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Comparative Study
. 2008 Jul;82(13):6734-46.
doi: 10.1128/JVI.00342-08. Epub 2008 Apr 16.

Kaposi's sarcoma-associated herpesvirus-encoded LANA can interact with the nuclear mitotic apparatus protein to regulate genome maintenance and segregation

Huaxin Si  1 , Subhash C Verma, Michael A Lampson, Qiliang Cai, Erle S Robertson
Affiliations
Comparative Study

Kaposi's sarcoma-associated herpesvirus-encoded LANA can interact with the nuclear mitotic apparatus protein to regulate genome maintenance and segregation

Huaxin Si et al. J Virol. 2008 Jul.

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) genomes are tethered to the host chromosomes and partitioned faithfully into daughter cells with the host chromosomes. The latency-associated nuclear antigen (LANA) is important for segregation of the newly synthesized viral genomes to the daughter nuclei. Here, we report that the nuclear mitotic apparatus protein (NuMA) and LANA can associate in KSHV-infected cells. In synchronized cells, NuMA and LANA are colocalized in interphase cells and separate during mitosis at the beginning of prophase, reassociating again at the end of telophase and cytokinesis. Silencing of NuMA expression by small interfering RNA and expression of LGN and a dominant-negative of dynactin (P150-CC1), which disrupts the association of NuMA with microtubules, resulted in the loss of KSHV terminal-repeat plasmids containing the major latent origin. Thus, NuMA is required for persistence of the KSHV episomes in daughter cells. This interaction between NuMA and LANA is critical for segregation and maintenance of the KSHV episomes through a temporally controlled mechanism of binding and release during specific phases of mitosis.

