Localization to the nucleolus is a common feature of coronavirus nucleoproteins, and the protein may disrupt host cell division

Abstract

The subcellular localization of transmissible gastroenteritis virus (TGEV) and mouse hepatitis virus (MHV) (group I and group II coronaviruses, respectively) nucleoproteins (N proteins) were examined by confocal microscopy. The proteins were shown to localize either to the cytoplasm alone or to the cytoplasm and a structure in the nucleus. This feature was confirmed to be the nucleolus by using specific antibodies to nucleolin, a major component of the nucleolus, and by confocal microscopy to image sections through a cell expressing N protein. These findings are consistent with our previous report for infectious bronchitis virus (group III coronavirus) (J. A. Hiscox et al., J. Virol. 75:506-512, 2001), indicating that nucleolar localization of the N protein is a common feature of the coronavirus family and is possibly of functional significance. Nucleolar localization signals were identified in the domain III region of the N protein from all three coronavirus groups, and this suggested that transport of N protein to the nucleus might be an active process. In addition, our results suggest that the N protein might function to disrupt cell division. Thus, we observed that approximately 30% of cells transfected with the N protein appeared to be undergoing cell division. The most likely explanation for this is that the N protein induced a cell cycle delay or arrest, most likely in the G(2)/M phase. In a fraction of transfected cells expressing coronavirus N proteins, we observed multinucleate cells and dividing cells with nucleoli (which are only present during interphase). These findings are consistent with the possible inhibition of cytokinesis in these cells.

FIG. 1

Indirect detection of TGEV (A and B) and MHV (C and D) N proteins in transfected cells and the IBV (E) N protein in transduced cells by immunofluorescence. LLC-PK1 cells (A), L cells (C), and Vero cells (B and D) were transfected with either pCi-TGEV-N or pCi-MHV-N or were transduced with BacIBVN (E). They were incubated for 24 h, fixed, and analyzed by indirect immunofluorescence using appropriate antibodies (green) (see text). Additionally, cells were stained with PI to directly visualize nuclear DNA (red). Differentially fluorescing images were gathered separately from the same 0.5-μm-thick optical section by using a confocal microscope and the appropriate filter. The two images were digitally superimposed to depict the distribution of the appropriate coronavirus N protein and nuclear DNA. Arrow, position of a nucleolus. Magnifications, ×62 (A to D) and ×16 (2.73 zoom) (E).

FIG. 2

Detection of TGEV N proteins by indirect immunofluorescence in transfected cells. Vero cells were transfected with pCi-TGEV-N, incubated for 24 h, fixed, and analyzed by indirect immunofluorescence using appropriate antibodies (green) (see text). Additionally, cells were stained with PI to visualize nuclear DNA (red). Differentially fluorescing images were gathered separately from the same 0.5-μm-thick optical sections by using a confocal microscope and the appropriate filter. The two images were digitally superimposed to depict the distribution of TGEV N protein and nuclear DNA (A). Arrow, position of a nucleolus. (B) The confocal microscope was used to take 0.1-μm-thick sections of the cell shown in panel A. The section on the top left is nearest the coverslip, and the section on the bottom right is nearest the media. Magnification, ×62 (and ×2 zoom).

FIG. 3

Detection of MHV N protein by indirect immunofluorescence in transfected cells. HeLa cells were transfected with pCi-MHV-N, incubated for 24 h, and analyzed by indirect immunofluorescence using appropriate antibodies to detect the N protein (red) and nucleolin (green) (see text). Differentially fluorescing images were gathered separately from the same 0.5-μm-thick optical section by using a confocal microscope and the appropriate filter. The two images (A and B) were digitally superimposed to depict the distribution of the MHV N protein and nucleolin (C). Yellow indicates colocalization. Magnification, ×61.

FIG. 4

Detection of MHV N proteins by indirect immunofluorescence in transfected cells. Vero cells were transfected with pCi-MHV-N, incubated for 24 h, fixed, and analyzed by indirect immunofluorescence using appropriate antibodies (green) (see text). Additionally, cells were stained with PI to visualize nuclear DNA (red). Differentially fluorescing images were gathered separately from the same 0.5-μm-thick optical sections by using a confocal microscope and the appropriate filter. The two images were digitally superimposed to depict the distribution of the MHV N protein and nuclear DNA. Squares, cells in which the MHV N protein localized to the cytoplasm; circles, cells where the MHV N protein localized to both the cytoplasm and the nucleolus. Magnification, ×16.

FIG. 5

Detection of TGEV N proteins (A, B, and E) and influenza B virus NP (F) by indirect immunofluorescence and detection of EGFP (D) (all green) and cell nuclei (A to F) (red) by direct immunofluorescence. Vero cells were transfected with either pCi-TGEV-N, pCDNA3-NP, or pEGFP, incubated for 24 h, fixed, and analyzed by indirect immunofluorescence using appropriate techniques (see text). Additionally, cells were stained with PI to directly visualize nuclear DNA. Differentially fluorescing images were gathered separately from the same 0.5-μm-thick optical sections by using a confocal microscope and the appropriate filter. The two images were digitally superimposed to depict the distribution of the appropriate protein and nuclear DNA. Magnification, ×62.