Investigating the Structure and Function of HSV-1 Tegument Proteins: UL7 and UL51
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Investigating the structure and function of HSV-1 tegument proteins: UL7 and UL51 Danielle Jane Owen Gonville and Caius College Supervisor: Dr Stephen Graham Division of Virology Department of Pathology September 2017 This dissertation is submitted for the degree of Doctor of Philosophy Summary Danielle Jane Owen Investigating the structure and function of HSV-1 tegument proteins: UL7 and UL51 Herpes simplex virus-1 (HSV-1) has a large double-stranded DNA genome encased within an icosahedral capsid. The capsid is surrounded by a protein-rich layer, termed tegument, and a membranous envelope containing viral glycoproteins. HSV-1 genome replication and encapsidation occurs in the nucleus, after which, DNA-loaded capsids enter the cytoplasm where they undergo tegumentation and assembly. This thesis presents a structural and functional investigation of two HSV- 1 tegument proteins, UL7 and UL51 that are conserved across all herpesviruses. Deletion of UL7 or UL51 results in impaired viral replication, a small plaque phenotype and an accumulation of unenveloped capsids in the cytoplasm, the latter of which is indicative of a defect in tegumentation and/or secondary envelopment. Similar phenotypes have been observed upon deletion of homologous proteins in pseudorabies virus and human cytomegalovirus, suggesting a conserved role for these proteins. This thesis presents evidence for the formation of a UL7-UL51 complex in transfected and infected cells. Pull-down experiments using recombinant UL7 and UL51 protein purified from E. coli demonstrated that the interaction is direct, and mapped the UL7-binding region within UL51. The interaction was shown to be conserved between UL7 and UL51 homologues from murid herpesvirus, ORF42 and ORF55, respectively. The UL7-UL51 complex was purified from E.coli and, after optimisation of the purification protocol and the UL51 construct, a pure protein sample was obtained that was suitable for crystallisation trials. Two conditions were identified that produced reproducible crystals. These crystals proved to be thin and fragile, preventing their analysis by X-ray diffraction. Optimisation of the crystallisation conditions to produce more robust crystals and/or in situ diffraction measurements may yet yield X-ray diffraction data for the complex. Host-cell binding partners for UL7 and UL51 were identified by yeast-two-hybrid screen and quantitative proteomics (SILAC). An interaction between UL51 and the G-Box domain of the centriole protein CPAP was identified by Y2H screen, and validated by immunoprecipitation from transfected cells and in pull-down experiments using recombinant proteins purified from E. coli. The CPAP-binding region within UL51 was mapped and shown to resemble a motif present in the cellular protein STIL that mediates an interaction between STIL and CPAP. Two UL51 point-mutations within the putative CPAP-binding motif blocked the interaction between UL51 and the CPAP G-box domain. A mutant HSV- 1 virus carrying these UL51 mutations was generated, but no difference was evident between single- step growth curves of wild-type HSV-1 and the UL51 mutant. Host-cell proteins pontin and reptin were identified as putative UL7/UL51 interaction partners in two SILAC screens. Purified GST-tagged UL7- UL51 complex was able to interact with pontin and reptin. However, it is likely that the interaction is non-specific since pontin and reptin were also found to bind a misfolded protein in a similar manner. Declaration Declaration This dissertation is the result of my own work and includes nothing which is the outcome of work done in collaboration except as declared in the text. It is not substantially the same as any that I have submitted, or, is being concurrently submitted for a degree or diploma or other qualification at the University of Cambridge or any other University or similar institution. I further state that no substantial part of my dissertation has already been submitted, or, is being concurrently submitted for any such degree, diploma or other qualification at the University of Cambridge or any other University or similar institution. In accordance with the Biology Degree Committee guidelines this thesis does not exceed 60,000 words excluding figure legends, tables, appendices and bibliography. Signed: Danielle Owen Acknowledgments Acknowledgements First and foremost, I would like to express my sincere gratitude to my supervisor Dr Stephen Graham for the opportunity to work on this project. Stephen has been generous with his time, advice and support during the last four years, even during sick leave and whilst on holiday. His knowledge and enthusiasm for science is inspiring and his mentorship was invaluable