Abstract
Epstein-Barr virus (EBV), a gamma herpes virus is associated with B-cell malignancies. EBNA-3C is critical for in vitro primary B-cell transformation. Interestingly, the N terminal domain of EBNA3C which contains residues 130–159, interacts with various cellular proteins, such as p53, Mdm2, CyclinD1/Cdk6 complex, and E2F1. In the current reverse genetics study, we deleted the residues 130-159 aa within EBNA3C open reading frame (ORF) by BACmid recombinant engineering methodology. Our experiments demonstrated that deletion of the 130-159 aa showed a reduction in cell proliferation. Also, this recombinant virus showed with higher infectivity of human peripheral blood mononuclear cells (PBMCs) compared to wild type EBV. PBMCs- infected with recombinant EBV deleted for 130-159 residues have differential expression patterns for the p53/Mdm2, CyclinD1/Cdk6 and pRb/E2F1 pathways compared to wild type EBV-infected PBMCs. PBMCs infected with recombinant virus showed increased apoptotic cell death which further resulted in activation of polymerase 1 (PARP1), an important contributor to apoptotic signaling. Interestingly, cells infected with this recombinant virus showed a dramatic decrease in chromosomal instability, indicated by the presence of increased multinucleation and micronucleation. In addition infection with recombinant virus have increased cells in G0/G1 phase and decreased cells in S-G2M phase when compared to wild type infected cells. Thus, these differences in signaling activities due to 29 amino acid residues of EBNA3C is of particular significance in deregulation of cell proliferation in EBV-infected cells.
Highlights
Epstein–Barr virus (EBV), a ubiquitous herpes virus, infects approximately 90-95% of the world’s population and is associated with various malignant tumors, including Burkitt’s lymphoma, nasopharyngeal carcinoma, and B-cell lymphoma in immune deficient individuals [1]
Infectious EBV can be produced by transfection of BACEBV-GFPWT into HEK-293T cells, selection followed by chemical induction [16]
EBV nuclear antigen 3C (EBNA3C) interacts with an array of transcription factors, cell cycle regulatory proteins, chromatin remodeling enzymes and ubiquitin-proteasome machinery that eventually leads to B cell lymphomagenesis [9, 12, 17, 24, 34, 35, 36]
Summary
Epstein–Barr virus (EBV), a ubiquitous herpes virus, infects approximately 90-95% of the world’s population and is associated with various malignant tumors, including Burkitt’s lymphoma, nasopharyngeal carcinoma, and B-cell lymphoma in immune deficient individuals [1]. The LCLs, which are produced in culture carry the viral genome as extra-chromosomal episomes and express nine latent EBV proteins including, the six nuclear antigens (EBNA1, 2, 3A, 3B, 3C & LP), an additional three membrane associated proteins (LMP1, LMP2A & 2B), and the twenty-nine EBV-encoded small RNAs and microRNAs BARTs [4]. These viral factors contribute to activation of the quiescent B-cells from G0 into the cell cycle, and to sustain proliferation and maintenance of the viral genome [5]
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