Developing genetic and biological systems for the study of Epstein Barr virus strain diversity

Abstract

Epstein-Barr virus is a ubiquitous gamma herpesvirus that can cause several types of lymphoma and carcinoma. Different viruses have different properties such as lytic reactivation, cell tropism, and oncogenesis, but the genetic determinants of these varied phenotypes are not fully known. There is a need for a better understanding of what constitutes wild-type EBV, as a background against which to identify mutations and/or polymorphisms associated with diseases. Most recent EBV sequencing projects through next-generation sequencing have ignored analysing large repeat regions as their assembly is technically challenging with short- read sequencing technologies. In this thesis, a strategy was developed to assemble and analyse in-depth repeat regions using short-read sequencing data. I have assembled and analysed internal repeat 1 (IR1) for 76 strains and the terminal repeat (TR) for 31 strains. The IR1 sequences showed that the latency promoter Wp, the EBNA-LP W1 and W2 exons, and the short intron between them are highly conserved, as are two regions of unknown function within BWRF1 open reading frame. The length of BWRF1 is not the same in all strains. I show that TR is highly variable across strains but includes conserved regions of known functions (e.g. packaging signals sites) and unknown function. Analysis showed that IR1 and TR are generally heterogeneous and this heterogeneity seems to arise from spontaneous mutation as well as from interstrain recombination. IR1 heterogeneity in tumour-derived strains suggested a larger degree of inter-strain recombination, raising the possibility that this may contribute to EBV pathology. We also identified a novel stop codon in one copy of the W1 exon of the commonly used laboratory strain of EBV, B95.8 and showed that this mutation reduced EBNA-LP protein production and the quality of transformation. Our strategy has highlighted the importance of studying the heterogeneity of repeat regions and shown some approaches that can improve the quality of the final sequence. To study the biological properties of EBV, I tried to develop a method to produce circulating EBV viruses by inducing lytic cycle in spontaneous LCLs (sLCLs) and also by cloning EBVs as BACs. sLCLs were very resistant to lytic induction stimuli. I succeeded in cloning the EBV strain P3HR1 as BAC, but was unable to repeat this consistently, highlighting the difficulty of generating EBV-BACs in latent B cells. The constructed P3HR1-BAC would potentially be useful for studying EBV type 2 biology.