Article Figures & Data
Figures
- FIG. 1.
Biochemical analysis of the subcellular distribution of CDC6, MCM4, MCM6, and MCM7 in lytic-program-induced Tet-BZLF1/B95-8 cells. (A) Tet-BZLF1/B95-8 cells were cultured in the presence of 2 μg/ml doxycycline, harvested at the indicated times, and subjected to biochemical fractionation as described in Materials and Methods. Tet-BZLF1/B95-8 cells were also treated with paclitaxel (20 μM) for 24 h to arrest cell cycle at the G2/M phase and processed similarly. The relative abundance of each protein in Triton X-100-extractable supernatants (S) and extracted nuclear pellets (P) was examined by immunoblotting with anti-CDC6, anti-MCM4, anti-phosphorylated Thr-110 of MCM4, anti-MCM6, anti-MCM7, and anti-BZLF1 antibodies. W, whole-cell lysate. (B) Subnuclear localizations of MCM4 and MCM7 in lytic-program-induced Tet-BZLF1/B95-8 cells. Cells were harvested at 24 h.p.i. and treated with 0.5% Triton X-100-mCSK buffer. Nonionic-detergent-extracted cells were fixed with methanol and then immunostained with anti-MCM4 or anti-MCM7 and anti-BMRF1 antibodies. Shown are merged images of MCM4 (red) or MCM7 (red) and BMRF1 (green) proteins.
- FIG. 2.
Phosphorylation of Thr-19 and Thr-110 residues of MCM4 upon induction of EBV lytic replication. B95-8, Tet-BZLF1/B95-8, and Tet-BZLF1/Akata cells were cultured in the presence of 2 μg/ml doxycycline and harvested at the indicated times. Equal amounts of proteins for each sample (∼20 to 50 μg) were subjected to immunoblot analysis with the specific antibodies indicated on the left side of each panel. Anti-CDK2 antibody was used to confirm equal protein loading.
- FIG. 3.
Expression of the EBV-PK encoded by the BGLF4 gene in HeLa cells results in phosphorylation of MCM4 at Thr-19 and Thr-110. (A) HeLa cells were transiently transfected with the BGLF4 protein expression vector pME-BGLF4(F) or a control vector, pME18S, and harvested after 2 days. Whole-cell extracts were prepared, and equal amounts of proteins for each sample (20 μg) were separated by gradient SDS-PAGE and subjected to immunoblot analysis with the specific antibodies indicated on the left side of each panel. (B) Effect of expression of the EBV-PK on the proliferation of HeLa cells. HeLa cells (0.6 × 106 cells/35-mm dish) were transfected with the BGLF4 expression plasmid pME-BGLF4(F) or the control plasmid pME18S and were counted with a hemacytometer at the indicated times.
- FIG. 4.
EBV-PK phosphorylates Thr-19 and Thr-110 residues on MCM4 of the MCM4-6-7 hexamer in vitro. (A) Purification of MCM4-6-7 hexamers. Sf21 cells were coinfected with recombinant baculoviruses, Bac-Mcm4-6 and Bac-Mcm7, and MCM4-6-7 complexes were purified as described in Materials and Methods, separated by SDS-7.5% PAGE, and stained with silver. The positions of the MCM4, Mcm6, and MCM7 proteins were determined by Western blot analyses (data not shown) and are indicated by arrows. (B) Wild-type or kinase-negative GST-BGLF4 proteins were isolated from Sf21 cells infected with Bac-GST-BGLF4 or Bac-GST-BGLF4K102I as described in Materials and Methods. Human MCM4-6-7 hexamer (100 ng) was incubated with increasing amounts of wild-type GST-BGLF4, kinase-negative GST-BGLF4K102I, or cyclin A/CDK2 (−, none). The samples were subjected to SDS-7.5% PAGE and analyzed by Western blotting using phosphospecific antibodies against MCM4 at Thr-19 and Thr-110.
- FIG. 5.
EBV-PK, like CDK2/cyclin A, inhibits DNA helicase (unwinding) activity associated with the MCM4-6-7 complex. (A) Substrate for DNA helicase assays. A 5′ 32P-labeled oligonucleotide (17-mer) annealed to M13 single-stranded DNA is depicted. (B) DNA helicase assays were performed with 100 fmol of the helicase substrate and human MCM4-6-7 hexamers (100 ng) in the presence of cyclin A/CDK2 (100 ng), BGLF4 protein (100 and 500 ng), or kinase-negative BGLF4K102I protein (200 and 500 ng) as described in Materials and Methods. Positions of the DNA substrate (17mer/M13) and displaced oligonucleotide (17mer) are indicated by arrows. The left two lanes show results for heat-denatured and native DNA substrates, respectively.
- FIG. 6.
EBV-PK, unlike CDK2/cyclin A, still inhibits helicase activity of MCM4-6-7 hexamers containing mutations in amino-terminal phosphorylation sites of MCM4. (A) MCM4-6-7 hexamers containing mutant MCM4 (MCM4a/6/7) (200 ng) or the wild type (MCM4-6-7) (100 ng) were examined for DNA helicase activity in the presence or absence of BGLF4 protein (500 ng) or cyclin A/CDK2 (500 ng) as described in Materials and Methods. The left two lanes show results for heat-denatured and native DNA substrates, respectively. Quantitative analysis of the DNA unwinding activities is shown in the graph. The percentage of 17-mer oligonucleotide displaced with each MCM complex in the presence of the BGLF4 EBV-PK or CDK2/cyclin A was calculated from the signal intensity, with that in the absence of the kinase taken as 100%. Data are means ± standard deviations of three independent experiments. (B) Human MCM4-6-7 or MCM4a-6-7 complexes (1 μg) were incubated with indicated amounts of CDK2/cyclin A (left) or the BGLF4 protein (right) in a 50-μl reaction mixture containing 20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM dithiothreitol, 10 mM MgCl2, 100 μM ATP, 2 μCi [γ-32P]ATP, and 0.2 mM sodium orthovanadate. 32P-labeled proteins were separated by SDS-7.5% PAGE followed by autoradiography. (C) Human MCM4-6-7 or MCM4a-6-7 complexes (1 μg) were phosphorylated by 500 ng of the BGLF4 protein in a 50-μl reaction mixture containing 20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM dithiothreitol, 10 mM MgCl2, 1 mM ATP, and 0.2 mM sodium orthovanadate, and products were separated by SDS-7.5% PAGE. Proteins were analyzed by Western blotting using MCM6 and MCM4 antibodies.
