Abstract
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.
Highlights
Epstein-Barr virus (EBV) is a human gammaher pesvirus that latently infects B-cells
The results showed that the PCR bands in from the EBNA3C open reading frame (ORF) shifted based on the presence of the remaining loxP site and HindIII site (Figure 1D)
Our data demonstrated that cell proliferation was significantly reduced with full length EBNA3C deletion as well as the ∆621–675 recombinant when compared with wildtype EBV infected cells
Summary
Epstein-Barr virus (EBV) is a human gammaher pesvirus that latently infects B-cells. We have www.impactjournals.com/oncotarget generated an EBV-BAC system with a GFP expression cassette for maintaining infection [2] This recombinant virus successfully infects human peripheral B-cells as seen by a strong GFP signal during early primary infection and activated CD40 in a time-dependent manner [2]. In this study we generated three EBV recombinants by BAC recombinant engineering, deleted for full-length EBNA3C, residues 621–675 which is the Nm23-H1 binding domain within EBNA3C, and the RBP-Jκ binding site residues 183–240 within EBNA3C, respectively These recombinant viruses were examined to evaluate the role of EBNA3C, and its binding domains for RBP-Jk and Nm23-H1 in B-cell activation and proliferation during latent and primary infection. These two mutants suggest that EBNA3C has a vital role in CD40 activation, MAP kinase activation and β-Catenin stabilization in B-cells infection and transformation
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