Identification of Epstein-Barr Virus Replication Proteins in

Preparing to load PDF file. please wait...

0 of 0
100%
Identification of Epstein-Barr Virus Replication Proteins in

Transcript Of Identification of Epstein-Barr Virus Replication Proteins in

A University of Sussex PhD thesis Available online via Sussex Research Online: http://sro.sussex.ac.uk/ This thesis is protected by copyright which belongs to the author. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the Author
The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the Author
When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given
Please visit Sussex Research Online for more information and further details

i
Investigating the role of Epstein-Barr virus lytic key regulator protein Zta in transcriptional regulation
By Rajaei Almohammed
A Thesis submitted for the degree of Doctor of Philosophy
School of Life Sciences University of Sussex September 2016

ii
I hereby declare that this thesis has not been and will not be, submitted in whole or in part to another University for the award of any other degree. Signature: ....................................................................................

iii
Acknowledgements
First and foremost, I would like to take the opportunity to thank my supervisor Professor Alison Sinclair for her invaluable and continuous support during this project. Alison has always been there for us and did not spare any effort to help us even when she was going through the hardest times in life. I am truly indebted for her patience, guidance and making me feel welcomed as a valuable member of her group.
I would also like to thank my co-supervisor Professor Michelle West for her help and for making a great effort to provide constructive feedback and insightful comments on my work.
Many thanks go to our lab members for their tremendous support. In particular, Dr. Kay Osborn and Dr. Sharada Ramasubramanyan for sharing their knowledge and expertise with me. I would also like to thank Chris Traylen, Barak Perez-Fernandez, Anja Godfrey, and Yaqi Zhou for being my best friends and colleagues at the same time. Besides our lab members, I would like to thank every member of the West’s group for their continuous encouragement.
I would like to thank all the undergraduate and master students who undertook their projects in our lab during my Ph.D. project. Being able to share my knowledge with them and observe their success had always been the source of my joy and inspiration.
I am so grateful to my scholarship donor, King’s Abdullah scholarship program, who offered me with generosity the opportunity to do a Ph.D. degree.
Last but not least, I would like to thank all my family for their endless support and understanding. Words cannot express how much I miss the feeling of being home in their presence. Many special thanks also go to Deema Jallad; I am truly indebted to her for all the help and thoughtful words of encouragement.

iv
UNIVERSITY OF SUSSEX RAJAEI ALMOHAMMED Ph.D. in BIOCHEMISTRY Investigating the role of Epstein-Barr virus lytic key regulator protein Zta in transcriptional regulation
Summary
Epstein-Barr virus (EBV) is a human herpes virus that, upon primary infection, establishes life-long persistence in B cells (latency). One viral protein, the immediate early lytic protein Zta (also known as BZLF1, ZEBRA, EB1, and Z) plays a significant role in disturbing this latency and inducing a viral productive (lytic) cycle. Expression of Zta, a basic leucine zipper transcription factor, induces a cascade of viral lytic cycle gene expression. The activation of many lytic genes requires the direct binding of Zta to its response elements (ZREs) within proximal promoters of these genes. Much research in recent years has focused on investigating Zta reactivation of latency and its role in lytic viral DNA replication. This has revealed a wealth of knowledge about Zta as a multifunctional transcription factor. However, a complete understanding of Zta transcriptional activation is still missing. Here, utilising ChIP-qPCR, we showed conserved binding patterns for Zta across several EBV lytic gene promoters in two different EBV systems including a non-B cell EBV-infected cell line. Also, using luciferase reporter assays, we show the first functional evidence for a possible role of Zta in controlling transcription regulation at distal regulatory elements (enhancers). Importantly, we identified BNLF2a, an essential viral immune evasion gene, as a direct target for Zta. We identified five ZREs and mapped functional ones within the BNLF2a promoter using mutational analysis and luciferase reporter assays. We also expressed and purified a recombinant GFP-bZIP Zta protein to address Zta binding to BNLF2a ZREs in vitro. Interestingly, using in silico approach, we also identified a conserved sequence in the ZRE flanking region of all five ZREs within BNLF2a promoter and uncovered a role for a possible repressor at ZRE2 flanking region. Our work not only adds to our understanding of Zta transcription regulation but characterises for the first time the regulation of a novel Zta target that has a role in evading the host immune system during EBV pre-latency and lytic cycle.

