Dissecting the contribution of EBNA3C domains important for EBV-induced B-cell growth and proliferation.
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.
Department 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.
Hem Chandra
HC
Jha
Department 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.
Sanket Kumar
SK
Shukla
Department 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.
Jie
J
Lu
Department 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.
Mahadesh Prasad
MP
Aj
Department 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.
Shuvomoy
S
Banerjee
Department 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.
Erle S
ES
Robertson
D001665:Binding Sites; D019013:CD40 Antigens / Q000378:metabolism; D049109:Cell Proliferation; D002472:Cell Transformation, Viral; D020031:Epstein-Barr Virus Infections / Q000276:immunology / Q000821:virology*; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D057809:HEK293 Cells; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000276:immunology / Q000378:metabolism* / Q000472:pathogenicity; D054884:Host-Pathogen Interactions; D006801:Humans; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000276:immunology / Q000821:virology*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D054778:NM23 Nucleoside Diphosphate Kinases / Q000378:metabolism; D016366:Open Reading Frames; D061251:Primary Cell Culture; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025842:SUMO-1 Protein / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection
D019013:CD40 Antigens; C498774:EBNA-3C, epstein-barr virus; D019309:Epstein-Barr Virus Nuclear Antigens; D054778:NM23 Nucleoside Diphosphate Kinases; D025842:SUMO-1 Protein; C495501:SUMO1 protein, human; D020928:Mitogen-Activated Protein Kinases; C517938:NME1 protein, human
BACmid; EBV; MAP kinase; Nm23-H1; homologous recombination