Deciphering the complex mechanisms that reprogram gene expression and promote Epstein Barr Virus replication.

Epstein-Barr virus (EBV) is an important human pathogen associated with cancer and other diseases (nasopharyngeal carcinoma, B-cell-lymphomas, Hodgkin's disease, lymphoproliferative diseases and infectious mononucleosis). Multiplication of EBV is essential to produce the virus particles that allow EBV to infect cells and spread between individuals. Infection of antibody-producing cells in the blood with EBV results in abnormal continual growth of the cells. Most of the time the virus hides in these cells, without replicating. Recently, the replication of the virus has been linked with its ability to cause these cells to grow continuously.
One viral gene has been nicknamed "The master regulator of lytic EBV replication". The gene generates a protein, known as Zta, which is required for replication of the viral genome. Zta alters the expression of viral and human genes by binding directly to DNA in the genomes. It recognises a very short region of DNA, containing only 7 DNA base-pairs - of 2041 potential combinations, there are 32 functional variations of this code and each is called a ZRE.
Zta increases the expression of some genes but decreases the expression of others. The ability of Zta to increase the expression of viral genes is key to its ability to promote viral replication and the molecules that allow Zta to increase gene expression are partly understood. In contrast little is known of the mechanism of Zta causing decreased gene expression. Two cellular genes known to be down-regulated by Zta are normally required by the immune system to function, so Zta may allow EBV to avoid destruction by the immune response.
Here, we aim to discover the molecular mechanisms that determine whether the expression of a gene is increased or decreased by Zta.
(i) We will employ state-of-the-art techniques (ChIP-Seq) to identify binding sites for Zta in the human genome. We will also determine whether genes near the Zta binding sites have increased or decreased gene expression. We will also investigate the other proteins that bind to genes near Zta and will investigate whether the DNA at Zta-binding sites is methylated or not. We can then select representative human genes to investigate further to identify which neighbouring DNA elements cause Zta interaction with a ZRE to either increase or decrease gene expression.
(ii) We will explore whether Zta-mediated increases or decreases in gene expression are influenced by the attachment of a small protein molecule (SUMO) to Zta or through Zta binding to other host proteins using a proteomics strategy.
(iii) This project will identify those host genes that are regulated by Zta during viral replication and so is likely to highlight further mechanisms used to reprogram human cells for viral replication. This will increase our understanding of the basic process of EBV replication and potentially these can be explored as targets for future drug discovery.

The aim of this research is to determine the molecular mechanisms that determine activation and repression of gene expression by Zta.
Two cell systems representing natural lineages for EBV replication will be used. (a) Modified B-lymphoma cells (Akata) that harbour latent EBV and a truncated CD2 gene under the control of an EBV lytic cycle promoter. Upon stimulation with anti-IgG, a subpopulation of cells express Zta and enter lytic cycle; those cells express the truncated surface CD2 protein that can be physically isolated using paramagnetic beads coated with anti-CD2 antibody. (b) HEK 293 cells harbouring latent EBV will be transfected with a Zta expression vector to stimulate lytic cycle. This process is efficient and no selection is necessary.
Cells will be harvested for several assays:
(i) Proteins and DNA will be cross-linked using formaldehyde, and chromatin prepared for chromatin precipitation assays (ChIPs) with commercial antibodies. Libraries will be generated using reagents from Illumina. Second-generation DNA sequencing, data processing and alignment to the genome will be undertaken at UCL Genomics.
(ii) RNA will be prepared, and cDNA generated for the quantitation of gene expression using Q-PCR with gene-sepcific TaqMan reagents from ABI.
(iii) DNA will be harvested and bisulphite converted. The Illumina Infinium Methylation Assay will be undertaken at UCL Genomics on a human gene regulatory region array.
Zta carrying a HaloTag (Promega) will be expressed transiently in cells and Zta and associated proteins purified using Halo-Link resin (Promega). Mass spectrometry of the purified protein will be undertaken using nano-scale liquid chromatography MS/MS at the University of Sussex proteomics facility (Dionex Ultimate U3000/ThermoScientific LTQ-Orbitrap Hybrid FT-MS/MS).
Immuneprecipitation/western blot analysis will be used to validate interactions, with further analysis using ChIP for Zta followed by re-CHiP for the associated protein.

The identification of genes regulated by Epstein-Barr virus and the mechanisms by which this is achieved will (i) increase the academic knowledge base in the UK and (ii) may be of interest to biotechnology companies attempting to generate antiviral reagents. These genes may provide targets against which to design drugs to interfere with the replication of EBV. In the future this may be beneficial for (iii) people who are particularly susceptible to EBV associated cancers (e.g., immunosuppressed transplant patients).
The identification of genes regulated by Epstein-Barr virus may also be of use to (i) the academic community and (ii) biotechnology companies who may want to promote expression of EBV replication genes specifically in cancer cells in order to expose them to the immune system. In the future this may be beneficial to (iii) people who have EBV associated cancers that are refractory to current treatments. At present in the UK, the most numerous patient group are those with Hodgkin's disease; however worldwide, the most numerous patient groups up those suffering from nasopharyngeal carcinoma (Far East) and Burkitt's lymphoma (sub-Saharan Africa).
The project will provide training in widely applicable skills to two biomedical researchers. One will gain valuable experience in state-of-the-art chromatin precipitation/next-generation sequencing and the second will gain valuable experience in proteomics. Both of these areas are widely applicable to many areas of biomedical and other biological research, and would equip the researchers to provide valuable contributions to research in the future in (i) biomedical research setting or (ii) a biotechnology company.