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Figures

FIG. 1.
FIG. 1.
The carboxy terminus of LANA binds with NuMA. (A) Schematic showing the domains of the LANA protein. LANA contains two nuclear localization sequences (NLS), a proline-rich domain (P-rich) and a glutamine-rich repeat region (Q-rich), an acidic domain (AD), and a putative leucine zipper (L-Zipper). C-terminal LANA mediates TR DNA binding, Brd2, MeCP2, pRb binding, and chromosome association. Brd2, bromodomain containing 2. (B) GST, GST-LANA-N, and GST-LANA-C fusion proteins were expressed in Escherichia coli and purified with glutathione-Sepharose beads. Nuclear extracts from BCBL-1 cells were incubated with either GST control or GST-LANA truncations normalized by Coomassie staining. The precipitated proteins were resolved by SDS-PAGE and detected with goat anti-NuMA antibody. In each case, 5% of the cell lysates were used as input for comparison. PC, precleared fraction. (C) Twenty million HEK293T cells were transfected with 20 μg of GFP-NuMA and 20 μg of either pA3M-LANA-C or pA3M-LANA-N expression plasmid. The cells were harvested at 36 h posttransfection, and the lysates were immunoprecipitated (IP) with 1 μg of anti-Myc antibody. Samples were resolved on SDS-6% PAGE and probed with anti-NuMA antibody. LANA truncations were detected with 9E10 Myc hybridoma supernatant. (D) Twenty million HEK293T cells were transfected with 20 μg of GFP-NuMA and 20 μg of pA3M-LANA expression plasmids as indicated. The cells were harvested at 36 h and were immunoprecipitated with 1 μg of anti-Myc antibody. Samples were resolved on SDS-6% PAGE and immunoblotted with anti-NuMA antibody to detect NuMA and 9E10 Myc hybridoma for LANA. (E) Fifty million KSHV-positive and negative BCBL1 and BJAB cells, respectively, were harvested and immunoprecipitated with 3 μl of polyclonal anti-NuMA antibody. Reverse immunoprecipitations were performed with 3 μl of rabbit anti-LANA antibody. LANA and NuMA were detected using the respective antibodies. (F) Twenty million HEK293T cells were transfected with 20 μg of GFP-NuMA and 20 μg of either pA3M-LANA aa 840 to 963 or pA3M-LANA aa 840 to 1067 expression plasmid. The cells were harvested at 36 h and immunoprecipitated with 1 μg of anti-Myc antibody.
FIG. 2.
FIG. 2.
NuMA and LANA colocalize in a cell cycle-dependent manner. U2OS cells cotransfected with GFP-NuMA and RFP-LANA were synchronized at specific cell cycle stages as indicated. GFP and RFP signals were visualized using confocal microscopy as described in Materials and Methods. The nuclei were stained with DAPI.
FIG. 3.
FIG. 3.
Colocalization of NuMA and LANA in U2OS cells. U2OS cells expressing GFP-NuMA and RFP-LANA were synchronized at interphase (A), cytokinesis (B), and metaphase (C). GFP-NuMA and RFP-LANA proteins were visualized by confocal microscopy. The nuclei were counterstained with DAPI. The images were analyzed with FLUOVIEW software (Olympus), and the colocalization of the two proteins is shown in both x and y sections.
FIG. 4.
FIG. 4.
NuMA and LANA separate during prophase. U2OS cells transfected with GFP-NuMA and RFP-LANA were synchronized at S phase. Cells released from treatment continued to grow in a chamber in L15 medium supplemented with 10% fetal bovine serum. U2OS cells were filmed on a Leica inverted microscope equipped with a 63× 1.4-NA PlanApo objective lens. Images were recorded at 5-minute intervals and analyzed with Image J software. Selected frames from the two-color time-lapse recording are shown. Arrows in Phase panels indicate the cells analyzed for GFP-NuMA and RFP-LANA.
FIG. 5.
FIG. 5.
NuMA and LANA reunite in daughter nuclei after cytokinesis. U2OS cells were transfected with GFP-NuMA and RFP-LANA and synchronized at mitosis. Cells released from treatment continued to grow in a chamber in L15 medium supplemented with 10% fetal bovine serum. U2OS cells were filmed on a Leica inverted microscope equipped with a 63× 1.4-NA PlanApo objective lens. Images were recorded at 5-minute intervals and analyzed with Image J software. Selected frames from the two-color time-lapse recording are shown. Bright field images are shown under the merged images. Arrows in Phase panels indicate the cells focused for the localization of GFP-NuMA and RFP-LANA.
FIG. 6.
FIG. 6.
The NuMA-dynein/dynactin complex is important in KSHV-TR maintenance. (A) Model of NuMA, dynein/dynactin, and microtubule complex. (B) Colocalization of NuMA (green) and microtubules in mitotic cells. BC-3 cells were synchronized at prometaphase (top) and metaphase (bottom). The cells were fixed and probed with anti-NuMA goat polyclonal antibody, anti-LANA rabbit polyclonal antibody, and anti-β-tubulin mouse monoclonal antibody. Staining was visualized by confocal microscopy with donkey anti-goat (green), goat anti-rabbit (red), and donkey anti-mouse antibodies. The nuclei were counterstained with DAPI. NuMA, green; tubulin, pink; LANA, red; DNA, blue. (C) HEK293 cells were transfected with pBSpuro-EGFP-3TR and expression constructs as indicated. Twenty hours posttransfection, the cells were subjected to selection with 1 μg/ml puromycin. Expression of GFP was measured and imaged at days 1, 3, 5, 7, and 9 posttransfection. The error bars indicate standard deviations.
FIG. 7.
FIG. 7.
Disruption of normal NuMA function inhibits KSHV maintenance. (A) Hirt's DNA was extracted on days 1, 3, 5, 7, and 9 posttransfection from cells with pBSpuro-EGFP-3TR with LANA, LANA plus p150-CC1, and LANA plus LGN. DNA from equal numbers of cells was subjected to real-time quantitation of the TR plasmids in these three sets. Representative CT values of the amplifications from these three samples at different time points are plotted. (B) Equal amounts of Hirt's DNA were amplified with TR-specific primers and normalized with GAPDH copies to get the relative number of copies of TR plasmid. Relative copy numbers of the TR plasmid in a representative experiment in these samples on day 1 and day 5 are plotted. Protein expression on days 1 and 5 was detected by immunoblotting. (C) siRNAs for NuMA cloned in pSIREN vector were introduced into 10 million HEK293 cells to generate si-NuMA stable cells. pSIREN vector with firefly luciferase siRNA was used as a control. NuMA knockdown stable cells were selected and maintained in 4 μg/ml puromycin. Expression of NuMA was examined by immunoblotting and immunofluorescence. (D) si-NuMA and si-Luc stable cells were transfected with pBSpuro-EGFP-3TR and subjected to selection. Expression of GFP was measured and imaged at days 1, 5, and 9 posttransfection.The error bars indicate standard deviations. (E) Hirt's DNA from si-Luc and si-NuMA with and without LANA-expressing cells containing pBSpuro-EGFP-3TR was extracted at days 1, 5, and 9 posttransfection. Equal amounts of Hirt's DNA were subjected to TR amplification, and the numbers of copies of TR plasmids in these cells relative to GAPDH are plotted.
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References

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