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. 2010 Oct;84(19):9718-32.
doi: 10.1128/JVI.00713-10. Epub 2010 Jul 21.

Bub1 and CENP-F can contribute to Kaposi's sarcoma-associated herpesvirus genome persistence by targeting LANA to kinetochores

Bingyi Xiao  1 , Subhash C Verma, Qiliang Cai, Rajeev Kaul, Jie Lu, Abhik Saha, Erle S Robertson
Affiliations

Bub1 and CENP-F can contribute to Kaposi's sarcoma-associated herpesvirus genome persistence by targeting LANA to kinetochores

Bingyi Xiao et al. J Virol. 2010 Oct.

Abstract

The latency-associated nuclear antigen (LANA) encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) is critical for segregation of viral episomes to progeny nuclei and allows for maintenance of the viral genome in newly divided daughter cells. LANA binds to KSHV terminal repeat (TR) DNA and simultaneously associates with chromatin-bound cellular proteins. This process tethers the viral episomes to host chromosomes. However, the mechanism of tethering is complex and involves multiple protein-protein interactions. Our previous proteomics studies which showed the association of LANA with centromeric protein F (CENP-F) prompted us to further study whether LANA targets centromeric proteins for persistence of KSHV episomes during cell division. Here we show that LANA colocalized with CENP-F as speckles, some of which are paired at centromeric regions of a subset of chromosomes in KSHV-infected JSC-1 cells. We also confirm that both the amino and carboxy termini of LANA can bind to CENP-F. Moreover, LANA associated with another kinetochore protein, Bub1 (budding uninhibited by benzimidazole 1), which is known to form a complex with CENP-F. Importantly, we demonstrated the dynamic association of LANA and Bub1/CENP-F and the colocalization between Bub1, LANA, and the KSHV episome tethered to the host chromosome using fluorescence in situ hybridization (FISH). Knockdown of Bub1 expression by lentivirus-delivered short hairpin RNA (shRNA) dramatically reduced the number of KSHV genome copies, whereas no dramatic effect was seen with CENP-F knockdown. Therefore, the interaction between LANA and the kinetochore proteins CENP-F and Bub1 is important for KSHV genome tethering and its segregation to new daughter cells, with Bub1 potentially playing a more critical role in the long-term persistence of the viral genome in the infected cell.

