polyclonal antibody (green). Additionally, cells were stained with PI to visualize the nuclear DNA (red) and were examined by confocal microscopy. The two colors were then merged and yellow color when it occurs is the region where green and red co-localized. (A)–(H) represents the merged panels of indicated deletion mutants. Arrow points to the cell nucleus or to the yellow region. Magnification 40×.

Fig. 5

Co-localization of SARS–CoV N deletion mutant N226–300 with nucleolar protein B23 in the nucleolus of transfected cells using indirect immunofluorescence. Vero E6 cells grown on coverslip were transfected with mutant segment N226–300 for 24 h. The cells were fixed then double-labeled with rabbit anti-N polyclonal antibody (green) (A) and mouse monoclonal antibody to the B23 (red) (B). The cells were examined by confocal microscopy. The two colors were then merged and the yellow region in panel (C) is the area where green and red co-localized. The arrow points to the cell nucleolus. Magnification 40×.

Fig. 6

Schematic representation of a summary of wild type EGFP-N fusion protein, different deletion mutants and their respective intracellular localization. The pEGFP-N1 vector was used to express all the constructs. Dash lines and numbers indicate the deleted amino acids and amino acids positions, respectively. The bars represent translated amino acids. N: nucleus; C, cytoplasm. (+) and (−) designate for presence and absence of protein expression, respectively.

Fig. 7

Intracellular localization of EGFP fusion proteins containing segments of N protein deletion mutants. Vero cells were transfected with pEGFP-N1 vector only or with different deletion mutants fused to N-terminal of EGFP. After 24 h cells were analyzed by direct fluorescence using confocal microscopy. The panels represent the distribution of fluorescence signals in the cells transfected with EGFP, EGFP-N and deletion mutants as indicated. ((C), (F), (I), (L), (N), (Q) and (T)) are phase-contrast of the fluorescence images ((B), (E), (H), (K), (M), (P) and (S)) of the same cells, respectively. Arrow points to the cell nucleus or to the nucleolus.

Fig. 8

SARS–CoV N expression inhibits the colony formation efficiency. Vero E6 cells of constitutively expressing N protein and neor control cells (1 × 102) were seeded on 35 mm culture dishes and cultured until colonies were visually seen. Colonies were stained as described under “Section 2”. The assay was carried out in triplicate and the results were reproducible.