IMPLICATIONS FOR XMRV INFECTIONS IN PROSTATE CANCER by SEUNGHEE HONG Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Thesis Advisor: Dr. Robert H. Silverman Department of Molecular Biology and Microbiology Molecular Virology Program CASE WESTERN RESERVE UNIVERSITY January, 2010 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of _____________________________________________________ candidate for the ______________________degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. TABLE OF CONTENTS LIST OF TABLES ..........................................................................................................4 LIST OF FIGURES .........................................................................................................5 ACKNOWLEDGEMENTS .............................................................................................7 LIST OF ABBREVIATIONS ..........................................................................................9 ABSTRACT .................................................................................................................. 12 CHAPTER 1. INTRODUCTION................................................................................... 14 IFN system in innate immunity .................................................................................. 14 2-5A/RNase L pathway in the interferon anti-viral system ......................................... 15 Role of RNase L in the biology of prostate cancer ...................................................... 18 Identification of xenotropic gammaretrovirus in prostate tumor tissues ...................... 19 Replication-competent XMRV genome has been constructed ..................................... 30 XPR1 as a receptor for xenotropic and polytropic murine leukemia viruses ................ 31 SEVI dramtically boosts HIV-1 infection ................................................................... 34 Retrovirus Restriction Factors .................................................................................... 35 Summary of Introduction ........................................................................................... 39 CHAPTER 2. MATERIALS AND METHODS ............................................................. 41 Cell Culture ............................................................................................................... 41 XPR1 Expression and XMRV Infection of MEFs and CHO Cells .............................. 41 Nested RT-PCR Assays for XPR1 .............................................................................. 42 XMRV preparations and infectivity assays ................................................................. 43 Effect of SEVI on viral infectivity .............................................................................. 43 Immunofluorescence assay ......................................................................................... 44 Effects of semen on viral infectivity ........................................................................... 44 Preparation of PSA, PSMA, PAP ............................................................................... 45 Plasmids and experiments with recombinant viruses .................................................. 45 Isolation of RNA in EPS ............................................................................................ 46 Depletion of XPR1 mRNA using siRNA .................................................................... 47 Quantitive RT-PCR .................................................................................................... 47 Nested RT-PCR ......................................................................................................... 49 Illumina cDNA microarray for gene expression profiles ............................................. 50 1 Preparation of in vitro co-cultures .............................................................................. 51 CHAPTER 3. XPR1 as a host cellular receptor for XMRV ............................................ 52 Summary ................................................................................................................... 52 Introduction ............................................................................................................... 52 Results ....................................................................................................................... 55 TCID50 of infectious XMRV supernatant was determined. ...................................... 55 Expression of human XPR1 in mouse cell does not render susceptible to XMRV infection. ................................................................................................................ 56 XPR1 expression renders hamster cells susceptible to XMRV Infection. ................ 57 Downregulation of XPR1 in prostate human cells reduces XMRV infectivity. ........ 62 Discussion.................................................................................................................. 68 CHAPTER 4. SEVI boosts infections by XMRV, a Human Retrovirus Associated with Prostate Cancer .............................................................................................................. 71 Summary ................................................................................................................... 71 Introduction ............................................................................................................... 71 Results ....................................................................................................................... 73 SEVI promotes XMRV infection of human prostatic and non-prostatic cell types. .. 73 SEVI potently increases the infectious titer of XMRV. ........................................... 80 SEVI and semen enhance infectivity at the level of virion attachment and fusion. ... 80 XMRV RNA isolated and sequenced from expressed prostatic secretions. .............. 88 Discussion.................................................................................................................. 98 Potent enhancement of XMRV infectivity mediated by SEVI or semen. ................. 98 Host restriction of XMRV replication by RNase L. ................................................. 99 The SEVI effect and presence of XMRV in EPS................................................... 100 CHAPTER 5. Gene expression profiles regulated by XMRV infection in human primary prostate cell cultures .................................................................................................... 102 Summary ................................................................................................................. 102 Introduction ............................................................................................................. 103 Results ..................................................................................................................... 105 Discussion................................................................................................................ 139 CHAPTER 6. Stromal-epithelial cell interaction and apoptosis affected by XMRV infection in prostate cancer progression ....................................................................... 144 2 Summary ................................................................................................................. 144 Introduction ............................................................................................................. 144 Results ..................................................................................................................... 146 Discussion................................................................................................................ 157 CHAPTER 7. DISCUSSION AND PERSPECTIVE .................................................... 160 Summary ................................................................................................................. 160 XPR1 as an XMRV receptor .................................................................................... 160 Host restriction factors ............................................................................................. 161 XMRV and prostate carcinogenesis .......................................................................... 163 XMRV as a sexually transmitted infection ............................................................... 164 Possible involvement of IL-8 in IFN systems ........................................................... 166 Apoptosis induced by Camptothecin upon XMRV infection..................................... 168 Antiviral effects of camptothecin ............................................................................. 168 BIBLIOGRAPHY ....................................................................................................... 169 3 LIST OF TABLES Table 4.1: Age, clinical parameters, geographical locations, RNASEL genotype, and presence of XMRV