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Comparative Analyses of Tumorigenic Mechanisms of Merkel Cell Polyomavirus T Antigens

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Comparative Analyses of Tumorigenic Mechanisms of Merkel Cell Polyomavirus T Antigens Title Page Comparative analyses of tumorigenic mechanisms of Merkel cell polyomavirus T antigens by Justin A. Wendzicki Bachelors of Science, Duquesne University, 2013 Submitted to the Graduate Faculty of the School of Medicine in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2019 Committee Pa ge UNIVERSITY OF PITTSBURGH SCHOOL OF MEDICINE This dissertation was presented by Justin A. Wendzicki It was defended on December 6, 2019 and approved by Neal A. DeLuca, Professor, Department of Microbiology and Molecular Genetics Saleem A. Khan, Professor, Department of Microbiology and Molecular Genetics Paul R. Kinchington, Professor, Department of Microbiology and Molecular Genetics Roderick J. O’Sullivan, Assistant Professor, Department of Pharmacology and Chemical Biology Dissertation Director: Yuan Chang, Professor, Department of Pathology ii Copyright © by Justin A. Wendzicki 2019 iii Abstract Comparative analyses of tumorigenic mechanisms of Merkel cell polyomavirus T antigens Justin A. Wendzicki, PhD University of Pittsburgh, 2019 The work described in this dissertation began in 2013 and is focused on characterizing the mechanisms by which Merkel cell polyomavirus (MCV) Large T (LT) and small T (sT) antigens induce tumorigenesis through comparative analyses with oncoproteins from other tumor viruses. A peptide motif in the C-terminal region of MCV LT that bears little sequence homology with other human polyomavirus LT proteins is shown to be critical for maintaining stability of the full-length LT protein (Chapter 3). Comparison between LT antigens of MCV and SV40 demonstrate that MCV LT in direct contrast to SV40 LT is incapable of avidly binding tumor suppressor p53 and inhibiting its transactivation capabilities (Chapter 4). Lastly, promiscuous E3 ligase targeting by MCV sT through its LT-stabilization domain (LSD) results in the formation of a genomically unstable phenotype, a known hallmark of cancer (Chapter 5). Many of the features of genomic instability induced by MCV sT such as centrosome overduplication parallel what has been observed previously for human papillomavirus 16 E7 oncoprotein. Overall, these comparative analyses have not only provided greater insight into MCV biology and how its T antigens function in causing an aggressive cancer like Merkel cell carcinoma (MCC), but they have also revealed new avenues for continued study involving MCV T antigens that will continue to move the field of tumor virology forward. iv Table of Contents Acknowledgments ......................................................................................................... xiv 1.0 Introduction ................................................................................................................. 1 1.1 Viruses and cancer ............................................................................................. 1 1.1.1 Origins of tumor virology ....................................................................... 1 1.1.2 Human tumor viruses .............................................................................. 2 1.2 Polyomavirus biology ........................................................................................ 7 1.2.1 History and discovery ............................................................................. 7 1.2.2 Phylogeny ................................................................................................. 9 1.2.3 Genome organization ............................................................................ 12 1.2.4 Life cycle ................................................................................................. 14 1.2.5 Association with disease in humans .................................................. 16 1.3 Mechanistic insights from SV40 T antigens ................................................. 19 1.3.1 Targeting of retinoblastoma family proteins ..................................... 19 1.3.2 Targeting of p53 ..................................................................................... 22 1.3.3 Targeting of p300 and CBP .................................................................. 23 1.3.4 Additional cellular targets of SV40 LT ................................................ 24 1.3.4.1 Heat shock cognate protein 70 (Hsc70) ................................ 24 1.3.4.2 Bub1 mitotic checkpoint serine/threonine kinase ............... 25 1.3.4.3 Cullin 7 ....................................................................................... 25 1.3.4.4 Insulin receptor substrate 1 (IRS1) ........................................ 26 1.3.4.5 F-box/WD repeat-containing protein 7 (Fbw7) ..................... 26 v 1.3.5 PP2A targeting by SV40 sT .................................................................. 26 1.3.6 Permissive vs. non-permissive infections ......................................... 27 1.4 Merkel cell polyomavirus................................................................................. 29 1.4.1 Discovery of MCV .................................................................................. 29 1.4.2 Genome structure .................................................................................. 31 1.4.3 Virus life cycle ........................................................................................ 31 1.4.4 Involvement in Merkel cell carcinoma ................................................ 33 1.4.5 Mutations in Merkel cell carcinoma .................................................... 35 2.0 Large T and small T antigens of Merkel cell polyomavirus ................................ 37 2.1 Introduction ....................................................................................................... 37 2.2 Large T antigen ................................................................................................. 39 2.3 Small T antigen ................................................................................................. 43 2.4 Conclusions ...................................................................................................... 46 3.0 The C-terminal domain of MCV Large T antigen is critical for its stability ....... 47 3.1 Introduction ....................................................................................................... 48 3.2 Materials and methods .................................................................................... 50 3.2.1 Generation of LT truncation mutants .................................................. 50 3.2.2 Alanine mutagenesis ............................................................................. 50 3.2.3 Cell culture and transfection ................................................................ 51 3.2.4 Immunoblotting ...................................................................................... 51 3.2.5 Immunofluorescence ............................................................................ 52 3.2.6 Ubiquitination assays ........................................................................... 52 3.2.7 Structural modeling ............................................................................... 53 vi 3.3 Results ............................................................................................................... 53 3.3.1 C-terminal truncations to MCV LT impact its stability ...................... 53 3.3.2 Alanine mutagenesis reveals hotspots important for LT stability .. 57 3.3.3 Unstable LT mutants possess higher levels of ubiquitination ........ 59 3.3.4 Attempts to stabilize LT mutants were unsuccessful ...................... 60 3.4 Discussion ......................................................................................................... 62 4.0 Differential targeting of p53 by polyomavirus Large T antigens ....................... 67 4.1 Introduction ....................................................................................................... 68 4.2 Materials and methods .................................................................................... 70 4.2.1 Cell culture and transfection ................................................................ 70 4.2.2 Plasmids ................................................................................................. 70 4.2.3 Immunoblotting and antibodies ........................................................... 71 4.2.4 Immunoprecipitation ............................................................................. 72 4.2.5 Proximity ligation assay ....................................................................... 72 4.2.6 RNA extraction and quantitative real-time PCR ................................ 73 4.2.7 Chromatin immunoprecipitation .......................................................... 73 4.2.8 Replication assays ................................................................................ 74 4.2.9 Luciferase assays .................................................................................. 75 4.2.10 Cycloheximide chase half-life studies .............................................. 75 4.3 Results ............................................................................................................... 76 4.3.1 MCV LT does not strongly interact with p53 or p300........................ 76 4.3.2 SV40 LT inhibits p53 DNA binding and transactivation ................... 78 4.3.3 Characterization of a p53 binding mutant SV40 LT .......................... 82 vii 4.3.4 SV40 LT mutation reverses p53 stabilization and inhibition
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