Host genes targeted by Epstein Barr virus during replication
Epstein-Barr virus (EBV) is an important human pathogen associated with nasopharyngeal
carcinoma, B-cell-lymphomas, Hodgkin’s disease, lymphoproliferative diseases and
infectious mononucleosis [1]. Lytic replication of EBV in B-cells and epithelial cells
produces the virions that allow EBV to spread between cells and individuals, and stimulation
of lytic replication of EBV within tumours is being explored as a mode of oncolytic cancer
therapy.
The master regulator of lytic EBV replication is the viral transcription and replication protein
Zta (also known as ZEBRA, BZLF1, EB1) encoded by the BZLF1 gene. Zta has the ability to
both activate and repress gene expression through direct interaction with a core 7-nucleotide
Zta response element (ZRE) in viral and host genes or via indirect cellular DNA binding
proteins (eg C/EBP alpha and p53) [2].
The PhD student will identify host genes directly regulated by Zta in epithelial cells
undergoing lytic cycle and determine their contribution to EBV replication. To achieve this
we have:
(i)
Established the technique of chromatin precipitation coupled to next-generation
sequencing (ChIP-Seq) for Zta [3].
(ii)
Established conditions where gene expression can be efficiently knocked-down using
lentiviral-based shRNA constructs.
Specifically, the student will initially generate a Zta ChIP-Seq library from epithelial cells
undergoing EBV lytic replication. Next generation DNA sequencing and alignment with the
human genome will be undertaken in collaboration with experts at UCL Genomics. The
student will use web-based bioinformatics tools to identify which host genes contain Zta
binding sites within their promoters, and will use high throughput Taq-Man PCR assays to
determine which of these genes are transcriptionally regulated. The functional relevance of
transcriptionally regulated genes will then be probed by subverting Zta regulation by either
overexpressing repressed target genes or ablating the expression of transcriptionally activated
target genes. The initial prioritization of the analysis of Zta targets will be based on gene
functions predicted to be relevant for EBV replication: for example genes known to be
involved in evading the immune response; epigenetic regulation of gene expression; DNA
replication and glycoprotein synthesis. The impact of the manipulation of expression of each
gene on EBV replication will be assessed initially using quantitative PCR measurements to
determine virion production - additional assays will depend on the function of the gene.
This project represents an opportunity to couple cell biology with state-of-the-art genomics.
The initial discovery phase will lead to an opportunity for the student to select which areas of
biology - identified within the Zta regulated target genes - they focus their detailed analysis.
1.
Rowe, M., et al., Burkitt's lymphoma: the Rosetta Stone deciphering Epstein-Barr
virus biology. Semin Cancer Biol, 2009. 19(6): p. 377-88.
2.
Sinclair, A.J., bZIP proteins of human Gamma Herpesviruses. Journal of General
Virology, 2003. 84: p. 1941-1949.
3.
Ramasubramanyan, S., et al., Genome-Wide Analyses of Zta Binding to the EpsteinBarr Virus Genome Reveals Interactions in both Early and Late Lytic Cycles and an
Epigenetic Switch Leading to an Altered Binding Profile. Journal of virology, 2012. 86(23): p.
12494-502.
Project enquiries should be sent to Prof. Alison Sinclair (A.J.Sinclair@sussex.ac.uk)