Subject(s)
Hematopoietic Stem Cell Transplantation , Herpesvirus 4, Human , Killer Cells, Natural/virology , Lymphoproliferative Disorders/therapy , T-Lymphocytes/virology , Adult , Cell Proliferation , Female , Humans , Lymphoproliferative Disorders/virology , Male , Middle Aged , Prognosis , Remission Induction , Time Factors , Transplantation Conditioning , Treatment Outcome , Young AdultSubject(s)
Antineoplastic Agents/administration & dosage , Blood Donors , Bone Marrow Transplantation , Epstein-Barr Virus Infections/therapy , Herpesvirus 4, Human , Lymphocyte Transfusion , Lymphoproliferative Disorders/therapy , Antibodies, Monoclonal, Murine-Derived , Humans , Lymphoproliferative Disorders/virology , Male , Middle Aged , Precursor Cell Lymphoblastic Leukemia-Lymphoma/therapy , Rituximab , Time Factors , Transplantation, HomologousABSTRACT
Latent Epstein-Barr virus (EBV) is maintained by the virus replication origin oriP that initiates DNA replication with the viral oriP-binding factor EBNA1. However, it is not known whether oriP's replicator activity is regulated by virus proteins or extracellular signals. By using a transient replication assay, we found that a low level of expression of viral signal transduction activator latent membrane protein 1 (LMP1) suppressed oriP activity. The binding site of the tumor necrosis factor receptor-associated factor (TRAF) of LMP1 was essential for this suppressive effect. Activation of the TRAF signal cascade by overexpression of TRAF5 and/or TRAF6 also suppressed oriP activity. Conversely, blocking of TRAF signaling with dominant negative mutants of TRAF5 and TRAF6, as well as inhibition of a downstream signal mediator p38 MAPK, released the LMP1-induced oriP suppression. Furthermore, activation of TRAF6 signal cascade by lipopolysaccharides (LPS) resulted in loss of EBV from Burkitt's lymphoma cell line Akata, and inhibition of p38 MAPK abolished the suppressive effect of LPS. These results suggested that the level of oriP activity is regulated by LMP1 and extracellular signals through TRAF5- and TRAF6-mediated signal cascades.
Subject(s)
Herpesvirus 4, Human/physiology , Mitogen-Activated Protein Kinases/metabolism , Proteins/metabolism , Viral Matrix Proteins/metabolism , Binding Sites , Burkitt Lymphoma , Capsid , Cell Line, Transformed , DNA Replication , Down-Regulation , HeLa Cells , Herpesvirus 4, Human/genetics , Humans , Lipopolysaccharides/pharmacology , Mutation , Plasmids , Signal Transduction/drug effects , TNF Receptor-Associated Factor 5 , TNF Receptor-Associated Factor 6 , Viral Matrix Proteins/genetics , Virus Latency , Virus Replication , p38 Mitogen-Activated Protein KinasesABSTRACT
Latent Epstein-Barr virus genome is maintained in cells by the viral oriP-binding factor EBNA1 and cellular replication factors. EBNA1 binds to the dyad symmetry (DS) element in oriP and initiates DNA replication once in a single S phase, but the mechanism by which this DS-dependent replication is initiated is unknown. Replication licensing of cellular chromatins occurs during early G1 phase. Because licensing is essential for the next round of replication in S phase, it facilitates once-in-a-cell-cycle replication of the cellular genome. Using the transient replication assay with HeLa/EB1 cell, we demonstrate that the oriP plasmid required a cell cycle window including early G1 phase for replication in the next S phase. The plasmid containing only the DS element had a similar requirement of early G1 phase for replication. Analysis using sucrose density gradient centrifugation revealed that the oriP minichromosome existed in two distinct states: one formed at late G1 and the other formed at G2/M. These results suggest that the DS-dependent DNA replication from oriP requires the replication licensing, implying a possible involvement of the cellular licensing factor MCM in the DNA replication from oriP.
