Intracellular localization of the severe acute respiratory syndrome coronavirus nucleocapsid protein: absence of nucleolar accumulation during infection and after expression as a recombinant protein in vero cells

Abstract

The nucleocapsid (N) protein of several members within the order Nidovirales localizes to the nucleolus during infection and after transfection of cells with N genes. However, confocal microscopy of N protein localization in Vero cells infected with the severe acute respiratory syndrome coronavirus (SARS-CoV) or transfected with the SARS-CoV N gene failed to show the presence of N in the nucleoplasm or nucleolus. Amino acids 369 to 389, which contain putative nuclear localization signal (NLS) and nucleolar localization signal motifs, failed to restore nuclear localization to an NLS-minus mutant Rev protein. These data indicate that nuclear localization is not a conserved property among all nidoviruses.

Figures

FIG. 1.

NLS motifs in the SARS-CoV N protein. Peptide sequence analysis of the SARS-CoV N protein Urbani isolate (GenBank accession no. AY278741) was performed using the Web-based program PSORT (17). The rules for defining pat4, pat7, and bipartite motifs are discussed in the introductory comments of the text.

FIG. 2.

Distribution of N in SARS-CoV-infected cells. (A) Confocal microscopy of a cluster of infected cells at 24 h after infection. Viral antigen was detected using AlexaFluor 594-labeled SA 46-4 anti-N MAb (red). Cells were counterstained with the nuclear stain TO-PRO-3 (blue). (B, C, and D) Images of a single representative infected cell at 24 h after infection. (B) AlexaFluor 594-labeled SA 46-4; (C) TO-PRO-3 staining; (D) merged image combining SA 46-4 and TO-PRO-3 staining. An 0.8-μm slice through the nucleus is shown in each image. Images were obtained with a 63× oil objective, and panels B, C, and D are magnified ×2.

FIG. 3.

Localization of SARS-CoV N-EGFP in Vero cells. (A, B, and C) Confocal microscopic images of a single cell at 24 h after transfection with pSARS-N-EGFP. (A) EGFP fluorescence; (B) TO-PRO-3 fluorescence; (C) merged image. (D) Image of a single cell transfected with pSARS-N-EGFP and exhibiting EGFP fluorescence in the nucleus. The cell was counterstained with AlexaFluor 594-labeled SA46-4 anti-N MAb (red). Below the image is a profile showing the intensity of EGFP (green line) and AlexaFluor 594 (red line) fluorescence along the axis shown by the dotted arrow in the image.

FIG. 4.

Effect of LMB on localization of SARS-N-EGFP in live cells. (A and B) Cells transfected with the control construct, pEGFP-ERev. (C and D) Cells transfected with pSARS-N-EGFP. (E and F) Cells transfected with the Rev-SARS chimeric construct pRev(NLS-minus)SARS-N (368-389). LMB, at a concentration of 10 nM, was added at 4 h after transfection. The results following LMB treatment are shown in the panels on the right. Photomicrographs were taken at 18 h after transfection.