The coronavirus infectious bronchitis virus nucleoprotein localizes to the nucleolus

Abstract

The coronavirus nucleoprotein (N) has been reported to be involved in various aspects of virus replication. We examined by confocal microscopy the subcellular localization of the avian infectious bronchitis virus N protein both in the absence and in the context of an infected cell and found that N protein localizes both to the cytoplasmic and nucleolar compartments.

FIG. 1

Vero cells were transfected with pCi-N, incubated for 24 h, fixed, and analyzed by indirect immunofluorescence using rabbit anti-IBV sera and FITC-labeled goat anti-rabbit antibodies. Additionally, cells were stained with propidium iodide (D to F) to visualize nuclear DNA. For images A to C, the fluorescing protein images were gathered from different 0.5-μm optical sections by using a confocal microscope and the appropriate filter; for images D to F, the differentially fluorescing IBV N protein (D) and nuclear DNA images (E) were gathered separately from the same 0.5-μm optical section by using a confocal microscope and appropriate filters. The two images were digitally superimposed to depict the distribution of the IBV N protein and nuclear DNA (E). Each arrow indicates the position of a nucleolus. Magnification, ×63.

FIG. 2

Vero cells were infected with IBV Beaudette at an multiplicity of infection of 1, incubated for 24 h, fixed, and stained in propidium iodide to visualize nuclear DNA. Indirect immunofluorescence using rabbit anti-IBV sera and FITC-labeled goat anti-rabbit antibodies was used to detect IBV proteins. For each image, the differentially fluorescing protein and nuclear DNA images were gathered separately from the same 0.5-μm optical section by using a confocal microscope and appropriate filters. The arrow indicates the position of a nucleolus. Magnification, ×63.

FIG. 3

Vero cells were transfected with pN-GFP (A to D) or pGFP (E and F) and incubated for 24 h; IBV N protein was detected by indirect immunofluorescence using rabbit anti-IBV sera and anti-rabbit Alexa Fluor 564. The differentially fluorescing IBV N protein (A) and GFP (B) images were gathered separately from the same 0.5-μm section and from different sections (D to F) by using a confocal microscope and appropriate filters; the two images were digitally superimposed (C to F). Each arrow indicates the position of a nucleolus. Magnification, ×63.

FIG. 4

(A) Amino acid sequence alignments to identify a potential NuLS and an RBS on the IBV Beaudette strain N protein, using the PRRSV NuLS (26) and eastern equine encephalitis virus (EEEV) RBS (37) as a basis for comparison. Locations of the two potential motifs on the IBV N protein are also shown (B). Similar amino acids are shown in boldface; the start position of the appropriate amino acid on the full-length protein is indicated in parentheses.