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, 87 (11), 6482-91

Nuclear Transport of Epstein-Barr Virus DNA Polymerase Is Dependent on the BMRF1 Polymerase Processivity Factor and Molecular Chaperone Hsp90

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Nuclear Transport of Epstein-Barr Virus DNA Polymerase Is Dependent on the BMRF1 Polymerase Processivity Factor and Molecular Chaperone Hsp90

Daisuke Kawashima et al. J Virol.

Abstract

Epstein-Barr virus (EBV) replication proteins are transported into the nucleus to synthesize viral genomes. We here report molecular mechanisms for nuclear transport of EBV DNA polymerase. The EBV DNA polymerase catalytic subunit BALF5 was found to accumulate in the cytoplasm when expressed alone, while the EBV DNA polymerase processivity factor BMRF1 moved into the nucleus by itself. Coexpression of both proteins, however, resulted in efficient nuclear transport of BALF5. Deletion of the nuclear localization signal of BMRF1 diminished the proteins' nuclear transport, although both proteins can still interact. These results suggest that BALF5 interacts with BMRF1 to effect transport into the nucleus. Interestingly, we found that Hsp90 inhibitors or knockdown of Hsp90β with short hairpin RNA prevented the BALF5 nuclear transport, even in the presence of BMRF1, both in transfection assays and in the context of lytic replication. Immunoprecipitation analyses suggested that the molecular chaperone Hsp90 interacts with BALF5. Treatment with Hsp90 inhibitors blocked viral DNA replication almost completely during lytic infection, and knockdown of Hsp90β reduced viral genome synthesis. Collectively, we speculate that Hsp90 interacts with BALF5 in the cytoplasm to assist complex formation with BMRF1, leading to nuclear transport. Hsp90 inhibitors may be useful for therapy for EBV-associated diseases in the future.

