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Cellular Transformation by Polyomavirus Oncoproteins By Cellular Transformation by Polyomavirus Oncoproteins by Tushar Gupta Bachelor in Engineering, University of Rajasthan, 2006 Master of Science, Northeastern University, 2008 Submitted to the Graduate Faculty of the Kenneth P. Dietrich School of Arts and Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2014 UNIVERSITY OF PITTSBURGH Dietrich School of Arts and Sciences This dissertation was presented by Tushar Gupta It was defended on November 13, 2014 and approved by Dr. Jon P. Boyle, Assistant Professor, Department of Biological Sciences Dr. Craig Peebles, Professor, Department of Biological Sciences Dr. Jeffrey L. Brodsky, Professor, Department of Biological Sciences Dr. Saleem A. Khan, Professor, Department of Microbiology and Molecular Genetics Dissertation Advisor: Dr. James M. Pipas, Professor, Department of Biological Sciences ii Copyright © by Tushar Gupta 2014 iii Cellular Transformation by Polyomavirus Oncoproteins Tushar Gupta, PhD. University of Pittsburgh, 2014 Polyomaviruses have contributed tremendously towards our understanding of molecular biology of the cell and especially in discovering cellular factors and pathways involved in cancer formation and progression. Polyomavirus encoded oncoproteins manipulate specific cellular molecular pathways to create cellular environment conducive for viral replication and persistence. In a non-productive infection, the alteration of such cellular pathways by polyomaviral oncoproteins leads to activation of certain "cancer hallmarks" and results into cell transformation. In one part of this study, I used polyomaviral oncoproteins as a molecular tool to understand and decode cellular pathways involved in cell transformation. In one part of this study, I used a well characterized oncoprotein of polyomavirus Simian Virus 40 (SV40), called the large tumor antigen (TAg), as a molecular and genetic tool to understand the role of RB/E2F pathway in oncogene mediated cell transformation. According to the current paradigm, activator E2Fs are considered essential for cell proliferation and oncogenic transformation. My results, contrary to the current paradigm, suggest that TAg activates an alternative molecular pathway to induce proliferation and transformation in the absence of activator E2Fs. In another project, I have studied oncoproteins encoded by a less studied polyomavirus, Lymphotropic Papovavirus (LPV). I have discovered previously unknown iv splice forms of LPV early region, and their comparative analysis with SV40 oncoproteins suggest distinct roles for the homologous proteins in cellular immortalization and transformation. Importantly, my research shows an essential role of LPV small tumor antigen (sT) in immortalization and transformation of primary murine embryonic fibroblasts. v TABLE OF CONTENTS PREFACE ................................................................................................................................. XIV 1.0 INTRODUCTION ........................................................................................................ 1 1.1 CANCER BIOLOGY: THE STUDY OF NATURE'S COMPLEXITY ................... 6 1.1.1 Polyomaviridae: A small DNA tumor virus family ...................................... 9 1.1.2 Polyomavirus oncoproteins: Molecular tools to study molecular biology of cancer .......................................................................................................................... 16 1.1.3 Cell cycle, RB-E2F pathway and Cancer: To cycle or not to cycle? ............ 19 1.1.4 Retinoblastoma family of tumor suppressors ................................................ 26 1.1.5 E2F family of transcription factors ................................................................. 33 1.1.6 p53-MDM2 tumor suppressor pathway cross-talks with the RB-E2F pathway ....................................................................................................................... 46 1.1.7 Protein phosphatase 2A tumor suppressor ..................................................... 51 1.1.8 The Large Tumor Antigen (TAg) .................................................................... 52 1.1.9 TAg inhibits the RB-E2F tumor suppressor pathway ................................... 57 1.1.10 TAg inhibits p53 tumor suppressor ............................................................... 59 1.1.11 The Small Tumor Antigen (sT) ...................................................................... 60 1.1.12 The Middle Tumor Antigen (mT) .................................................................. 65 vi 1.1.13 Polyomavirus oncoproteins co-operate with other oncoproteins in cellular transformation and tumorigenesis ........................................................................... 70 1.2 INTRODUCTION SUMMARY .................................................................................. 71 2.0 MATERIALS AND METHODS .............................................................................. 74 2.1.1 Cell culture, Transfection and Transduction .............................................. 74 2.1.2 SDS-PAGE and Western Blot ...................................................................... 76 2.1.3 Polymerase Chain Reaction (PCR) .............................................................. 79 2.1.4 Immunofluorescence ..................................................................................... 82 2.1.5 Transformation assays .................................................................................. 83 2.1.6 Proliferation assays........................................................................................ 84 2.1.7 Cloning and Plasmids:................................................................................... 85 2.1.8 Site directed mutagenesis:............................................................................. 87 2.1.9 Extraction of SV40 genomic DNA from CV1 cells and quantification ..... 88 3.0 ROLE OF ACTIVATOR E2F'S IN SV40 TAG MEDIATED PROLIFERATIOIN AND TRANSFORMATION. ................................................................. 90 3.1 INTRODUCTION ............................................................................................. 90 3.2 SV40 TAG DOES NOT REQUIRE ACTIVATOR E2F'S TO INDUCE PROLIFERATION AND TRANSFORMATION IN MEF'S ........................................ 92 3.2.1 Development of cell culture model system .................................................. 92 3.2.2 TAg rescues the proliferation defects of TKO MEFs ................................. 95 3.2.3 LTAg does not require activator E2Fs to induce transformation in MEFs .........................................................................................................................101 vii 3.3 SV40 TAG LXCXE MOTIF IS REQUIRED FOR INDUCING PROLIFERATION AND TRANSFORMATION OF TKO MEF'S ........................... 110 3.3.1 TAg mediated inhibition of RB and p53 pathways is intact in the absence of activator E2Fs ...................................................................................................... 111 3.3.2 TAg requires LXCXE motif and a C-terminus activity to rescue the proliferation defect in TKO MEF's ........................................................................ 115 4.0 CHARACTERIZATION OF LYMPHOTROPIC PAPOVAVIRUS (LPV) EARLY REGION (ER). ........................................................................................................... 122 4.1 INTRODUCTION ........................................................................................... 122 4.2 LPV ER TRANSFORMS PRIMARY MEFS IN CELL CULTURE. ........ 126 4.2.1 LPV ER inhibits RB/E2F and p53/MDM2 tumor suppressive pathways .........................................................................................................................129 4.2.2 LPV ER encodes four differentially spliced products .............................. 131 4.3 DIFFERENTIAL EFFECTS OF LPV ER SPLICE PRODUCTS ON CELL ............................................................................................................................138 PROLIFERATION AND TRANSFORMATION ......................................................... 138 4.3.1 LPV LT' induces growth arrest in MEFs. ................................................. 139 4.3.2 LPV TAg or sT expression is sufficient to immortalize MEFs ................ 141 4.3.3 LPV sT is essential and sufficient for growth in low serum .................... 143 5.0 CONCLUSIONS AND DISCUSSION ................................................................... 146 5.1 CONCLUSIONS AND DISCUSSION I ........................................................ 146 5.1.1 How are TKO;TAg MEFs proliferating in the absence of activator E2Fs? .........................................................................................................................147 viii 5.1.2 How are the E2F target genes up-regulated in TKO;TAg MEFs in the absence of E2F activators? ...................................................................................... 151 5.1.3 Why there are discrepancies in the results obtained in previous studies as compared to this study? ........................................................................................... 155 5.2 CONCLUSIONS AND DISCUSSION II ....................................................... 163 5.3 IMPLICATIONS ............................................................................................. 167 5.3.1 What activity of TAg is sufficient to rescue
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