Route of Entry-Dependent Blocks to Retroviral Replication A thesis submitted to University College London in part fulfilment o f the requirements for the degree o f Doctor of Philosophy July 2008 Eleanor Ruth Gray Division of Virology National Institute for Medical Research The Ridgeway M ill H ill London NW7 1AA UMI Number: U591600 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U591600 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 I, Eleanor Ruth Gray, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. Abstract Restriction factors are endogenous cellular proteins that block retroviral replication at specific points in the life cycle. Those identified so far include F vl, Trim5a and TrimCyp. Their characterisation has extended knowledge o f retroviral and cellular functions, and has added a new branch to innate immunity. Retroviral susceptibility to Fvl and Trim5a is determined by its capsid, and is manifested in a pre- (Trim5a, TrimCyp) or post- (F vl) reverse transcription block to replication. Other blocks to replication have been postulated. For example, a novel anti-viral factor, Lv2, is thought to block replication o f several primary isolates of HIV-2 in some cell lines. Knowledge o f the early steps o f virus replication, between entry and nuclear import, is critical to understanding restriction. The intention o f the studies described in this thesis was to determine whether alternative routes of virus trafficking might affect susceptibility to Fvl and Trim5a, as well as to the putative Lv2. A system of two receptors was used, Tva800 and Tva950; both permit entry via ASLV envelope protein, but take the virus into the cell by two different endocytic mechanisms. The pathways traversed after binding to Tva800 and Tva950 were investigated and shown not to reroute virions around restriction mediated by Fvl and Trim5a. When virus titration curves were analysed, a distinctive pattern emerged suggesting that entry via Tva800, but not Tva950, requires engagement of more than one receptor- envelope pair. The block to replication caused by the putative factor Lv2 was also analysed. It was concluded that a combination o f low surface CD4 expression and poor receptor engagement are the cause o f low viral titres in some cell lines, rather than a cellular anti-viral factor per se. Acknowledgements I owe so much to the support o f so many. Firstly to Jonathan, for so much patience, for putting up with me and for encouraging excellent science. A huge debt of gratitude is also owing to the rest o f the Stoye lab, past and present, to Melvyn Yap, Mark Dodding, Sada Okhura, Rebecca Butcher, Seti Grambas and Laura Hilditch, for teaching me so much about being a good scientist, for being a pleasure to work with, and also for some cracking recipes. Outside o f the lab and inside o f science I am also grateful to M olly Strom, Barry Ely, Steve Wharton, and Srividya Sriskantharajah for useful advice on vectors, immunology, and random odd chemicals. Outside of science the whole thesis process was made so much easier by the Lisbume Ladies who never moved my papers around the lounge, so thanks to Anna-Lea Cooke and Kate Davenport, and I am also particularly grateful to Caroline Goringe who made the day to day process of writing up pass far more enjoy ably than it really should have done. After it was all done, Anna Hughes was extremely tactful and utterly correct in her proof-reading. And how could I forget any o f my friends who have listened to me talk about my work over the last few years without having a clue what I was going on about - 1 honestly really appreciated it! The support o f my family has been so important to me. Thanks to Morag and Serge for being so encouraging and for the beautiful picture - this is for you (Simpson et al. 2006); I told you I ’d manage it somewhere! Thank you to Julia and Hazel, and to Cameron and Margaret. And thanks are finally and most importantly due to my parents, for rock-solid faith in me, and for being themselves so inspirational. I am among those who think that science has great beauty. A scientist in his laboratory is not only a technician: he is also a child placed before natural phenomena which impress him like a fairy tale. Marie Curie May the words of my mouth and the meditation of my heart be pleasing in your sight. Ps 19vl4 Table of Contents Abstract ............................................................................................................................... 3 Acknowledgements ........................................................................................................... 4 Table o f Contents................................................................................................................7 Index o f Figures and Tables ............................................................................................ 11 Abbreviations .................................................................................................................... 14 Introduction ...................................................................................................................... 16 1.1 Virus Taxonomy ...............................................................................................17 1.2 Genome Organisation ...................................................................................... 18 1.3 Structure ............................................................................................................20 1.4 Proteins .............................................................................................................21 1.4.1 PR .............................................................................................................21 1.4.2 R T .............................................................................................................22 1.4.3 IN ..............................................................................................................22 1.4.4 M A ............................................................................................................23 1.4.5 N C .............................................................................................................23 1.4.6 C A .............................................................................................................24 1.4.7 p 6 ..............................................................................................................24 1.4.8 Env ............................................................................................................25 1.4.9 Accessory proteins ................................................................................... 26 1.5 Virus Infectivity Cycle .................................................................................... 27 1.5.1 Binding .....................................................................................................29 1.5.2 Fusion and Entry ...................................................................................... 30 1.5.3 Reverse Transcription ..............................................................................32 1.5.4 Nuclear Import ......................................................................................... 35 1.5.5 Integration .................................................................................................36 1.5.6 Assembly and E xit ...................................................................................38 1. 6 Vectors and Viral Pseudotypes ........................................................................39 1.7 Entry o f A S L V -A .............................................................................................40 1.7.1 Tva800 and Tva950 .................................................................................40 1.7.2 A S L V E n v ............................................................................................... 42 1.7.3 Mechanism o f entry via ASLV-A Env...................................................43 1. 8 Inhibition of viral replication ...........................................................................46 1.8.1 Fv Susceptibility Genes ...........................................................................46 1.8.2 Trim 5a ......................................................................................................47 1.8.3 APOBEC3G ............................................................................................. 49 1.8.4 Lv2 ............................................................................................................ 50 1.8.5 Other Blocks to Retroviral Infection ......................................................51 1.9 Other factors influencing viral entry .............................................................. 52 1.9.1 Endocytosis ...............................................................................................53