Figures

Fig 1
Fig 1
Nuclear transport of the BALF5 DNA polymerase catalytic subunit requires the BMRF1 polymerase accessory protein. HeLa cells were transfected with pcDNA-FLAG-BALF5 (A) or pcDNA-BMRF1 (B) or cotransfected with both expression vectors (C). Cells were fixed at 24 h posttransfection, immunostained with anti-FLAG (green) and anti-BMRF1 (red) antibodies, and examined by laser-scanning confocal microscopy. In each sample, nuclei were stained with DAPI (blue). The right panels are merged images. (D) Interaction between BALF5 and BMRF1. HEK293T cells were transfected with pcDNA-FLAG-BALF5, pcDNA-BMRF1, or both. Cells were harvested at 24 h posttransfection, and immunoprecipitation (IP) analysis was carried out, as described in Materials and Methods. The antibodies used for IP and subsequent immunoblot (IB) analysis are indicated.
Fig 2
Fig 2
A BMRF1 mutant lacking the nuclear localization signal (NLS) does not support nuclear transport of the BALF5 DNA polymerase catalytic subunit. (A) Functional domains of BMRF1 protein. BMRF1 protein, consisting of 404 amino acids, has an NLS in its C terminus (amino acids 372 to 404). A BMRF1 mutant lacking the C-terminal 90 amino acids (aa 1 to 317; BMRF1ΔC) is indicated. (B) Subcellular localization of BMRF1ΔC. HeLa cells were transfected with pcDNA-BMRF1ΔC, fixed at 24 h posttransfection, immunostained with anti-BMRF1 (red), and examined by laser-scanning confocal microscopy. Nuclei were stained with DAPI (blue). (C) Physical interaction between BALF5 and BMRF1ΔC. HEK293T cells were transfected with pcDNA-FLAG-BALF5 alone or both pcDNA-FLAG-BALF5 and pcDNA-BMRF1ΔC. Cells were harvested at 24 h posttransfection, and immunoprecipitation (IP) analysis was carried out, as described in Materials and Methods. The antibodies used for IP and subsequent immunoblot (IB) analysis are indicated. (D) Subcellular localization of BALF5 in the presence of BMRF1ΔC. HeLa cells were transfected with pcDNA-FLAG-BALF5 and pcDNA-BMRF1ΔC, fixed at 24 h posttransfection, immunostained with anti-FLAG (green) and anti-BMRF1 (red), and examined by laser-scanning confocal microscopy. Nuclei were stained with DAPI (blue). The right panels are merged images.
Fig 3
Fig 3
Prevention of BMRF1-dependent nuclear transport of BALF5 DNA polymerase catalytic subunit by the Hsp90 inhibitor radicicol. HeLa cells were transfected with the indicated combinations of pcDNA-FLAG-BALF5, pcDNA-BMRF1, and pcDNA-HA-Hsp90 in the absence or presence of 1 μM radicicol. After fixation 24 h posttransfection, the cells were immunostained with anti-FLAG (green) and anti-HA (yellow) antibodies (A), with anti-BMRF1 (red) and anti-HA (yellow) antibodies (B), and with anti-FLAG (green), anti-BMRF1 (red), and anti-HA (yellow) antibodies(C). Nuclei were stained with DAPI (blue). (D) Numerical analysis of the localization pattern of BALF5 in transfected cells. The proportions of cells showing each localization pattern are expressed as percentages out of the examined cells (more than 100). The images in the upper panels represent the typical BALF5 localization patterns. (E) Effect of Hsp90 inhibitor. HeLa cells were treated with 0.5 μM radicicol for 24 h and harvested. Clarified cell lysates were prepared, separated by SDS–10% PAGE, and applied for Western blot analyses with anti-Chk1 protein polyclonal antibody.
Fig 4
Fig 4
Effects of Hsp90 inhibitors on the subcellular localization of BALF5 and BMRF1 proteins in lytic replication-induced cells. Tet-BZLF1/B95-8 cells were treated with doxycycline (4 μg/ml) to induce lytic replication in the absence or the presence of 1 μM radicicol or 1 μM 17-AAG, and the cells were processed for immunostaining at 24 h postinduction with anti-BALF5 (A; red) or anti-BMRF1 (B; red) antibodies. DAPI-stained images (upper panels) and merged images (lower panels) are also shown.
Fig 5
Fig 5
Interaction of Hsp90 with the BALF5 DNA polymerase catalytic subunit. (A) Coimmunoprecipitation of Hsp90 with BALF5. HEK293T cells were transfected with pcDNA-FLAG-BALF5 alone, pcDNA-HA-Hsp90 alone, or both pcDNA-FLAG-BALF5 and pcDNA-HA-Hsp90. Cells were harvested at 24 h posttransfection, and immunoprecipitation (IP) analysis was carried out as described in Materials and Methods. The antibodies used for IP and subsequent immunoblot (IB) analysis are indicated. (B) Disruption of the interaction between BALF5 and Hsp90 by radicicol. HEK293T cells were transfected with pcDNA-FLAG-BALF5 and pcDNA-HA-Hsp90 in the absence or the presence of 0.5 μM radicicol. Cells were harvested at 24 h posttransfection, and IP was carried out, as described in Materials and Methods. Antibodies used for IP and subsequent IB are indicated. (C) The indicated amounts of input samples of panel B were electrophoresed and analyzed by immunoblotting with anti-FLAG, anti-cdc2, and antitubulin antibodies. (D) Stabilization of the interaction between BALF5 and Hsp90 by molybdate. HEK293T cells transfected with pcDNA-FLAG-BALF5 and pcDNA-HA-Hsp90 were lysed in 0.5% mCSK buffer in the presence or absence of 20 mM molybdate. Lysates were subjected to IP and subsequent IB analyses. The antibodies used for IP and IB are indicated. (E) Hsp90 does not associate with BMRF1. HEK293T cells were transfected with pcDNA-BMRF1 and pcDNA-HA-Hsp90 in the absence or presence of 20 mM molybdate. Cells were harvested at 24 h posttransfection, and IP was carried out as described in Materials and Methods. The antibodies used for IP and subsequent IB analyses are indicated. (F) Hsp90 contributes to BALF5-BMRF1 complex formation. HEK293T cells were transfected with pcDNA-FLAG-BALF5, pcDNA-HA-Hsp90, and pcDNA-BMRF1. Cells were harvested at 24 h posttransfection, and IP was carried out as described in Materials and Methods. The antibodies used for IP and subsequent IB analyses are indicated. On each panel, the input lane contains 10% of the amount of the soluble fractions that were subjected to immunoprecipitation.
Fig 6
Fig 6
Effects of radicicol on viral DNA synthesis in lytic replication-induced cells. (A) Lytic replication was induced in B95-8 cells with the addition of doxycycline in the presence or the absence of 1 μM radicicol, and the cells were harvested at the indicated hours postinduction. The levels of viral DNA synthesis were determined by quantitative real time-PCR assay and plotted as ratios to the value at 0 h ± the standard error of the mean from three independent experiments. (B) The expression levels of BZLF1 were determined by Western blotting analyses using anti-BZLF1 protein polyclonal antibody. (C) Cell viabilities were determined by trypan blue dye exclusion test. The percentages of viable cells out of total cells (more than 100 cells counted for each sample) are indicated. (D) The growth curves of the cells in panel C are shown.
Fig 7
Fig 7
Hsp90β knockdown prevents viral DNA synthesis in lytic replication-induced AGS-CR2/EGFP-EBV cells. (A) AGS-CR2/EGFP-EBV cells expressing either luciferase shRNA (shLuc) or Hsp90β shRNA (shHsp90β) were subjected to immunoblotting analysis with anti-Hsp90β and antitubulin antibodies. (B) Cells transduced with shLuc or shHsp90β were transfected with either pcDNA or pcDNA-BZLF1 to induce lytic replication and were harvested at 72 h posttransfection. Levels of viral DNA synthesis were determined by quantitative real time-PCR assay. Bars indicate ratios compared to the value of the pcDNA-transfected shLuc cells at 0 h.
Fig 8
Fig 8
Hsp90β knockdown blocks nuclear transport of BALF5 DNA polymerase catalytic subunit. (A) AGS-CR2/EGFP-EBV cells expressing either luciferase shRNA (shLuc) (a) or Hsp90β shRNA (shHsp90β) (b) were subjected to immunofluorescence analysis. Each cell was transfected with pcDNA-BZLF1 to induce lytic replication and harvested 24 h posttransfection. Harvested cells were fixed and immunostained with anti-BALF5 (green), anti-Hsp90β (red), and anti-BMRF1 (yellow) antibodies. Nuclei were stained with DAPI (blue). (B) HeLa cells expressing either luciferase shRNA (shLuc) (a) or Hsp90β shRNA (shHsp90β) (b) were transfected with pcDNA-FLAG-BALF5 and pcDNA-BMRF1. At 24 h posttransfection, cells were fixed and immunostained with anti-FLAG (green), anti-Hsp90β (red), and anti-BMRF1 (yellow) antibodies. Nuclei were stained with DAPI (blue). Hsp90β expression levels of each cell were confirmed by immunoblot analysis (c).

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