Manipulation of host cell signaling pathways and translation factors during poxvirus infection A thesis submitted for the degree of Ph.D. Dublin City University by Izabela Zaborowska M.Sc. The research work described in this thesis was carried out under the supervision of Dr. Derek Walsh National Institute for Cellular Biotechnology Dublin City University Republic of Ireland 2010 I hereby certify that this material, which I now submit for assessment on the programme of study leading to the award of Ph.D. is entirely my own work, that I have exercised reasonable care to ensure that the work is original, and does not to the best of my knowledge breach any law of copyright, and has not been taken from the work of others save and to the extent that such work has been cited and acknowledged within the text of my work. Signed: ______________________ ID No.: _______________ Date: ______________ ABSTRACT Poxviruses are large double-stranded DNA viruses that replicate exclusively in the cytoplasm of infected cells in distinct sites termed viral factories, with Vaccinia Virus (VacV) acting as the laboratory prototype for those that infect humans. Despite encoding proteins responsible for DNA replication and mRNA transcription, like all viruses they remain dependent on the host-cell translational machinery to produce viral proteins. Although recent studies have shown that VacV stimulates mTOR activity and redistributes translation factors to viral factories, how this is accomplished and its importance to VacV replication remain unknown. Here, we demonstrate that VacV activates the PI3 kinase (PI3K) to stimulate its downstream substrate mTOR, thereby inactivating the translational repressor protein, 4E-BP1. This provides the first evidence that VacV activates PI3K signal pathway in a manner distinct from other poxviruses studied, such as myxoma virus, which directly stimulates Akt activity in a PI3K-independent mode. Using specific inhibitors we dissect the role of various components of PI3K/Akt pathway and show that rapamycin- insensitive mTORC1 and mTORC2 activity is responsible for stimulating the formation of the translation initiation factor, eIF4F, which drives cap-dependent translation in infected and uninfected cells. We also show that eIF4F is important but not absolutely essential for viral protein synthesis. In addition, we demonstrate that inactivation of the PI3K by either LY294002 or wortmannin does not cause apoptosis in human or monkey cell lines. In an attempt to understand how VacV causes the redistribution of host translation initiation factors to viral factories, we performed immunoprecipitation-based screens and identified the product of the VacV I3L gene, the single-stranded DNA (ssDNA) binding protein, I3 as interacting with eIF4F. We demonstrate that I3 binds eIF4G directly in vivo and in vitro within the eIF4G C-terminal domain. PAA, a DNA polymerase inhibitor that blocks viral factory formation and entry into late stages of infection, did not significantly affect the ability of I3 to interact with eIF4F, although it did block re-localization of these factors. Cloned I3 was able to mediate the binding of initiation factors to ssDNA in cell extracts, suggesting that I3 sequesters host translation factors within viral factories as they form. Our attempt to recover VacV ∆I3L mutants using the Vac-Bac/ λ system was unsuccessful, suggesting that the I3L gene product (I3) is essential during VacV infection. Overall, our results provide new insights into how VacV manipulates host signaling and translation initiation factor function and their importance in viral protein synthesis and replication, highlighting a novel approach by a DNA virus to manipulate the host-cell translation initiation machinery. Acknowledgements First of all I would like to thank my supervisor, Dr. Derek Walsh, who introduced me to the magic world of science. With his enthusiasm, optimism and brilliant ideas he provided me support and encouragement during these years of my research, his guidance has been invaluable. Thanks also to Prof. Martin Clynes and all others in the Centre who contributed to this thesis, especially Mick for helping with protein identification, and Finbarr for assistance with the microscopes and “lending” the antibodies. Many thanks to Joe for his excellent technical assistance. I am also grateful to Mairead, Carol, Yvonne, and Sree for being incredibly kind, helpful and having things in hand. I appreciatively thank Prof. Michael Clemens and Dr. Niall Barron for their constructive comments and critical insights on my thesis, and for being the members of my examining committee. Thanks to Rob for the helpful discussions, Kerstin and Patricia for all the girly talks and gossip. Many thanks to all my friends, especially