In silico analysis of pathways targeted by EBV infection and malignant transformation

Abstract: Epstein-Barr virus (EBV) is a ubiquitous γ-herpes virus with dual cell tropism for human B-lymphocytes and epithelial cells. EBV infection is linked to several malignancies such as Burkitt s lymphoma (BL) and nasopharyngeal carcinoma (NPC). In vitro EBV is a potent transforming virus that converts resting B-lymphocytes into indefinitely proliferating lymphoblastoid cells (LCLs). The overall aim of this study was to develop and utilize bioinformatics methods to dissect the molecular mechanisms by which EBV modulates the cellular environment. EBV is a large DNA virus that encodes about 100 open reading frames (ORFs) expressed at various times during infection. Using established sequence search methods such as patterns and hidden Markov models (HMM), we have identified catalytic domains of ubiquitin specific proteases (deubiquitinating enzymes, DUBs) in a limited number of EBV ORFs. The DUB activity of three high scoring candidates: BPLF1, BSLF1 and BXLF1, was confirmed by functional and mutational analysis (Paper-I). EBV establishes distinct programs of viral gene expression in latently infected and malignant cells. The EBV nuclear antigen (EBNA)-1 is expressed in all EBV carrying proliferating cells. EBNA-1 has been associated with the induction of cellular oxidative stress due to the production of reactive oxygen species (ROS) and DNA damage. To search for the cellular genes involved in these effects we used gene expression data sets from public databases. By analyzing the expression of genes involved in ROS metabolism in EBV positive and negative Burkitt's lymphoma (BL) cell lines and lymphoblastoid cell lines (LCL) we found that the NADPH oxidase (NOX)-2 is induced in cells expressing EBNA-1. Activation of the NOX2 gene by EBNA-1 is associated with the induction and/or maintenance of genomic instability, a critical step in malignant transformation (Paper-II). The expression of EBNA-1 is associated with a global rearrangement of cellular transcription. To investigate the primary and secondary targets of this transcriptional effect we have analyzed the gene expression profiles of stable and inducible EBNA-1 expressing cells. Functional analysis of the regulated transcripts revealed that EBNA-1 influences the expression of genes involved in the maintenance of chromatin architecture. Several subunits of chromatin remodeling complexes were down-regulated on EBNA-1 expression (Paper-III). Microarray analysis and systems biology approaches were implemented to investigate the cellular pathways modulated by tripeptidyl peptidase II (TPPII) a cellular protein that is highly expressed in BL cells and participates in the induction of the malignant phenotype. Comparison of the gene expression profiles of control and TPPII knockdown BL cells and systems level analysis of differentially regulated genes demonstrated that the MAPK signaling pathway is selectively inactivated by TPPII knockdown (Paper-IV). In summary, this work demonstrated the potential of bioinformatics tools and highthroughput genomic approaches in identifying the novel strategies of EBV.