Journal: Journal of Virology

Article Title: S-Like-Phase Cyclin-Dependent Kinases Stabilize the Epstein-Barr Virus BDLF4 Protein To Temporally Control Late Gene Transcription

doi: 10.1128/JVI.01707-18

Figure Lengend Snippet: CDK inhibitors regulate the expression of late genes during EBV lytic replication. (A) qPCR of viral RNAs (center) and viral replication levels (right) in HEK293/EBV(WT) cells treated with CDK inhibitors at 48 h after the induction of lytic replication. Cells were transfected with the BZLF1 expression plasmid and were treated with CDK 2/9 inhibitor (CDK2/9i; 500 nM) or alsterpaullone, 2-cyanoethyl (A2CE; 1 μM) as indicated on the schedule (left). Results shown are means ± SDs from three independent experiments. Double asterisks, P < 0.01; n.s., not significant. (B) qPCR analyses of viral RNA levels (center) and viral replication levels (right) in HEK293/EBV(WT) cells treated with CDK inhibitors 48 h after the induction of lytic replication. Cells were transfected with a BZLF1-expressing plasmid and were treated with CDK2/9i (500 nM) or A2CE (1 μM) as indicated on the schedule (left). Results shown are means ± SDs from three independent experiments. Double asterisks, P < 0.01. (C and D) HEK293/EBV(WT) cells were transfected with a TATT-oriLyt-Luc reporter plasmid together with pSV40-Rluc as an internal control and a BZLF1 expression plasmid as indicated. DMSO or a CDK inhibitor was added to the medium at 24 h p.t. (C) Lysates harvested from the cells at 48 h p.t. were subjected to immunoblot analysis with the indicated antibodies. BZLF1 is an IE gene; BMRF1 and BALF2 are E genes; and BRRF2, BALF4 (gB), and BKRF4 are L genes (11, 48, 55, 56). (D) Lysates were also analyzed using luciferase reporter assays. The results are presented as means ± SDs from three independent transfections after normalization to the internal control (Renilla luciferase activity). Double asterisks, P < 0.01. (E) HEK293/EBV(WT) cells were first transfected with BZLF1 expression vectors and then treated with CDK inhibitors. The virus yields were determined by counting GFP-positive Akata(–) cells. The results are shown as means ± SDs from three independent experiments and are relative to the virus yield of BZLF1 with DMSO treatment (infectivity value, 1). Double asterisks, P < 0.01.

Article Snippet: Anti-CDK1 and anti-CDK2 rabbit polyclonal antibodies were obtained from Bethyl Laboratories (Montgomery, TX, USA).

Techniques: Expressing, Transfection, Plasmid Preparation, Control, Western Blot, Luciferase, Activity Assay, Virus, Infection

Journal: Journal of Virology

Article Title: S-Like-Phase Cyclin-Dependent Kinases Stabilize the Epstein-Barr Virus BDLF4 Protein To Temporally Control Late Gene Transcription

doi: 10.1128/JVI.01707-18

Figure Lengend Snippet: CDK2 is responsible for the phosphorylation-mediated stabilization of BDLF4. (A) The cell cycle of HEK293/FLAG-BDLF4 cells was synchronized using a double thymidine block (61). Cells were harvested at the indicated times after release of the block and were analyzed by immunoblotting using the indicated antibodies. AS, asynchronized; h.p.r., hours postrelease. (B and C) HEK293/FLAG-BDLF4 cells were transfected with plasmids expressing shRNA targeting CDK1 or CDK2 mRNA. Cells were lysed at 48 h p.t. for immunoblotting using the indicated antibodies. (D) HEK293/FLAG-BDLF4 cells were transfected with FLAG-BZLF1 and/or shRNA expression plasmids as indicated, followed by CHX treatment (50 mg/ml). The protein levels of FLAG-BDLF4 and FLAG-BZLF1 (control) were detected using anti-FLAG antibodies. Results are presented as means ± SDs from three independent experiments and are shown relative to the protein levels in the absence of CHX treatment. Asterisks, P < 0.05; n.s., not significant. (E) HEK293/FLAG-BDLF4 cells transfected with shCDK2 were treated with MG132 for 24 h before harvesting. Lysates were analyzed by immunoblotting using the indicated antibodies. (F) (Left) Lytic replication was induced by transfection of 293/EBV(WT) cells with a BZLF1-expressing plasmid, followed by transfection with plasmids carrying shCDK2 at 8 h p.t.; the cells were harvested at 48 h p.t. (Right) Lysates were subjected to immunoblotting using the indicated antibodies.

Article Snippet: Anti-CDK1 and anti-CDK2 rabbit polyclonal antibodies were obtained from Bethyl Laboratories (Montgomery, TX, USA).

