Function and transport of a herpesvirus encoded Ubiquitin-specific protease in virus entry and assembly A thesis submitted to the degree of Doctor of Philosophy Imperial College London, Department of Medicine by Thomas Hennig Supervised by Professor Peter F. O’Hare Supported by Imperial College London and the Wellcome Trust September 2011 - June 2015 - 1 - Acknowledgements Acknowledgements Foremost, I would like to thank Professor Peter O’Hare for his support throughout my PhD and during the post-doctoral application process. There was not a single day when you could not ask him a question or discuss results. I think I speak for everyone in the group (and outside) in that he was always approachable, patient and that I felt well supported at every point during my PhD. The last four years working with present and past members of the group have been a great experience, allowed me to learn many new techniques and made the learning process more manageable. I even started appreciating the calming influence of classical music. I would also like to thank Dr Fernando Abaitua for his help during my first year and the very detailed discussions during the early morning hours, even after having left our group over two years ago. A big ‘thank you’ and ‘sorry’ also goes to Dr Sonia Barbosa, who could usually not escape these elaborate discussions in a timely fashion, to Dr Remi Serwa, who performed the mass spectrometry, and Nora Schmidt, who had the pleasure of reading and commenting on my thesis. I was supported by two very helpful assessors, Dr Goedele Maertens and Professor Wendy Barclay, who were always happy to advise me on any issues that arose from the work presented here. Dr Maertens kindly provided the retrovirus backbones which enabled the creation of the two inducible cell lines used for the identification of VP1-2 interacting proteins. This work would have also taken much longer without the help of Dr Anna Stockum (Dr Maertens group) who helped with the retrovirus infections. During the write-up period I also received serveral helpful comments on my thesis by my PhD examiners for which I am very grateful. There is an extensive list of additional people both at St Mary’s and outside whose help should be acknowledged here but they cannot all be listed here. I am eternally grateful for all that they have done to make my work easier and overall a very pleasant experience. I would like to thank Professor Søren Paludan for enabling a short exchange to his laboratory at Aarhus University to learn valuable skills and for discussing and supporting my application to the Wellcome Trust for further funding. Finally, I was lucky enough to be funded by the Wellcome Trust, which enabled me to present and publish some of my data and attend the laboratory of Professor Søren Paludan in Aarhus (Denmark) through provision of generous grants. Substantial additional funds towards travel and accommodation were granted by the IHW and the SGM. - 2 - Declarations Declaration of Originality I confirm that this thesis has been written and the work conducted by myself unless collaborators were acknowledged. This thesis contains the work on which I expect to be examined for the degree of Doctor of Philosophy. All work that was not conducted by myself is referenced appropriately and permissions were obtained when necessary and in accordance with Imperial College policy. Copyright declaration The copyright of this thesis rests with the author and is made available under a creative commons attribution non-commercial no derivatives licence. Researchers are free to copy, distribute or transmit that thesis on the conditions that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work. - 3 - Abstract Abstract Herpes simplex virus type I (HSV-1), the prototype α-herpesvirus (HV), is a double stranded DNA virus that replicates in the nucleus of infected cells. The nuclear pore represents a gateway that must be engaged and navigated immediately after cell entry for successful infection by many classes of human viruses. For herpesviruses, capsid-tegument assemblies must be targeted to the pore where the viral genome exits and transport into the nucleus occurs. We currently have little mechanistic knowledge of this fundamental step of infection. A swath of evidence indicates that the conserved tegument protein VP1-2 is essential for early capsid transport and pore binding, and that it contains a conserved nuclear localisation signal (NLS) required for pore docking. In this thesis I undertook a detailed analysis to dissect functional determinants within the NLS from herpes simplex virus, to examine putative NLSs in VP1-2 homologues from representatives of all sub-families, to characterise protein interactions