Dissecting the contribution of EBNA3C domains important for EBV-induced B-cell growth and proliferation
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Hem Chandra Jha1,*, Sanket Kumar Shukla1,*, Jie Lu1, Mahadesh Prasad Aj1, Shuvomoy Banerjee1, Erle S. Robertson1
1Department of Microbiology and the Tumor Virology Program, Abramson Comprehensive Cancer Center, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, 19104, United States of America
*These authors have contributed equally to this work
Erle S. Robertson, e-mail: [email protected]
Keywords: EBV, BACmid, MAP kinase, homologous recombination, Nm23-H1
Received: May 19, 2015 Accepted: August 07, 2015 Published: August 18, 2015
Epstein-Barr virus (EBV) is an oncogenic gammaherpes virus which is linked to pathogenesis of several human lymphatic malignancies. The EBV essential latent antigen EBNA3C is critical for efficient conversion of primary human B-lymphocytes to lymphoblastic cell lines and for continued LCL growth. EBNA3C, an EBV latent antigen with oncogenic potential can bind and regulate the functions of a wide range of cellular transcription factors. In our current reverse genetics study, we deleted the full length EBNA3C, and independently the RBP-Jκ and Nm23-H1 binding sites within EBNA3C using BACmid recombinant engineering methodology. Our experiments demonstrated that deletion of the EBV EBNA3C open reading frame (ORF) and more specifically the residues 621–675 which binds Nm23H1 and SUMO-1 showed a significant reduction in the ability of the cells to proliferate. Furthermore, they exhibited lower infectivity of human peripheral blood mononuclear cells (PBMCs). We also showed that recombinant EBV with deletions of the EBNA3C ORF, as well as a recombinant with residues 621–675 within EBNA3C ORF deleted had diminished abilities to activate CD40. Our study also revealed that the full length (1–992) and 621–675 aa deletions of EBNA3C when compared to wild type EBV infected PBMCs had differential expression patterns for the phosphorylation of MAP kinases specifically p38, JNK and ERK. Regulation of β-catenin also differed among wild type and EBNA3C deleted mutants. These temporal differences in signaling activities of these recombinant viruses in PBMCs is likely important in defining their functional importance in EBV-mediated B-cell transformation.
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