Techniques: Phospho-proteomics, Blocking Assay, Western Blot, Transfection, Expressing, shRNA, Control, Plasmid Preparation

Journal: Journal of Virology

Article Title: S-Like-Phase Cyclin-Dependent Kinases Stabilize the Epstein-Barr Virus BDLF4 Protein To Temporally Control Late Gene Transcription

doi: 10.1128/JVI.01707-18

Figure Lengend Snippet: Phosphorylation of BDLF4 by CDK2 complexes enhances L gene expression. (A) In vitro phosphorylation assay showing that BDLF4 protein was phosphorylated by recombinant Cyc A1/CDK2 and Cyc E1/CDK2, but not by Cyc B1/CDK1. Histone H1 served as the control. The phosphorylation assay used ATP-γ-S as the phosphodonor and an anti-thiophosphate ester (anti-ThioP) antibody to detect substrate phosphorylation. Immunoblotting (IB) using an anti-BDLF4 or anti-histone H1 antibody shows the levels of the recombinant protein used for the assay. (B) List of the serine/threonine residues in BDLF4 phosphorylated by Cyc A1/CDK2 and Cyc E1/CDK2. These residues were identified by LC–MS-MS analysis. (C) Schematic representation of the S/T sites mutated in the A×6 and A×4 mutants. (D) HEK293/EBV(dBDLF4) cells were transfected with the pTATT-oriLyt-Luc reporter plasmid together with pSV40-Rluc as an internal control and BZLF1 and BDLF4 (WT or mutant) expression plasmids as indicated. Luciferase activities were measured at 48 h posttransfection. The results are presented as means ± SDs from three independent transfections after normalization against the internal control (Renilla luciferase activity). Lysates were subjected to immunoblotting using the indicated antibodies. Asterisks, P < 0.05. (E) HEK293/EBV(dBDLF4) cells were transfected with BZLF1 and BDLF4 (WT or mutant) expression plasmids as indicated. The viral yields were determined by counting GFP-positive Akata(–) cells. The results are shown as means ± SDs from three independent experiments relative to the viral yield of BZLF1 plus WT BDLF4 (infectivity value, 1). Double asterisks, P < 0.01.

Article Snippet: Anti-CDK1 and anti-CDK2 rabbit polyclonal antibodies were obtained from Bethyl Laboratories (Montgomery, TX, USA).

Techniques: Phospho-proteomics, Gene Expression, In Vitro, Recombinant, Control, Western Blot, Liquid Chromatography with Mass Spectroscopy, Transfection, Plasmid Preparation, Mutagenesis, Expressing, Luciferase, Activity Assay, Infection

Journal: Journal of Virology

Article Title: S-Like-Phase Cyclin-Dependent Kinases Stabilize the Epstein-Barr Virus BDLF4 Protein To Temporally Control Late Gene Transcription

doi: 10.1128/JVI.01707-18

Figure Lengend Snippet: Phosphorylation of BDLA4 at T91 plays an important role in the stabilization of BDLF4 protein. (A) Lysates from HEK293 cells transfected with WT BDLF4 or BDLF4 A mutants were analyzed by immunoblotting using anti-FLAG and anti-GAPDH antibodies. (B) HEK293 cells were first transfected with a BDLF4 WT or T91A mutant expression plasmid and then treated with MG132 or BTZMB. Lysates were analyzed by immunoblotting using the indicated antibodies. (C) HEK293/EBV(dBDLF4) cells were transfected with the pTATT-oriLyt-Luc reporter plasmid, pSV40-Rluc (internal control), and plasmids expressing BZLF1 and BDLF4 (WT, T91A mutant, or T91E mutant) as indicated. Luciferase activities were measured at 48 h posttransfection. The results are presented as means ± SDs from three independent transfections after normalization to the internal control (Renilla luciferase activity) levels. Lysates were subjected to immunoblotting using the indicated antibodies. Asterisks, P < 0.05. (D) HEK293/EBV(dBDLF4) cells were transfected with the pTATT-oriLyt-Luc reporter plasmid, pSV40-Rluc (internal control), and plasmids expressing BZLF1 and BDLF4 (WT or T91A mutant). Luciferase activities in cells treated with CDK inhibitors (CDK2/9i or A2CE) were assayed. The effects of CDK inhibitors on reporter expression are expressed as percentages of decrease from the level of activity for samples treated with DMSO. The results are means ± SDs from three independent experiments. Asterisks, P < 0.05; double asterisks, P < 0.01, respectively. (E) HEK293/EBV(dBDLF4) cells were transfected with BZLF1 and BDLF4 expression vectors as indicated. The viral yields were determined by counting GFP-positive Akata(–) cells. The results are shown as means ± SDs from three independent experiments relative to the viral yield of BZLF1 plus WT BDLF4 (infectivity value, 1). Double asterisks, P < 0.01.

Article Snippet: Anti-CDK1 and anti-CDK2 rabbit polyclonal antibodies were obtained from Bethyl Laboratories (Montgomery, TX, USA).

Techniques: Phospho-proteomics, Transfection, Western Blot, Mutagenesis, Expressing, Plasmid Preparation, Control, Luciferase, Activity Assay, Infection