Analysis of viral and cellular gene expression patterns in cells latently infected with EBV by suppression subtractive hybridization

Abstract

Epstein-Barr virus is a ubiquitous γ-herpes virus and causes the self-limiting lymphoproliferative disease, called infectious mononucleosis. Its presence is associated with several malignancies, including Burkitt lymphoma, gastric cancer and other malignant diseases. EBV has a powerful ability to transform human B lymphocytes in vitro into continuously growing immortalized lines.

The aim of this thesis was to develop and utilize high throughput methods to identify and analyze cellular genes that play roles in EBV induced transformation. Suppression subtractive hybridization combined with high-density DNA arrays were chosen as methods to identify EBV regulated genes. SSH is a very efficient subtractive hybridization technique that combines normalization and suppression PCR effect in a single cycle. We modified the original SSH protocol with a magnetic bead separation step that increases the efficiency of subtraction considerably.

To test the efficiency of our combination of the modified SSH with high throughput array hybridization, we compared the EBV positive Burkitt's lymphoma cell line Akata EBV-Neo with its EBV negative parental cell line. We could identify lytic EBV genes and cellular genes that were differentially expressed. Among these genes, showing the largest expression difference was the T cell leukemia I oncogene (TCL-1). Two other independently established EBV positive BLs (MUTU and Oma) that have also thrown off EBV showed a similar decrease in TCL-1 expression after virus loss. Considering the high expression of TCL-1 in all BLs, in contrast to its lack of expression in the GC progenitor cells, we postulate that TCL-1 activation is a necessary event in the development of the BL phenotype. We suggest that EBV negative BLs switch on TCL-1 constitutively during their neoplastic development. In the virus carriers, EBV is responsible for the upregulation.

The expression pattern of an EBV-infected gastric carcinoma cell line, Nu-GC-3 and its uninfected control line has been compared by SSH. We have found that EBV infection upregulated the truncated human basic hair keratin 1 (hHb1-ΔN). hHb1-ΔN is the first example of an upregulated cellular gene after EBV infection of carcinoma cells that do not express LMP1. However, its role in EBV-positive carcinoma cells is obscure. Our findings are consistent with the possibility that EBV acts by upregulating hHb1-ΔN that leads to the inhibition of epithelial differentiation and supports the anaplastic status of the carcinomas that carry the virus.

We combined SSH with DOP-PCR (DOP-SSH) in order to be able to perform subtractive hybridization on minute amount of samples. DOP-PCR has been optimized for capillary PCR machine to speed up the amplification process and make it more specific. Combining DOP-PCR with SSH enabled us to perform subtraction hybridization on cDNAs isolated from resting B cells and on its freshly (2-week) EBV-infected counterpart. The identified differentially expressed genes were confirmed by ratio PCR and a novel method, termed DOP-Northern. Two cellular genes were identified among the subtracted clones. One of them was the DCPS gene, which is a scavenger pyrophosphatase. The other upregulated gene was the BLNK (B cell linker protein).The BLNK adaptor is expressed throughout B cell ontogeny and plays an essential role in signal transduction from the BCR. The activation of the BLNK by EBV might indicate the virus's strategy to prevent apoptosis of the activated B cell in the absence of CD40 ligand.

The roles of the newly identified genes in EBV-associated malignancies are mostly unknown. Experiments are under way to identify the viral genes that activate them. This might help us to understand better the role of EBV in trumorigenesis.