during this PhD project. I have learnt a lot, thank you! Special thanks goes to my friends and colleagues in Stephen’s lab and numerous members of the Division of Virology, past and present. It has been a delight to work and socialise with so many friendly, funny and extremely helpful people. In particular, fellow PhD student Julia for being a true friend and always being prepared to offer her assistance, and occasionally cake. Thank you to post docs Morag, Chen and Lyudmila for answering my many lab-based questions and conundrums. Thanks are also due to Susanna Colaco (another source of delicious cake), Dr Heather Coleman and Deb Walsh for all they do to support the work in the Division. I am also grateful to Dr Janet Dean for critiquing my first year report, her advice and humour during lab meetings and assistance with SEC-MALS experiments. Thank you to my mentor Dr Colin Crump and members of his lab, especially Viv Conner for generating the mutant viruses used in this work, also Dr Tim Soh and Firoz Ahmed for their friendship and help all things herpes. I am grateful to Dr Edward Emmott for his assistance with the SILAC experiments and Giles Lewis for running the crystallisation facility in CIMR. To my parents, Harry and Julia, without your encouragement and support I simply would not have been able to do any of this, thank you. And to my family at home and in Australia who have always been on hand for advice and a giggle. Last but by no means least, to my wonderful partner Mark for making me happy. It’s mad that you’ve spent the last four years driving between Manchester and Cambridge on the weekends but I’m extremely glad you did. Abbreviations Abbreviations AH – amphipathic helix CCS – cell-to-cell spread Cryo-EM - cryo-electron microscopy CVSC – capsid vertex-specific component DMEM – Dulbecco’s Modified Eagle Medium DSF – differential scanning fluorimetry DTT – dithiothreitol E genes – early genes EBV – Epstein-Barr virus EDTA – ethylenediaminetetraacetic acid ELISA – enzyme-linked immunosorbent assay EYFP – enhanced yellow fluorescent protein FAB – UL7-UL51 complex FITC – fluorescein isothiocyanate FPLC – fast protein liquid chromatography GAB – ORF42-ORF55 complex GFP – green fluorescent protein GSH – glutathione GST – glutathione S-transferase h – hour(s) h.p.i. – hours post infection HCMV – human cytomegalovirus HSV – herpes simplex virus HVEM – herpesvirus entry mediator IE genes – immediate early genes INM – inner nuclear membrane IP – immunoprecipitation IPTG – isopropyl β-D-1 thiogalactopyranoside kpsi – kilopound per square inch KSHV – Kaposi sarcoma-associated herpesvirus L genes – late genes LC-MS/MS – liquid chromatography-mass spectrometry m – minute(s) MALS – multi-angle light scattering MHV – murid herpesvirus MOI – multiplicity of infection MQW – Milli-Q® water MTOC – microtubule organising centre Mw – molecular weight NEC – nuclear egress complex (UL31-UL34) NPC – nuclear pore complex ONM – outer nuclear membrane PAGE – polyacrylamide gel electrophoresis PBS – phosphate-buffered saline PBS-T – phosphate-buffered saline with 0.1% Tween-20 PCR – polymerase chain reaction PDB – Protein Data Bank PFU – plaque forming unit PM – plasma membrane PNPP - p-Nitrophenyl phosphate Abbreviations PrV – pseudorabies virus rpm – rotation per minute RPMI – Roswell Park Memorial Institute s – second(s) SAXS – small-angle X-ray scattering SDS – sodium dodecyl sulphate SEC – size exclusion chromatography SILAC – stable isotope labelling with amino acids in cell culture TGN - trans-Golgi network UL – unique long US – unique short VAC – virus assembly compartment VZV – varicella-zoster virus Y2H – yeast two-hybrid screen Table of contents Table of contents 1 INTRODUCTION ....................................................................................................................... 1 1.1 HISTORY AND TAXONOMY 1 1.2 HUMAN HERPESVIRUSES 2 1.3 HERPES SIMPLEX VIRUS (HSV)-1 4 1.3.1 HERPES SIMPLEX VIRUS-1 GENOME ORGANISATION ............................................................................... 5 1.3.2 HSV-1 VIRION STRUCTURE ................................................................................................................ 6 1.3.3 HSV-1 LIFE CYCLE .......................................................................................................................... 10 1.3.4 HSV-1 TEGUMENT PROTEINS: UL7 AND UL51 ................................................................................... 25 1.4 OBJECTIVE AND AIMS OF THIS STUDY 28 2 MATERIALS AND METHODS ................................................................................................... 29 2.1 MOLECULAR BIOLOGY 29 2.1.1 POLYMERASE CHAIN REACTION (PCR) ............................................................................................... 29 2.1.2 DNA ANALYSIS BY AGAROSE