v
Table of Contents
Chapter 1 Introduction ------------------------------------------------------------------------ 1-14
1.1 Viruses and cancer--------------------------------------------------------------------------------------1-14 1.2 Herpesviruses --------------------------------------------------------------------------------------------1-18 1.3 Epstein- Barr virus (EBV) ------------------------------------------------------------------------------1-20
1.3.1 EBV structure-----------------------------------------------------------------------------------------------------1-20 1.3.2 EBV genome ------------------------------------------------------------------------------------------------------1-23 1.3.3 EBV strains and subtypes -------------------------------------------------------------------------------------1-27 1.3.4 EBV life cycle -----------------------------------------------------------------------------------------------------1-28 1.3.5 EBV cell tropism ------------------------------------------------------------------------------------------------- 1-44 1.3.6 EBV-associated diseases---------------------------------------------------------------------------------------1-46
1.4 Zta (BZLF1) ------------------------------------------------------------------------------------------------1-60
1.4.1 Zta structure and DNA binding ------------------------------------------------------------------------------ 1-61 1.4.2 Zta promoter (Zp) ----------------------------------------------------------------------------------------------- 1-65 1.4.3 Zta protein interactions and functional diversity-------------------------------------------------------1-67 1.4.4 Zta transcriptional regulation function--------------------------------------------------------------------1-69
1.5 EBV immune evasion-----------------------------------------------------------------------------------1-71
1.5.1 The role of BNLF2a in immune evasion -------------------------------------------------------------------1-73
1.6 Project Aims ----------------------------------------------------------------------------------------------1-75 Chapter 2 Materials and Methods -------------------------------------------------------- 2-76
2.1 Materials, and reagents -------------------------------------------------------------------------------2-76
2.1.1 DNA constructs (plasmids)------------------------------------------------------------------------------------2-76 2.1.2 Cell lines -----------------------------------------------------------------------------------------------------------2-77 2.1.3 Antibodies---------------------------------------------------------------------------------------------------------2-77 2.1.4 Primers (oligonucleotides)------------------------------------------------------------------------------------2-78

vi
2.1.5 Solutions and buffers ------------------------------------------------------------------------------------------2-80 2.1.6 Kits and reagents ------------------------------------------------------------------------------------------------2-82
2.2 Methods ---------------------------------------------------------------------------------------------------2-84
2.2.1 General nucleic acid methods -------------------------------------------------------------------------------2-84 2.2.2 General protein methods ------------------------------------------------------------------------------------- 2-92 2.2.3 Tissue culture ----------------------------------------------------------------------------------------------------2-93 2.2.4 Luciferase reporter assays ------------------------------------------------------------------------------------2-95 2.2.5 Chromatin immunoprecipitation (ChIP-qPCR) ----------------------------------------------------------2-96 2.2.6 Protein expression and purification (His-GFP-bZIP Zta)-----------------------------------------------2-99 2.2.7 HeLa cell nuclear extract ------------------------------------------------------------------------------------ 2-101 2.2.8 Electrophoretic mobility shift assay ---------------------------------------------------------------------- 2-101 2.2.9 Computational methods ------------------------------------------------------------------------------------ 2-103
Chapter 3 Zta binding across the viral genome -----------------------------------3-104
3.1 Introduction -------------------------------------------------------------------------------------------- 3-104
3.2 Results --------------------------------------------------------------------------------------------------- 3-106
3.2.1 ChIP-qPCR approach to analyse Zta binding to EBV genome-------------------------------------- 3-106 3.2.2 Zta binds similarly to EBV OriLyt -------------------------------------------------------------------------- 3-112 3.2.3 Zta binds similarly to promoter regions of the immediate-early genes ------------------------ 3-114 3.2.4 Zta binds similarly to promoter regions of various early lytic genes ---------------------------- 3-116 3.2.5 Zta binds similarly to promoter regions of various late lytic genes------------------------------ 3-118 3.2.6 Zta binds to a novel site with unknown regulatory function -------------------------------------- 3-120
3.3 Discussion ----------------------------------------------------------------------------------------------- 3-124 Chapter 4 ZREs as long-distance enhancer elements--------------------------4-129
4.1 Introduction -------------------------------------------------------------------------------------------- 4-129
4.2 Results --------------------------------------------------------------------------------------------------- 4-132
4.2.1 Luciferase reporter assay to investigate ZREs as potential enhancer elements ------------- 4-132

