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Identification and Functional Analysis of Micro-Rnas Encoded by Kaposi’S Sarcoma-Associated Herpesvirus

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Identification and Functional Analysis of Micro-Rnas Encoded by Kaposi’S Sarcoma-Associated Herpesvirus IDENTIFICATION AND FUNCTIONAL ANALYSIS OF MICRO-RNAS ENCODED BY KAPOSI’S SARCOMA-ASSOCIATED HERPESVIRUS By MARK ATIENZA SAMOLS Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Adviser: Dr. Rolf Renne Department of Molecular Biology and Microbiology CASE WESTERN RESERVE UNIVERSITY August, 2007 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of ______________________________________________________ candidate for the Ph.D. degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. This work is dedicated to my parents, David and Sui Bi Samols. iii Table of Contents Title Page…………………………………………………………....………...…...….…...i Signature Sheet....................................................................................................................ii Dedication Page..................................................................................................................iii Table of Contents................................................................................................................iv List of Tables.....................................................................................................................vii List of Figures ..................................................................................................................viii Preface .................................................................................................................................x Acknowledgements............................................................................................................xi List of Abbreviations........................................................................................................xii Abstract..............................................................................................................................1 Chapter 1............................................................................................................................3 Introduction Kaposi’s sarcoma-associated herpesvirus as an agent for disease...........................3 KSHV latency and latency proteins.........................................................................8 MicroRNAs are a novel class of cellular regulators..............................................10 MicroRNAs exist in many organisms and have multiple functions......................12 Cellular differentiation and development can be controlled by miRNAs.............16 MicroRNA biogenesis...........................................................................................17 MicroRNA target determination and recognition .................................................25 MicroRNAs and viruses.........................................................................................28 Methods to identify and molecularly clone viral miRNAs....................................33 Identification of viral miRNA targets and functions.............................................37 iv Does KSHV encode miRNAs? What could their role be for the virus?................39 Chapter 2..........................................................................................................................40 Cloning and Identification of a MicroRNA Cluster Within the Latency-Associated Region of Kaposi’s Sarcoma-Associated Herpesvirus Abstract..................................................................................................................40 Cloning of small RNAs from KSHV-infected cells..............................................41 Identification of 11 KSHV encoded candidate miRNAs.......................................42 KSHV encoded candidate miRNAs fold into hairpin structures...........................49 Addendum..............................................................................................................53 Chapter 3..........................................................................................................................57 Identification of Cellular Genes Targeted by KSHV-Encoded MicroRNAs Abstract..................................................................................................................57 Author Summary....................................................................................................58 Introduction............................................................................................................59 Results....................................................................................................................63 Ectopic expression of KSHV miRNAs in 293 cells..............................................63 Expression profiling revealed 81 gene expression changes in miRNA expressing cells..........................................................................................67 qRT-PCR and bio-statistical analyses validate potential targets...........................73 Protein levels of THBS1 are greatly reduced in the presence of KSHV miRNAs.....................................................................................................88 KSHV miRNAs repress THBS1 in BJAB cells....................................................88 THBS1 is targeted by multiple KSHV miRNAs...................................................88 v Decreased THBS1 protein levels translate into reduced TGF-β activity..............92 Discussion..............................................................................................................95 Materials and Methods.........................................................................................101 Chapter 4........................................................................................................................108 Conclusions and Future Directions KSHV miRNAs may function to aid pathogenesis..............................................108 What do viral miRNAs regulate?.........................................................................110 Expression of viral miRNAs................................................................................112 Viral miRNAs and evolutionary conservation.....................................................119 Viral miRNAs and cancer....................................................................................124 Future directions for KSHV miRNA study.........................................................129 What’s next for viral microRNAs?......................................................................139 Appendix.........................................................................................................................141 Protocols microRNA cloning...............................................................................................141 Northern Blot.......................................................................................................148 2-D PAGE............................................................................................................152 Silver Stain...........................................................................................................156 microRNA target cloning.....................................................................................157 Luciferase assays.................................................................................................161 Bibliography...................................................................................................................167 vi List of Tables Table 2-1............................................................................................................................44 Distribution of cloned small RNA molecules Table 2-2............................................................................................................................56 Conversion table for KSHV miRNA nomenclature Table 3-1............................................................................................................................71 List of changed genes with 100% cross validation Table 3-2............................................................................................................................75 Fold changes for selected genes Table 3-3............................................................................................................................77 Seed matching data using an ad hoc scanning algorithm Table 3-4..........................................................................................................................104 List of primers for the cloning on miRNA expression and sensor constructs Table 4-1..........................................................................................................................123 Summary of patient and cell line characteristics and subtyping vii List of Figures Figure 1-1.............................................................................................................................7 Schematic representation of the genome structure of KSHV Figure 1-2...........................................................................................................................20
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