The immunocomplexes were separated by 10% SDS-PAGE. was induced with100 ng/ml Dox for 24 h for BGLF4 expression. HeLa cells were seeded at a concentration of 2106 cells per 10-cm dish and transfected with 5 g GFP-BGLF4 and harvested at 24 h post transfection. The protein expression levels of BGLF4 were resolved by 10% SDS-PAGE and immunoblotted with specific antibodies. GAPDH served as a loading control.(TIF) pone.0039217.s001.tif (384K) GUID:?34B2951A-D5D2-4DC1-96F4-2C1B06996C2A Figure S2: The kinase activity of viral protein kinase in with protein expression as indicated HMGCS1 was cultured in 10 ml Ura-SC broth to an OD at 600 nm of 1 1.0. The yeast extracts were collected for IP-kinase assay. EBV BGLF4, HCMV UL97 and human Cdc2 were immunoprecipitated with HA antibody (HA.11, Covance). The precipitated proteins were detected using HA antibody. (B) For kinase assay, the immunoprecipitates were incubated with kinase buffer (20 mM Tris-HCl, 1 mM EDTA, 1 mM DTT, 10 mM MgCl2, 0.2 mM Na3VO4, 100 mM ATP) containing[/?32P]ATP with 1 g histone H1 (Calbiochem) at 30C for 30 min. After kinase reaction, proteins were resolved by 12% SDS-PAGE. Gels were dried and subjected Diazepam-Binding Inhibitor Fragment, human to autoradiography for 12 h.(TIF) pone.0039217.s002.tif (260K) GUID:?FD47FF99-7F51-4FFD-8353-D54353B4D47B Figure S3: Expression kinetics of BGLF4 in 293 T-REx BGLF4 inducible cells. (A) Various 293 T-REx BGLF4 inducible clones, B9, B10, B17, B19, B20 and B22, were treated with 100 ng/ml doxycycline (Dox) for 24 h. The BGLF4 and GAPDH proteins were resolved by 10% SDS-PAGE and immunoblotted with specific antibodies. GAPDH served as a loading control. (B) B22 cells were induced with 100 ng/ml Dox for the times indicated. The proteins were displayed by SDS-PAGE and detected with specific antibodies. (C) Slide cultured B9, B20 and B22 cells were incubated with 100 ng/ml Dox. At 24 h post induction (hpi), cells were fixed with 4% paraformaldehyde and stained for BGLF4 with monoclonal antibody 2224 and DNA with Hoechst 33258. (D) Slide cultured B22 cells were incubated with 100 ng/ml Dox, harvested at the time points indicated and stained for BGLF4 and DNA. Chromosome condensation was observed at 6 hpi in B22 cells. (E) B9, B20 and B22 cells were seeded in 96-well plate in a triplicate manner and induced with 100 ng/ml Dox for the expression of BGLF4. At 24, 48, 72, 96 and 120 hpi, an MTT assay was performed and the optical densities (OD) were determined by spectrophotometry at 550 nm.(TIF) pone.0039217.s003.tif (1.2M) GUID:?756496CC-AB26-4BB8-82ED-7697EDF492A3 Figure S4: Expression kinetics of BGLF4 in NPC-TW01 T-REx BGLF4 inducible cells. (A) The NPC-TW01 T-REx BGLF4 inducible clones KIT1, KIT2, KIT3, KIT20, KIT21 and KIT22 were treated with 50 ng/ml doxycycline (Dox) for 24 h. The BGLF4 protein was resolved by 10% SDS-PAGE and immunoblotted with specific antibodies. GAPDH served as a loading control. (B) KIT2 cells were induced with 50 ng/ml Dox and cell extracts were collected at the time points indicated. The protein expression was displayed by SDS-PAGE and detected with specific antibodies. (C) Slide cultured KIT2 and KIT21 cells were incubated with 50 ng/ml Dox. At 60 h post induction (hpi), cells were fixed with 4% paraformaldehyde and stained for BGLF4 with monoclonal antibody 2224 and DNA with Hoechst 33258. More than 95% of the cells expressed BGLF4.(TIF) pone.0039217.s004.tif (870K) GUID:?949D0BE3-B32F-4DF2-93CB-9B761B98B241 Abstract Epstein-Barr virus (EBV) induces an uncoordinated Diazepam-Binding Inhibitor Fragment, human S-phase-like cellular environment coupled with multiple prophase-like events in cells replicating the virus. The EBV encoded Ser/Thr kinase BGLF4 has been shown to induce premature chromosome condensation through activation of condensin and topoisomerase II and Diazepam-Binding Inhibitor Fragment, human reorganization of the nuclear lamina to facilitate the nuclear egress of nucleocapsids in a pathway mimicking Cdk1. However, the observation that RB is hyperphosphorylated in the presence of BGLF4 raised the possibility that BGLF4 may have a Cdk2-like activity to promote S-phase progression. Here, we investigated the regulatory effects of BGLF4 on cell cycle progression and found that S-phase progression and DNA synthesis were interrupted by BGLF4 in mammalian cells. Expression of BGLF4 did not compensate Cdk1 defects for DNA replication in under asynchronous culture conditions at the non-permissive temperature [15]. To determine whether BGLF4 displays Diazepam-Binding Inhibitor Fragment, human S-phase like Cdk activity in yeast, we monitored the function of BGLF4 in the yeast system. The yeast cell cycle is controlled by a single Cdk with various cyclin partners. Yeast Cdk promotes bud emergence, spindle pole body duplication, DNA replication, spindle formation and cell division [20]. Its homologues in prokaryotic and mammalian cells have been identified and proved to compensate the kinase activity in the ts mutant yeast strain is arrested at G2/M phase.