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Figures

FIG. 1.
FIG. 1.
Endogenous LANA colocalizes with CENP-F and associates with kinetochores in KSHV-positive JSC-1 cells. (A) JSC-1 cells were immunostained for CENP-F (green) and LANA (red), and nuclei were stained with DAPI (blue). The merged image of these two proteins showed distinct colocalization of CENP-F and LANA in JSC-1 cells as bright yellow dots. (B) LANA protein associates with centromeres on a metaphase chromosome spread. Immunolocalization of LANA protein (red) and centromeric ACA antigen (green) on a metaphase chromosome spread of JSC-1 cells is shown. (C) Quantification of the numbers of LANA and ACA dots and their colocalization dots in a single chromosome spread. Twenty cells was counted.
FIG. 2.
FIG. 2.
Full-length LANA binds with the amino and carboxy termini of CENP-F. (A) Schematic illustration of CENP-F full-length protein and the individual truncations used to map the LANA binding domain. (B) The cell lysates from 20 million BC-3 cells were subjected to immunoprecipitation with anti-LANA antibody, followed by SDS-PAGE and immunoblotting with anti-CENP-F antibody. (C) The cell lysates from HEK293 cells transfected with LANA-Flag were incubated with purified GST-truncated CENP-F fusion proteins. Precipitated proteins were resolved by SDS-PAGE and detected with mouse anti-Flag antibody. (D) 35S-labeled LANA was prepared using the TNT-T7 quick-coupled transcription-translation system and incubated with GST or GST-truncated CENP-F in binding buffer, and bound proteins were resolved by SDS-10% PAGE. They were detected by autoradiography. (E) Fifteen million HEK293 cells were transfected with 15 μg of pA3F-LANA and 15 μg of GFP-Myc-NLS-CENP-F-truncation expression plasmids as indicated. The cells were harvested at 36 h after transfection and immunoprecipitated with 1 μg of anti-Myc antibody. Samples were resolved on SDS-PAGE and immunoblotted with the indicated antibody to detect LANA and CENP-F.
FIG. 3.
FIG. 3.
Both the N-terminal domain of LANA (residues 1 to 340) and the LANA C terminus (residues 842 to 1162) bind to CENP-F. (A) A co-IP assay for LANA (residues 1 to 340) and CENP-F truncation interaction was conducted using cell lysates containing overexpressed N-LANA (residues 1 to 340) and CENP-F truncation proteins. Coimmunoprecipitated LANA was determined by immunoblotting using anti-Flag antibody. (B and C) Purified GST-CENP-F truncation fusion proteins were incubated with overexpressed N-terminal LANA (B) or 35S-radiolabeled in vitro-translated LANA residues 1 to 340 (C) for GST pulldown assays. The pulldown of N-LANA was detected by immunoblotting using anti-Flag antibody or autoradiography. (D to F) C-terminal LANA (residues 842 to 1162) is sufficient for interaction with CENP-F. GST-tagged CENP-F was incubated with the cell lysates from HEK293 cells transfected with C-LANA-Flag (D) or 35S-radiolabeled in vitro-translated LANA residues 842 to 1162 (E) in a GST pulldown assay. The presence of C-LANA in the protein complex was detected by immunoblotting using anti-Flag antibody (D) or autoradiography (E). (F) The cell lysates from HEK293 cells transfected with pA3F-C-LANA and truncated CENP-F expression plasmids were immunoprecipitated with anti-Flag antibody. The co-IP protein was determined by immunoblotting.
FIG. 4.
FIG. 4.
LANA can interact with Bub1. (A) Fifteen million HEK293 cells were transfected with pA3F-LANA and GFP-Bub1 constructs. The cells were harvested at 36 h after transfection and were immunoprecipitated with 1 μg of anti-Flag antibody. Samples were resolved on SDS-PAGE and immunoblotted with anti-Flag antibody to detect LANA and Bub1 antibody. (B and C) Twenty million JSC-1 cells (B) or BC-3 cells (C) were lysed, and cell exacts were incubated with LANA monoclonal antibody. Precipitated proteins were resolved by SDS-PAGE and transferred to a nitrocellulose membrane, followed by detection with Bub1 antibody. (D) Purified GST and GST-LANA-N and GST-LANA-C fusion proteins were incubated with cell extracts from HEK293 cells. The precipitated proteins were resolved by SDS-PAGE and detected with Bub1 antibody.
FIG. 5.
FIG. 5.
LANA colocalizes with Bub1 on host mitotic chromosomes in KSHV-infected cells. JSC-1 cells (A and B) and BC-3 cells and BCBL-1 cells (C) were assayed for Bub1/LANA (A and C) or Bub1/EBNA1 (B) colocalization by indirect immunofluorescence. Cells were also counterstained with DAPI to identify nuclei and mitotic chromosomes. Colocalization of Bub1 and LANA proteins appears as yellow in the merged images as shown by the greater than 50% overlapping dots. Numbers indicate the percentage of LANA protein dots which colocalized with Bub1 proteins. ND, no detection of colocalization.
FIG. 6.
FIG. 6.
Analysis of the kinetics of LANA interaction with CENP-F and Bub1 during specific phases of the cell cycle. Cells were stained with an anti-CENP-F goat polyclonal antibody, the anti-LANA monoclonal antibody, and an anti-Bub1 rabbit polyclonal antibody. The staining was detected by incubation with an Alexa Fluor 488 chicken anti-goat IgG, an Alexa Fluor 594 donkey anti-mouse IgG, and an Alexa Fluor 647 donkey anti-rabbit IgG. Cells were also counterstained with DAPI to identify nuclei and mitotic chromosomes. (A) Colocalization of CENP-F, Bub1, and LANA in JSC-1 cells was distinctly observed on mitotic chromosomes as bright white dots. (B) Representation of a confocal XYZ stack of JSC-1 cells with LANA, CENP-F, and Bub1 staining. (C) Kinetics of LANA (red) association with CENP-F (green) and Bub1 (light blue) during specific phases of the cell cycle in BC-3 cells. Although LANA associated with CENP-F from interphase to metaphase, LANA maintained its interaction with Bub1 throughout the cell cycle.
FIG. 7.
FIG. 7.
Bub1 associates with the KSHV genome on host chromosomes. JSC-1 cells were hybridized with biotinylated KSHV LANA cDNA probe, followed by incubation with specific antibodies individually against LANA and Bub1. The staining was detected by incubation with Alexa Fluor 488 donkey anti-rabbit IgG, Alexa Fluor 594 streptavidin conjugate, and Alexa Fluor 647 donkey anti-mouse IgG. Cells were also counterstained with DAPI.
FIG. 8.
FIG. 8.
Disruption of normal Bub1 function significantly reduces maintenance of KSHV episomes. (A) JSC-1 cells were transduced with lentivirus containing specific (sh-Bub1) and control (sh-luc) shRNA. Genomic DNA and cell lysates from sh-control and sh-Bub1 JSC-1 cells were extracted at 1, 2, 3, 4, and 5 days after puromycin selection. Relative copy numbers of TR in a representative experiment in these samples were plotted, and Bub1 protein levels were also shown by immunoblotting. (B) Western blots showed expression of Bub1 and LANA in JSC-1-sh-control, and JSC-1-sh-Bub1 stable cell lines. Beta-actin was used as the loading control. (C) Immunofluorescence assay of Bub1 and LANA in shRNA-treated JSC-1 cells showed a significant knockdown of Bub1 levels in the Bub1 shRNA-treated cells compared with control cells. LANA proteins were also dramatically reduced in the Bub1 shRNA-treated cells. (D) The number of LANA protein dots was counted in a single cell, which was represented by a symbol on the scatter plot, and 40 cells in each group were quantitated. The Bub1-knocked-down cells showed significantly reduced LANA protein staining, but depletion of CENP-F did not significantly affect the LANA protein level (C and D). (E) Genomic DNA from JSC-1-sh-control, JSC-1-sh-Bub1, and JSC-1-sh-CENP-F stable cell lines were extracted, and the number of KSHV episome copies was calculated by real-time qPCR with the terminal repeat as the target for amplification. Bub1 shRNA-treated JSC-1 cells showed about an 80% decrease in viral genome copy numbers, but depletion of CENP-F did not significantly affect the number of KSHV genomic copies. (F) Western blots showed protein levels of Bub1 and LANA, and KSHV episome copies were calculated by real-time qPCR in BC-3-sh-bub1, and BCBL-1-sh-Bub1 cell lines. (G) Stable cell lines were harvested and fixed with ethanol, and cell cycle progression was analyzed by flow cytometry. DNA content was quantified by staining the cells with propidium iodide (PI). Numbers indicate the percentage of cells in the cell cycle.
FIG. 9.
FIG. 9.
Proposed model of KSHV genome tethering to the host chromosome during mitosis. LANA most likely tethers the KSHV viral episomes to host chromosomes through binding of LANA to Bub1 in a complex with CENP-F, NuMA, and other cellular proteins. Once the cell enters anaphase, LANA association with CENP-F, NuMA, and others may be lost, but it can maintain its association with Bub1 throughout mitosis. Thus, this is likely to be critical for segregation of KSHV episomes to daughter cells.
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