vii
4.2.2 Zta activates a heterologous minimal promoter through distal ZREs --------------------------- 4-136 4.2.3 Zta activates a known target promoter through distal ZREs -------------------------------------- 4-138
4.3 Discussion ----------------------------------------------------------------------------------------------- 4-140 Chapter 5 Zta activates viral immune evasion gene BNLF2a through direct binding to proximal promoter ZREs ----------------------------------------------------5-143
5.1 Introduction -------------------------------------------------------------------------------------------- 5-143 5.2 Results --------------------------------------------------------------------------------------------------- 5-146
5.2.1 Zta binds at the BNLF2a promoter during lytic cycle ------------------------------------------------ 5-146 5.2.2 BNLF2a predicted ZRE sequences.------------------------------------------------------------------------ 5-150 5.2.3 BNLF2a promoter constructs to investigate each ZRE function ---------------------------------- 5-152 5.2.4 Zta activates BNLF2a through promoter ZREs in different cell lines ---------------------------- 5-159 5.2.5 The proximal ZREs to the TSS are essential for BNLF2a activation.------------------------------ 5-161 5.2.6 ZREs functional redundancy-------------------------------------------------------------------------------- 5-165 5.2.7 In vitro analysis of Zta binding to BNLF2a ZREs ------------------------------------------------------- 5-172
5.3 Discussion ----------------------------------------------------------------------------------------------- 5-180 Chapter 6 Other elements play a role in BNLF2a gene regulation --------6-187
6.1 Introduction -------------------------------------------------------------------------------------------- 6-187
6.2 Results --------------------------------------------------------------------------------------------------- 6-189
6.2.1 The conservation of a motif flanking BNLF2a ZREs -------------------------------------------------- 6-189 6.2.2 Investigating other regulatory elements within BNLF2a promoter------------------------------ 6-191 6.2.3 A single point mutation in the TATA box enhances Zta activation of BNLF2a ---------------- 6-197 6.2.4 The KLF4 site plays a role in BNLF2a promoter regulation----------------------------------------- 6-201 6.2.5 Mutational analysis of the motif flanking ZREs revealed a repressor binding site ---------- 6-204 6.2.6 Zta binding to ZRE2 is not affected by mutation in the flanking motif ------------------------- 6-209 6.2.7 Specific complex binds to ZRE2 flanking sequence (E-box) in HeLa cells nuclear extract - 6-211

viii 6.3 Discussion ----------------------------------------------------------------------------------------------- 6-213 Chapter 7 General discussion ------------------------------------------------------------7-219 References -------------------------------------------------------------------------------------------7-225 Appendix A. Publications -------------------------------------------------------------------------7-255

ix
Table of Figures
Figure 1-1 EBV structure................................................................................................................... 1-22 Figure 1-2 EBV genome .................................................................................................................... 1-26 Figure 1-3 EBV life cycle ................................................................................................................... 1-32 Figure 1-4 Zta (BZLF1) structure ....................................................................................................... 1-64 Figure 1-5 BZLF1 locus and promoter ............................................................................................... 1-66 Figure 3-1 Differences in Zta binding to the EBV genome. ...............................................................3-109 Figure 3-2 Experimental design of Zta ChIP-qPCR ............................................................................3-111 Figure 3-3 Zta binding at the origin of lytic replication (OriLyt). ......................................................3-113 Figure 3-4 Zta binding at the immediate-early lytic promoters........................................................3-115 Figure 3-5 Zta binding at the early lytic promoters..........................................................................3-117 Figure 3-6 Zta binding at the late lytic promoters............................................................................3-119 Figure 3-7 Zta binding at a region with unknown regulatory function (~82kbp). .............................3-122 Figure 3-8 Sequence variations between EBV strain at the ~82kbp region. .....................................3-123 Figure 4-1 Investigating a long-range ZRE element effect on promoter activation. .........................4-134 Figure 4-2 DNA sequences of the long-range ZREs element and heterologous promoters...............4-135 Figure 4-3 A long-range ZRE element drives Zta activation of a heterologous minimal promoter. ..4-137 Figure 4-4 A long-range ZRE element drives Zta activation of a known Zta target promoter. ..........4-139 Figure 4-5 Increasing evidence suggesting a role for Zta at enhancer elements. .............................4-142 Figure 5-1 Zta binding at BNLF2a promoter. ....................................................................................5-148 Figure 5-2 Zta binding at BNLF2a promoter region in different cell lines. ........................................5-149 Figure 5-3 Zta response elements (ZREs) upstream of BNLF2a gene. ...............................................5-151 Figure 5-4 Investigating BNLF2a promoter activity. .........................................................................5-154 Figure 5-5 BNLF2a promoter sequence along with the predicted ZRE motifs. .................................5-155 Figure 5-6 A schematic diagram of BNLF2a promoter......................................................................5-156 Figure 5-7 BNLF2a ZREs luciferase promoter constructs. .................................................................5-158 Figure 5-8 Zta activates BNLF2a through ZREs. ................................................................................5-160 Figure 5-9 The proximal ZREs contribute the most to BNLF2a activity. ............................................5-164
RoleZresZtaThesisAuthor