Abstract
Epstein-Barr virus (EBV) productive replication occurs in an S-phase-like cellular environment with high cyclin-dependent kinase (CDK) activity. The EBV protein kinase (PK), encoded by the viral BGLF4 gene, is a Ser/Thr protein kinase, which phosphorylates both viral and cellular proteins, modifying the cellular environment for efficient viral productive replication. We here provide evidence that the EBV PK phosphorylates the CDK inhibitor p27(Kip1), resulting in ubiquitination and degradation in a proteasome-dependent manner during EBV productive replication. Experiments with BGLF4 knockdown by small interfering RNA and BGLF4 knock-out viruses clarified that EBV PK is involved in p27(Kip1) degradation upon lytic replication. Transfection of the BGLF4 expression vector revealed that EBV PK alone could phosphorylate the Thr-187 residue of p27(Kip1) and that the ubiquitination and degradation of p27(Kip1) occurred in an SCF(Skp2) ubiquitin ligase-dependent manner. In vitro, EBV PK proved capable of phosphorylating p27(Kip1) at Thr-187. Unlike cyclin E-CDK2 activity, the EBV PK activity was not inhibited by p27(Kip1). Overall, EBV PK enhances p27(Kip1) degradation effectively upon EBV productive replication, contributing to establishment of an S-phase-like cellular environment with high CDK activity.
Highlights
Tein that functions as the receptor component of an SCF (Skp1/ Cullin/F-box protein)-type ubiquitin ligase complex (4 – 8)
We document evidence that, during Epstein-Barr virus (EBV) productive replication, EBV protein kinase (PK) phosphorylates p27Kip1 so that it is ubiquitinated by SCFSkp2 ubiquitin ligase and degraded in a proteasome-dependent manner
EBV lytic replication occurs in an S-phase-like cellular environment with high cyclin-dependent kinase (CDK) activity [20, 31]
Summary
Cells—HeLa and HEK293T cells were grown and maintained at 37 °C in Dulbecco’s modified Eagle’s medium (Sigma) supplemented with 10% fetal calf serum. An EBV producer cell line, Akata(ϩ) [32], was grown in RPMI medium supplemented with 10% fetal calf serum and mixed with polyclonal rabbit anti-human IgG (Dako) at a final concentration of 28.5 g/ml in the culture medium to induce lytic replication. Immunoblot Analysis—Cells were suspended in lysis buffer (20 mM Tris-HCl (pH 7.4), 0.5% Triton X-100, 300 mM NaCl, 1 mM EDTA, 0.1% SDS, 100 mM NaF, 2 mM Na3VO4, protease inhibitor mixture (Sigma)) and incubated on ice for 40 min followed by centrifugation. For siRNA transfection, 293/EBV-WT cells (1 ϫ 106) and HEK293T cells (1.2 ϫ 106) were transfected with 100 pmol of Skp siRNA or non-targeting/control siRNA using a microporator and Lipofectamine 2000, respectively. The immune complex was washed four times with wash buffer (50 mM Tris-HCl (pH 8), 150 mM NaCl, 0.1% Nonidet P-40, and 1 mM EDTA) and eluted with SDS gel loading buffer
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
