Adenovirus protein V induces redistribution of nucleolin and B23 from nucleolus to cytoplasm

Abstract

Adenovirus infection inhibits synthesis and processing of rRNA and redistributes nucleolar antigens. Adenovirus protein V associates with nucleoli in infected cells. This study delineates regions of protein V independently capable of nucleolar targeting. Also, evidence is presented that protein V has the unique property of relocating nucleolin and B23 to the cytoplasm when transiently expressed on its own in uninfected cells. Point mutation analysis indicates a role for the C terminus of protein V in the redirection of nucleolin and B23 to the cytoplasm. This is the first time an adenovirus protein has been shown to have a direct effect on nucleolar antigens in isolation from viral infection. Moreover, adenovirus protein V is the first protein demonstrated to be capable of redirecting nucleolin and B23 to the cytoplasm.

FIG. 1

Identification of regions of protein V involved in nuclear and nucleolar targeting. (A) Schematic showing the regions of protein V fused to EGFP. On the left, each clone is identified by the amino acids from the full-length protein V sequence expressed N terminal to the EGFP sequences. On the right, the subcellular distribution of each fusion protein is indicated as No. (nucleolar), Np. (nucleoplasmic), Cyt. (cytoplasmic) or diffuse (distribution throughout the cell, with no particular subcellular localization). (B) Sequences at the N and C termini of protein V which are capable of independently directing EGFP to the nucleus and nucleolus. (C) Subcellular localization of five representative fusion proteins. The images represent the five patterns of fluorescence seen in these experiments. All images were scanned from slide film and labeled using Adobe Photoshop 4.0.

FIG. 2

Protein V induces relocalization of nucleolin and B23. In all color images, V-EGFP stains green, DAPI stains blue, and nucleolin or B23 is in red. Images A and B represent 20- by 0.36-μm focal planes which start below and end above the cells. They are superimposed so that all layers of the cell can be visualized simultaneously. The scale bars in images A and B represent 10 μm. (A) Distribution of nucleolin (red) in cells expressing various levels of V-EGFP. The cell marked with an arrowhead shows expression of low levels of protein V. (B) Distribution of B23 (red) in cells expressing various levels of V-EGFP. (C) A 0.36-μm section (approximately halfway through the cell) of the cell marked with an arrow in image B. (D) Close-up of the nucleolus marked with an arrow in image C, with DAPI staining deleted for clarity. The diameter of the large nucleolus is approximately 3 μm. Images C and D were annotated by Adobe Photoshop 4.0. (E) Deletion mutants from Fig. 1, assessed for redistribution of nucleolin and B23. Text on the right indicates whether the mutant could relocate B23 and nucleolin to the cytoplasm (N.D., not determined). (F) Amino acid sequences of the C-terminal seven amino acids of protein V from Ad2 along with the analogous C-terminal sequences of protein V from canine Ad1, murine Ad1, human Ad40, and human Ad12. (G) The three point mutants of protein V which were generated by PCR. Each fusion expressed amino acids 1 to 369 (full-length protein V) fused to EGFP but with a single amino acid alteration as indicated. Abilities of mutants to redirect nucleolin or B23 to the cytoplasm are indicated on the right. (H) Total protein extracts from cells transfected with the point mutants described in panel G. Expression of the mutants and V-EGFP was assayed by Western blotting using rabbit polyclonal antiserum against protein V. Also included is a mock transfection. Duplicate transfections were assayed to confirm the data presented in panel G.

FIG. 3

rRNA synthesis is not affected by V-EGFP, nor does inhibition of RNA synthesis affect the ability of V-EGFP to relocalize nucleolin to the cytoplasm. (A to C) Cells, some expressing V-EGFP, in which RNA synthesis has been detected by a standard in situ assay as outlined in the text. (A) Cells expressing V-EGFP; (B) phase-contrast image of the same cells (the dark nucleoli are clearly visible); (C) nucleoli that fluoresce due to the detection of Br-UTP incorporated into nascent RNA. (D to F) HeLa cells transfected with V-EGFP and then treated with actinomycin D to inhibit RNA synthesis. (D) V-EGFP expression pattern; (E) DAPI-stained cells; (F) distribution of nucleolin. Slides were scanned and annotated by Adobe Photoshop 4.0.

FIG. 4

Adenovirus infection causes redistribution of nucleolin to the cytoplasm, but B23 is redistributed within the nucleus at 18 h postinfection. All images show adenovirus-infected HeLa cells at 18 h postinfection. (A) Polyclonal anti-V serum reveals the accumulation of protein V (note that cells at the bottom left express lower levels of protein V); (B) the same field stained with nucleolin MAb; (C) DAPI staining. Images A and B are overexposed to allow visualization of the faint staining of cells with protein V (A) or nucleolin (B). (D) Polyclonal anti-V serum reveals the accumulation of protein V in one cell; (E) the same field stained with B23 MAb; (F) DAPI staining. Images D to F are higher magnifications of images A to C to clearly show the patterns of B23 and protein V localization in the infected nucleus. Slides were scanned and annotated by Adobe Photoshop 4.0.