Modulation of cellular and viral functions in Epstein-Barr virus infected cells

Abstract

The aim of the study was to investigate the interplay of cellular and viral genes in Epstein-Barr virus (EBV) infected cells. Two aspects of this interaction have been investigated. The first aspect is related to the expression of major histocompatibility complex (MHC) class I molecules in virus infected malignant cells. MW class I expression plays a key role in the regulation of immune responses and is frequently altered in human tumors. We analyzed the mechanisms of this MW class 1 downregulation in Burkitt's lymphomas (BLs) that arise in HLA. All positive individuals by comparing five pairs of BL lines and Epstein-Barr virus (EBV) transformed lymphoblastoid cell lines (LCL) derived from the normal B cells of the same individuals. The cell lines were compared for the presence of HLA A11 gene and A11-specific mRNA, the reactivity to IFN-alpha treatment, the restoration of HLA All expression by transfection or hybridization, the activity of HLA All promoter driven chloramphenicol acetyl transferase reporter gene (pA l 1 CAT). Our results suggest that genetic defects and lack of transcription factors may contribute to the selective down- regulation of HLA AI 1 in BL cells. Furthermore we analysed the involvement of EBV LMP1 in the process. The BL phenotype-dependent transcriptional defect observed for the HLA AI 1 promoter was shown to involve other WC class I promoters including HLA A2. Since the virus LMP1 was shown to be mainly responsible for changing the phenotype of BL cells towards LCL-like characteristics, we have tested how the class I promoter activity is affected by expression of LMP1. We can conclude, that expression of LMP1 correlates with activation of the HLA All promoter and up-regulation of several components of the transcription factor family NF-kappa-B/Rel.

The second aspect of this thesis deals with viral strategies that promote escape from immune recognition. EBNA1 is expressed in all EBV associated malignancies and is the only viral protein expressed in BL. Endogenously expressed EBNA1 is not recognized by MHC class P restricted CTLs. This phenomenon is correlated with the presence of an internal glycine- alanine repeat (GAr) in the structure of the protein. The GAr generates a cis-acting inhibitory signal that interferes with antigen processing and MW class I restricted presentation by inhibiting the generation of antigenic peptides by ubiquitin-proteasome/dependent proteolysis. The mechanism of action of the GAr was investigated using as model a known target of the ubiquitin-poteasome system, the NF-kappa-B inhibitor I-kappa-B-alpha. Insertion of a minimal GA repeat of eight amino acids in different positions of I-kappa-B-alpha was sufficient to prevent tumor necrosis factor (TNF-alpha) induced ubiquitin-proteasome dependent degradation, and decrease its basal turnover in vivo. The chimeras are phosphorylated and ubiquitinated in response to TNF-alpha but than released and failed to associate with the proteasome. This explains how functionally competent I-kappa-N-alpha is protected from proteasomal disruption and identifies the GAr as a regulator of proteolysis. The analysis of the impact of length and amino acid composition on the capacity of various repeat sequences to inhibit the TNF-alpha-induced and physiological turnover of I-kappa-B-alpha demonstrated that inhibition is achieved by insertion of octamer peptides containing 3 hydrophobic amino acids, interspersed by no more then three consecutive glycines. The inhibitory activity was abolished by increasing the length of the spacer to four glycines, by elimination of the spacers, or by substitution of a single hydrophobic residue with a polar or charged residue. These findings suggest a model where inhibition of proteolysis requires the interaction of at least dime alanine residues of the GAr in a beta-strand conformation with adjacent hydrophobic binding pockets of a putative receptor.

The multifunctional EBNA1 protein is involved also in the replication of the EBV genome as an extra chromosomal element, and plays important role in the maintenance of the viral episome and is the key transcriptional regulator of latent viral gene expression including its own transcriptional regulation. We have investigated whether presence of the GAr modulates the functions of EBNA1. The GAr containing EBNA1 protein has an extremely slow turnover in virus infected cells while a GAr deleted EBNA1 is degraded by the ubiquitin-proteasome system. To assess the contribution of protein stability on EBNA l functions, destabilized variants were generated and studied for their capacity to promote the maintenance of oriP containing plasmids and regulate the viral Qp promoter. Both functions were strongly influenced by EBNA1 stability suggesting the GAr plays an important role in modulating the function of EBNA1 in virus infected cells.