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First published online 3 April 2007
doi: 10.1242/jcs.03434

Journal of Cell Science 120, 1529-1539 (2007)
Published by The Company of Biologists 2007
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Symmetrical localization of extrachromosomally replicating viral genomes on sister chromatids

Teru Kanda1,*, Masato Kamiya1,2, Seiji Maruo2, Dai Iwakiri2 and Kenzo Takada2

1 Center for Virus Vector Development, Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815, Japan
2 Department of Tumor Virology, Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815, Japan


Figure 1
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  		Fig. 1. Characterization of cells harboring the recombinant EBV expressing HA-tagged EBNA1. (A) Expression of HA-tagged EBNA1 protein in the cells that were infected with the recombinant EBV. Whole cell extracts of Akata cells harboring naturally infected EBV (lane 1, referred to as wild-type EBV hereafter) and four independent cell clones harboring the recombinant EBV (lanes 2-5) were analyzed by western blot analyses. EBV-immune human serum (left panel) and anti-HA antibody (right panel) were used as primary antibodies. (B) Detection of EBV genomes by FISH analyses. Akata cells harboring wild-type EBV (top panels) or cells harboring the recombinant EBV (bottom panels) were processed for FISH analyses before (left panels) and after 5 weeks of hydroxyurea (HU) treatment (right panels). An EBV genome-derived FISH probe was used for the analyses. Note that the majority of HU-treated cells are free of FISH signals (right panels). (C) EBV copy number estimation by Southern blotting. Akata cells harboring wild-type EBV or the recombinant virus were treated with HU for 5 weeks. Genomic DNAs were prepared before (lanes 1 and 4) and after HU treatment [either after 3 weeks (3 wk) or 5 weeks (5 wk) of treatment]. The genomic DNAs (10 µg) were digested by NcoI and analyzed by Southern blotting using a part of BamHI-C fragment (EcoRI-BamHI fragment) as a probe. The NcoI fragments of 21-kb size are shown. The copy number controls, which were prepared from a BAC clone DNA of the EBV genome, are also indicated.

 

Figure 2
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  		Fig. 2. Subnuclear localization of HA-tagged EBNA1 protein. (A) Cells were processed for immunofluorescence analyses with anti-HA antibody. Images of interphase nuclei counterstained with DAPI (left panel) and anti-HA antibody staining (middle panel) are shown. The merged image is also shown (right panel). (B) Punctate signals of EBNA1 overlap the signals of EBV genomes in the recombinant EBV-infected cells. A combined method of FISH and immunofluorescence was used to visualize EBV genomes (FISH) and HA-tagged EBNA1 simultaneously. Merged images of FISH and immunofluorescence signals are also shown. The images of wild-type EBV-infected cells subjected to the same type of analysis are also shown as controls (bottom). Bars, 5 µm.

 

Figure 3
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  		Fig. 3. Localization of fluorescent dots of EBNA1 on mitotic chromosomes. (A) Cells were processed for immunofluorescence analyses with anti-HA antibody. Chromosomes are counterstained with DAPI. Note that fluorescent dots of EBNA1 (red) associate with prophase (left panel) and anaphase (right panel) chromosomes. (B) Dual-color immunofluorescence analyses to visualize the localization of EBNA1 (red) and CENP-C (green) on mitotic chromosome spreads of hyptonically swollen cells. Higher magnification images demonstrating paired EBNA1 dots symmetrically localizing on sister chromatids are shown in the accompanying sub-panels. (C) Simultaneous FISH and immunofluorescence to visualize EBV genomes (FISH) and HA-tagged EBNA1 on a mitotic chromosome spread. Merged images of FISH and immunofluorescence signals are also shown. Arrows indicate symmetrical localization on sister chromatids. Bars, 5 µm. (D) Distribution of EBV genomes on mitotic chromosome spreads of Akata cells. EBV genomes were detected by FISH analyses with an EBV genome-derived FISH probe. Chromosomes were counterstained by propidium iodide. EBV genomes (yellow signals) that symmetrically localize on sister chromatids are indicated by arrowheads. Bars, 5 µm.

 

Figure 4
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  		Fig. 4. Localization of EBNA1 dots in G2-phase-enriched cells. (A) Cells harboring the recombinant EBV were synchronized at the G1-S boundary by first thymidine and then aphidicolin block. Cells were then released by removing aphidicolin, and cell cycle progression was monitored by FACS analyses. (B) Immunofluorescence images (stained by anti-HA antibody) of two representative interphase cells that were harvested at 6 hours after release. Closely spaced double-dots of EBNA1 (red signals) are indicated by arrowheads. (C) Immunofluorescence images of two representative G2-PCCs (out of more than 100 acquired images of G2-PCCs). Calyculin A-treated cells were harvested at 6 hours after release. Note that there are many symmetrical double dots on sister chromatids (arrowheads). (D) Quantitative analysis to determine the frequency of symmetrical EBNA1 doublets in individual cells. The signals of EBNA1 (red) and CENP-C (green) are shown. Number of paired EBNA1 dots with paired CENP-C dots are assumed to be symmetrically localized EBNA1 dots (surrounded by a red rectangle). Number of symmetrically localized EBNA1 dots are indicated in each sub-panel. Additional EBNA1 double dots that do not fit the above criteria are also shown. Question marks indicate obscure CENP-C staining. Bars, 5 µm.

 

Figure 5
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  		Fig. 5. Simultaneous visualization of EBNA1 and EBV episomes in G2-phase-enriched cells. (A) Simultaneous FISH and immunofluorescence images of two interphase cells (out of more than 50 acquired images of interphase nuclei harboring EBNA1 doublets). Cells were harvested at 6 hours after release and processed for simultaneous FISH and immunofluorescence. The signals of EBV episomes (green signals revealed by FISH) and those of EBNA1 protein (red signals revealed by immunofluorescence) are shown as merged images. Chromosomes are counterstained with DAPI (left panels). Higher magnification images of the EBNA1 double-dots, merged with the signals of EBV episomes, are shown in the sub-panels (1-6). Scale bars are as indicated. (B) Simultaneous FISH and immunofluorescence images of two representative G2-PCCs (out of more than 20 acquired images of G2-PCCs). Higher magnification images of the EBNA1 double-dots, merged with the signals of EBV episomes, are also shown in the accompanying sub-panels. Scale bars are as indicated.

 

Figure 6
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  		Fig. 6. Dumbbell-like structures in G2-arrested cells treated with a topoisomerase II inhibitor. (A) Cells harboring the recombinant EBV were synchronized at the G1-S boundary and then released. Cell cycle progression, in the presence or absence of ICRF-193, was monitored by FACS analyses. (B) A representative image (out of more than 20 acquired images) of ICRF-193-treated cells (10 hours after release) that were processed for simultaneous FISH and immunofluorescence. Note many dumbbell-like structures consisting of EBNA1 doublets (red) and EBV genome signals (green). Merged images are also shown. Bar, 5 µm.

 

Figure 7
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  		Fig. 7. Model of EBV episome segregation onto sister chromatids. Clusters of EBNA1 molecules mediate noncovalent association of EBV episomes with cellular chromatin. In S-phase cells, approximately equal amounts of EBNA1 molecules are distributed to sister chromatids while cellular and viral replication are synchronously replicating. Replicated sister EBV genomes are initially catenated, and then get decatenated by the action of topoisomerase II (topo II). Asymmetrically localized EBV episomes in mitotic cells could also be segregated symmetrically during S phase, although dynamic movement of EBV episomes in interphase nuclei is speculative.

 

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© The Company of Biologists Ltd 2007