ABSTRACT
Primary infection of the immunocompromised host with the oncovirus Epstein-Barr virus (EBV) that targets mainly B-cells is associated with an increased risk for EBV-associated tumors. The early events subsequent to primary infection with potential for B-cell transformation are poorly studied. Here, we modeled in vitro the primary infection by using B-cells isolated from tonsils, the portal of entry of EBV, since species specificity of EBV hampers modeling in experimental animals. Increasing evidence indicates that the host DNA damage response (DDR) can influence and be influenced by EBV infection. Thus, we inoculated tonsillar B-cells (TBCs) with EBV-B95.8 and investigated cell proliferation and the DDR during the first 96 hours thereafter. We identified for the first time that EBV infection of TBCs induces a period of hyperproliferation 48-96 hours post infection characterized by the activation of ataxia telangiectasia and Rad3-releated (ATR) and checkpoint kinase-1 (Chk1). Whereas inhibition of Chk1 did not affect B-cell transformation, the specific inhibition of ATR robustly decreased the transformation efficiency of EBV. Our results suggest that activation of ATR is key for EBV-induced B-cell transformation. Thus, targeting the interaction between ATR/Chk1 and EBV could offer new options for the treatment of EBV-associated malignancies.
Subject(s)
B-Lymphocytes/virology , Cell Transformation, Viral , Checkpoint Kinase 1/metabolism , Epstein-Barr Virus Infections/virology , Herpesvirus 4, Human/pathogenicity , Palatine Tonsil/enzymology , Palatine Tonsil/virology , Antigens, CD19/metabolism , Ataxia Telangiectasia Mutated Proteins/analysis , Ataxia Telangiectasia Mutated Proteins/metabolism , B-Lymphocytes/drug effects , B-Lymphocytes/enzymology , B-Lymphocytes/immunology , CD40 Ligand/metabolism , Cell Proliferation , Cells, Cultured , Checkpoint Kinase 1/analysis , DNA Damage , DNA Repair , Enzyme Activation , Epstein-Barr Virus Infections/enzymology , Epstein-Barr Virus Infections/immunology , Epstein-Barr Virus Infections/pathology , Herpesvirus 4, Human/immunology , Host-Pathogen Interactions , Humans , Palatine Tonsil/drug effects , Palatine Tonsil/immunology , Protein Kinase Inhibitors/pharmacology , Signal Transduction , Time FactorsABSTRACT
Lymphoproliferative diseases (LPDs) associated with Epstein-Barr virus (EBV) infection cause significant morbidity and mortality in bone marrow and solid organ transplant recipients. To gain insight into LPD pathogenesis and to identify potential effective therapeutic approaches, we investigated early molecular events leading to B-cell transformation by gene expression profiling of EBV-infected B-cells from tonsils by Affymetrix microarray 72 hr postinfection when the B-cells hyperproliferation phase starts. Cell cycle and apoptosis were the most significantly affected pathways and enriched gene sets. In particular, we found significantly increased expression of cyclin-dependent kinase (CDK)1 and CCNB1 (cyclin B1) and of one of their downstream targets BIRC5 (survivin). Importantly, the strong upregulation of the antiapoptotic protein survivin was confirmed in lymphoblastoid cell lines (LCLs) and 71% of EBV-positive post-transplant EBV-LPD lesions scored positive for survivin. The validity of early transforming events for the identification of therapeutic targets for EBV-LPD was confirmed by the marked antiproliferative effect of the CDK inhibitor flavopiridol on LCLs and by the strong induction of apoptosis by survivin inhibition with YM155 or terameprocol. Our results suggest that targeting of CDKs and/or survivin in post-transplant EBV-LPD by specific inhibitors might be an important approach to control and eliminate EBV-transformed B-cells that should be further considered.