Monika and Marta for their support during my every crisis. A special thank you goes to Suraj for his patience, motivational lectures and a company in every joy and sadness I’ve been through during the past 3 years of my studies. Lastly, and most importantly, I would like to thank my parents, Wanda and Adam for supporting me in all my decisions and always believing in me. To them I dedicate this dissertation. To my parents Dla Rodziców Abbreviations 4E-BP - eIF4E-binding protein Ab - antibody ATP - adenosine triphosphate BrdU - bromodeoxyuridine BSA - bovine Serum albumin BSC40 - monkey epithelial kidney cells C2 domain - protein kinase C homology domain 2 cDNA - complementary DNA C-terminus - carboxy-terminus DEPC - diethyl pyrocarbonate DMEM - Dulbecco’s modified Eagle’s medium DMSO - dimethyl sulfoxide DNA - deoxyribonucleic acid ds - double stranded DNAse - deoxyribonuclease dNTP - deoxynucleotide triphosphate DTT - dithiothreitol EDTA - ethylene diamine tetracetic acid eIF - eukaryotic initiation factor FBS - fetal bovine serum FKBP12 - FK506-binding protein, molecular mass of 12 kDa g - gram GST - glutathione-S-transferase HEK - human embryonic kidney HEPES - N-[2-Hydroxyethyl]piperazine-N’-[2-ethanesulphonic acid] IC 50 - inhibitory concentration 50% IPTG - isopropyl β-D-1-thiogalactopyranoside IRES - internal ribosome entry site kDa - kilo Daltons kb - kilobase kbp - kilobase pair M - mole MAb - monoclonal antibody MAPK - microtubule associated protein kinase Met - methionine Met-tRNA i - methionyl-initiator transfer ribonucleic acid min - minute mRNA - messenger RNA miRNA - micro ribonucleic acid ml - mililitre mm - milimetre mM - mili mole Mnk - MAP kinase interacting kinase mTOR - mammalian target of rapamycin MW - molecular weight µ - micro NHDF - normal human diploid fibroblast N-terminus - amino-terminus OD - optical density P - phospho- PAA - phosphonoacetic acid PABP - poly(A)-binding protein PBS - phosphate buffered saline PCR - polymerase chain reaction PDK1 - phosphoinositide-dependent kinase 1 PI - phosphoinositide PI3K - phosphoinositide-3 kinase PIP 2 - phosphatidylinositol-4,5-biphosphate PIP 3 - phosphatidylinositol-3,4,5-triphosphate PtdIns - phosphatidylinositol PTEN - phosphatase and tensin homolog deleted on chromosome 10 raptor - regulatory-associated protein of mTOR rictor - rapamycin-independent companion of mTOR RNA - ribonucleic acid RNAse - ribonuclease RNAi - RNA interference RNAsin - ribonuclease inhibitor rpm - revolution(s) per minute SDS - sodium dodecyl sulphate SDS-PAGE - sodium dodecyl sulphate-polyacrylamide gel electrophoresis sec - second siRNA - small interfering ribonucleic acid SSB - single-strand DNA-binding ssDNA - single-stranded DNA ssRNA - single-stranded RNA TBS - tris buffered saline TCA - trichloroacetic acid TE - Tris-EDTA TEMED - N, N, N’, N’-tetramethyl-ethylenediamine UHP - ultra high purity UTR - untranslated region V - volt VacV - Vaccinia virus VarV - Variola virus WB - Western blot xg - times gravity Table of contents INTRODUCTION 1 1.1 The Poxviridae family 2 1.1.1 Classification of Poxviruses 2 1.1.2 Smallpox disease 4 1.1.2.1 Smallpox in human history 7 1.1.2.2 Poxviruses still endanger 8 1.1.3 Vaccinia virus 9 1.1.3.1 The origin of VacV 9 1.1.3.2 Virion structure and entry 10 1.1.3.3 Life cycle 13 1.1.3.4 Virion assembly 14 1.1.3.5 Genome organization 15 1.1.3.6 DNA replication 19 1.2 Translation initiation in eukaryotes 22 1.2.1 The structure of eukaryotic mRNA 23 1.2.1.1 Cap structure 23 1.2.1.2 Poly(A) Tail 25 1.2.2 Mechanism of cap-dependent translation initiation 26 1.2.3 eIF4F initiation complex 28 1.2.3.1 eIF4G 29 1.2.3.1.1 eIF4G structure 30 1.2.3.1.1.1 eIF4G – PABP interaction 31 1.2.3.1.1.2 eIF4G – eIF4E interaction 31 1.2.3.1.1.3 eIF4A, eIF3 and Mnk1/2- binding sites 32 1.2.3.2 eIF4E 32 1.2.3.2.1 Structure of eIF4E 33 1.2.3.2.2 Function of eIF4E 34 1.2.3.2.3 eIF4E-Binding Proteins 35 1.2.3.2.4 eIF4E regulation 38 1.2.3.2.4.1 Regulation of eIF4E abundance and turnover 38 1.2.3.2.4.2 Regulation by phosphorylation 39 1.2.3.2.4.3 Regulation by translational inhibitors 41 1.2.4 Signaling pathways regulating protein synthesis 43 1.2.4.1 The PI 3K/Akt/mTORC1 pathway 44 1.2.4.1.1 Inhibitors of the PI3 kinase 48 1.2.4.2 mTOR 50 1.3 Manipulation of cellular translational apparatus by viruses 53 1.3.1 RNA viruses 53 1.3.1.1 The closed-loop model of translation initiation 53 1.3.1.2 IRES 54 1.3.2 DNA viruses 55 1.4 Aims of thesis 57 MATERIAL AND METHODS 58 2.1 Equipment 59 2.2 Antibodies 60 2.3 Water 61 2.4 Sterilization 61 2.5 Cell culture 61 2.5.1 Medium 61 2.5.2 Cell lines 61 2.5.3 Cell passaging 63 2.5.4 Cell starvation 63 2.5.5 Cell harvesting 63 2.5.6 Cell freezing 64 2.5.7 Cell thawing 65 2.6 Cell fixation 65 2.7 Virus 65 2.7.1 Growing