with VP1-2 and finally to construct a GFP expressing entry defective recombinant virus to study the consequences of infection. I show that the HSV NLS can function as a mono-or bipartite motif and has a particular organisation conserved in the -herpesvirus homologues but distinct from those in the - and - herpesviruses. The representatives of all 3 classes contain a functional NLS at approximately the same position. All bi-partite motifs were able to rescue the HSV VP1-2ΔNLS virus defect albeit to varying extent whereas the mono-partite HHV-8 motif did not. I constructed and purified chimeric recombinant viruses for the VZV, HCMV and EBV motifs and show distinct differences in their ability to replicate in non-complementing cells. In HSV, NLS function in the context of protein nuclear import or of viable virus replication, was dependent on lysine 428 and the integrity of the full bi-partite motif. Mutations which reduced NLS activity generally caused reduced fitness of recombinant viruses. For the analysis of interaction partners of VP1-2 and the NLS, I developed a one-step approach to analyse the capsid interactome during entry. Additionally, I constructed mammalian GST-VP1-2.NLS fusion proteins and cell lines which inducibly express the N-terminal region of VP1-2 for analysis of NLS interacting proteins. Using mass spectrometry (MS) I identified a number of VP1-2-interacting, cellular proteins including DTX3L, an important regulator of the DNA damage response. - 4 - Abbreviations Abbreviations BES N-bis(2-hydroxyethyl)-2-aminoethane sulfonic acid BHV-1 bovine Herpesvirus-1 BoHV-4 bovine Herpesvirus-4 bpNLS bi-partite NLS BSA bovine serum albumin CA Capsid protein (of HIV) cNLS classical NLS CPE cytopathic effect CV Crystal violet DAB Diaminobenzidine DABCO diazabicyclooctane DAPI 4′,6-Diamidin-2-phenylindol ddH2O double distilled H2O DDR DNA damage response DMEM Dulbecco's Modified Eagle's Medium DMSO dimethyl sulfoxide dsDNA/RNA double stranded DNA/RNA EBV Epstein Barr Virus EHV equine Herpesvirus EM electron microscopy ER endoplasmatic reticulum ERAD ER associated protein degradation FBS fetal bovine serum GAG glycosaminoglycan gD (or similar) HV glycoprotein D GST Glutathione-S transferase HA hemagglutinin HBS HEPES buffered saline HCMV human Cytomegalovirus HHV human Herpesvirus HIV human immunodeficiency virus HP1 heterochromatin protein 1 HR homologous recombination HRP horseradish peroxidase HS human serum HSV herpes simplex virus HV herpesvirus HVEM HV entry mediator HVS Herpesvirus Saimiri IAV influenza A virus IBB Importin-β binding domain ICP infected cell protein IN integrase (of HIV) INM inner nuclear membrane - 5 - Abbreviations IP immunoprecipitation ISG interferon stimulated genes lTag large T antigen (of SV40) M1/2 matrix protein 1/2 (of IAV) MCS multiple cloning site MLV murine leukemia virus MOI multiplicity of infection mpNLS mono-partite NSL MS mass spectrometry MTOC microtubule-organising centre MuHV murid Herpesvirus MyoNLS NLS of Myopodin NCS new-born calf serum NE nuclear envelope NEAA non-essential amino acids NEBD nuclear envelope breakdown NES nuclear export signal NHEJ non-homologous end joining NLS nuclear localisation signal NPC nuclear pore complex NP nucleoprotein (of IAV) Nups Nucleoproteins ONM outer nuclear membrane ORF open reading frame PAA phosphono acetic acid PBS phosphate buffered saline Pen/Strep Penicillin/Streptomycin PFA paraformaldehyde PFU plaque forming unit PIC pre-integration complex (of HIV) PMSF Phenylmethanesulfonyl fluoride PRV Pseudorabies Virus pUL37 protein product of open reading frame 37 of unique long genome segment pUS3 protein product of open reading frame 3 of the unique short genome segment SDS sodium dodecyl sulfate SDS-PAGE SDS polyacrylamide gel electropophoresis ssDNA/RNA single stranded DNA/RNA SV40 Simian virus 40 TGN trans Golgi network ts mutant temperature-sensitive mutant USP ubiquitin-specific protease Vhs virion host shutoff protein VP virion protein Vpr viral protein R (of HIV) VZV Varicella zoster virus β-gal β-galactosidase - 6 - Table of contents 1 INTRODUCTION ................................................................................................... - 16 - 1.1 Introduction to herpesviruses ............................................................................................... - 16 - 1.1.1 Discovery and classification of Herpesviruses .................................................................... - 16 - 1.1.2 Introduction to Herpes Simplex Virus ................................................................................ - 17 - 1.1.3 Virion structure..................................................................................................................