Changes in nucleolar morphology and proteins during infection with the coronavirus infectious bronchitis virus

Abstract

The nucleolus is a dynamic subnuclear structure involved in ribosome subunit biogenesis, cell cycle control and mediating responses to cell stress, among other functions. While many different viruses target proteins to the nucleolus and recruit nucleolar proteins to facilitate virus replication, the effect of infection on the nucleolus in terms of morphology and protein content is unknown. Previously we have shown that the coronavirus nucleocapsid protein will localize to the nucleolus. In this study, using the avian infectious bronchitis coronavirus, we have shown that virus infection results in a number of changes to the nucleolus both in terms of gross morphology and protein content. Using confocal microscopy coupled with fluorescent labelled nucleolar marker proteins we observed changes in the morphology of the nucleolus including an enlarged fibrillar centre. We found that the tumour suppressor protein, p53, which localizes normally to the nucleus and nucleolus, was redistributed predominately to the cytoplasm.

Figure 1

A. Confocal analysis of the subcellular localization of EGFP‐nucleolin (green) and DsRed‐B23 (red) in mock‐infected cells, and the morphology of the nucleolus as revealed by transmission phase contrast microscopy (labelled transmission).
B. Distribution of EGFP (green) in mock and IBV‐infected cells (red). The nucleolus (No), fibrillar centre (FC), dense fibrillar component (DFC) and nuclear envelope (NE) are indicated. Size bar is 10 µm.

Figure 2

Confocal microscopy analysis of the distribution of DsRed‐B23 in cells infected with IBV at 12, 18 and 24 h pi. Merged images are presented as well as individual images for the detection of IBV proteins (green) and DsRed‐B23 (red). The transmission phase contrast images (labelled TPC) of the cells are shown below. Various features are indicated for orientation, examples of nucleoli are indicated (No), the fibrillar centre (FC) and nuclear envelope (NE). Size bar is 10 µm.

Figure 3

A. Confocal analysis of the distribution of EGFP‐nucleolin (green) in IBV (red)‐infected cells at 24 h pi; separate images are shown for each label and the merged image.
B. Z‐sections were taken through the nucleus of the cell shown in (A) in 1.00 µm increments. Merged images are shown.
C. Confocal images of an infected cell (red) at 24 h pi expressing EGFP‐nucleolin (green) showing an enlarged FC, separate images are shown for each label and the merged image.
D. Confocal and transmission phase contrast of a nucleolus in mock‐infected cells expressing EGFP‐nucleolin (green).
E. The diameter of the FC was measured using the intensity profile of EGFP‐nucleolin (green) across the nucleolus. Both merged and fluorescent profiles are presented for different cells exhibiting the large FC phenotype. Various features are indicated for orientation, examples of nucleoli are indicated (No), the fibrillar centre (FC) and nuclear envelope (NE). Size bar is 10 µm.

Figure 4

A. Western blot analysis of consecutive stages in the preparation of nucleolar extracts. Nucleolin is used as a marker for the nucleolus and Lamin B as a maker of the nucleus and nucleoplasm.
B. Top image, Coomassie stained acrylamide gel of nucleolar extracts from IBV and mock‐infected cells separated on a 10% NuPage Bis‐Tris precast polyacrylamide gel (Invitrogen) in MOPs running buffer. Arrows indicated both novel protein species (1) and reduced cellular proteins (2) in the nucleolar extracts from infected and mock‐infected cells respectively. Below are shown Western blots for N protein and nucleolin.
C. Live cell imaging of cells expressing EGFP‐IBV N protein. The nucleolus is indicated (No).

Figure 5

A. Distribution of p53 in mock‐infected cells.
B. Confocal images of cells expressing IBV proteins (green) and showing the distribution of p53 (red) and merged at 24 h pi.
C. Enlarged images of IBV‐infected cells with IBV proteins labelled in green and p53 in red at 24 h pi. Transmission phase contrast (TPC) of the lower cell is shown below. Nucleoli are indicated (No) and the size bar is 10 µm.
D. Western blot analysis of p53 in mock and infected cells at 0 and 24 h pi. Protein concentrations were standardized and compared with GAPDH.

Figure 6

A. Confocal analysis to investigate the potential colocalization of p53 (red) in cells expressing EGFP‐IBV N protein (green).
B and C. Confocal analysis of the distribution of IBV proteins (blue), EGFP‐IBV N protein (green) and p53 (red) in IBV‐infected cells at 24 h pi. Colocalization between EGFP‐IBV N protein plus p53, EGFP‐IBV N protein plus IBV proteins, p53 plus IBV proteins, and between the three complexes, appears yellow, cyan, purple and white respectively. The nucleolus is indicated (No). Size bar is 10 µm.

Figure 7

A. Confocal analysis of cells expressing EGFP‐IBV N protein (green) showing low levels of protein in the nucleus but not nucleolus, which has been stained with PI (red), at 24 h post transfection. Separate channels are shown as well as the merged image. The right‐hand images are enhanced magnification images of sections of the nuclear/cytoplasmic boundary showing the nucleoplasm (Np), nucleolus (No) and nuclear envelope (NE).
B. Upper images are examples of differential stained nucleoli in cells expressing EGFP‐IBV N protein (green) and PI to stain the nucleolus (red), and merged. The lower image shows the apparent circumferences of the nucleoli when visualized using EGFP and PI.
C. Orthogonal reconstruction through the cell in (B) by Z‐sectioning. The orthogonal section corresponds to the vertical bar in the lower left‐hand cell shown in (B).
D. Z‐sectioning through the nucleus/nucleolus in a cell expressing ECFP‐NR1+2 and DsRed‐B23 at 24 h post transfection. Also shown is the apparent maximum diameter (indicated) of the FC using the nucleolus shown in the 2.92 µm section. The nucleolus, nucleoplasm and nuclear envelope are indicated No, NP and NE respectively. The horizontal size bar is 10 µm.