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Elucidation of the Tissue Tropism Determinants of Jaagsiekte Sheep Retrovirus and Enzootic Nasal Tumor Virus

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Elucidation of the Tissue Tropism Determinants of Jaagsiekte Sheep Retrovirus and Enzootic Nasal Tumor Virus Elucidation of the Tissue Tropism Determinants of Jaagsiekte Sheep Retrovirus and Enzootic Nasal Tumor Virus by María Carla Rosales Gerpe A Thesis Presented to The University of Guelph In partial fulfillment of the requirements for the degree of Doctor in Philosophy in Pathobiology Guelph, Ontario, Canada © María Carla Rosales Gerpe, August 2018 ABSTRACT TROPISM DETERMINANTS OF JAAGSIEKTE SHEEP RETROVIRUS AND ENZOOTIC NASAL TUMOR VIRUS María Carla Rosales Gerpe Advisor: University of Guelph, 2018 Sarah K. Wootton Jaagsiekte Sheep Retrovirus (JSRV) and Enzootic Nasal Tumor Virus (ENTV) cause adenocarcinomas of the lung and nasal tract, respectively, in sheep and goats. Nevertheless, JSRV and ENTV share a high degree of nucleotide (nt) (89%) and amino acid (a.a.) (80%) identity. They also enter cells via the same cell receptor, Hyaluronoglucosaminidase 2 (Hyal2), an enzyme involved in epithelial, endothelial and chondrocytic hyaluronan metabolism. Finally, they transform tissues using their envelope (Env) glycoprotein. JSRV and ENTV genome sequence comparisons reveal two regions of dissimilarity: the U3 of the Long Terminal Repeats (LTRs) (35% nt), and the cytoplasmic tail of the Envelope glycoprotein (50% a.a.). To uncover genomic regions important for tropism, we developed JSRV-ENTV chimeras within a JSRV backbone, containing ENTV’s LTRs, structural proteins and Env. Because JSRV and ENTV lack a reporter gene and have difficulty propagating in vitro, we also employed lentivectors (LV) pseudotyped with the JSRV Env (Jenv) and ENTV Env (Eenv), and developed a tissue slice ex vivo model to study entry of JSRV, ENTV and the chimeras. Our data demonstrated that, unlike JSRV and Jenv LV, ENTV and Eenv LV could not infect or transduce ovine lung tissue slices. Furthermore, JSRV LTRs were statistically significantly less active than ENTV LTRs in primary ovine chondrocyte cells. We also observed strong staining against Env in ENTV-infected ovine nasal turbinate slices, particularly in chondrocytes. Our in vitro data also showed that lentivectors pseudotyped with Eenv best transduced ovine primary chondrocytes, similarly to cells overexpressing Hyal2, revealing new aspects of ENTV pathogenesis. These results suggest that JSRV and ENTV tissue selectivity requires both Env and the LTRs. This marked tissue specificity allowed us to explore Jenv as an LV pseudotype in lung gene therapy. We generated two novel Jenv mutants to pseudotype LV, yielding titers similar to Vesicular Stomatitis Virus glycoprotein pseudotyped LV, capable of efficiently transducing both ovine and Hyal2-expressing murine lung slices. Overall, this thesis proposes that JSRV and ENTV tropism is orchestrated by both their promoters and Env glycoproteins, and that their tissue specificity can be re-purposed for gene therapy to the lung. iv “In 2003, Dolly the sheep, the first mammal cloned from an adult cell, died after being diagnosed with an incurable pulmonary adenocarcinoma caused by JSRV.” Leroux et al. 20061 v DEDICATION Para mamá, papá, Mary, y Ryan vi ACKNOWLEDGEMENTS This thesis’ efforts would not have been possible without the support, encouragement and empowerment that my supervisor Dr. Sarah Wootton provided. I have both grown as a scientist and as a person under her supervision. For these reasons, I am deeply thankful to her. I would also like to thank my Thesis Advisory Committee members, Dr. Peter Krell, Dr. George Harauz and Dr. Alicia Viloria-Petit, whose support, guidance and critical input on my research allowed me to make informed assessments on my thesis work. Scott Walsh, the staff at Ponsonby General Animal Facility, Pathobiology Isolation Unit, and the Food Science’s Abattoir, all deserve the deepest gratitude for their help to this thesis’ research. Furthermore, I would like to show my appreciation to the Ontario Veterinary College who supported me financially throughout my PhD with an OVC PhD Scholarship, and who showed me the benefits of being surrounded by a great research and graduate student community. Finally, I am indebted to the NSERC PGS-D and University of Guelph Tri-Council Top-up scholarships that allowed me to pursue this degree. I am also grateful for my peers, Laura van Lieshout, Lisa Santry, Jake van Vloten, Joelle Ingrao, Thomas McAusland, Betty-Anne McBey, Jondavid de Jong, Jake Domm and Jodre Datu. I give extra thanks to the latter two for showing the meaning of teaching and proving that mentoring is full of rewards. I sincerely appreciate everyone’s technical assistance and advice during my time at the Wootton lab. Although not in this lab, I would still like to thank Kasandra Bélanger, whose mentorship and friendship during my Masters cemented the foundations of my scientific journey. vii Finally, I would like to thank my parents and sister, my love, Ryan Kim, and friends – especially, Carmen Wong, Stephanie Coit, Eleni Armenakis, Joe Burns, and Kasandra Bélanger – for pushing and motivating me when times were tough. This work was funded by an NSERC grant awarded to Dr. Sarah K. Wootton. viii LIST OF CONTRIBUTIONS I performed the work outlined in this thesis under the guidance of Dr. Sarah K. Wootton and my advisory committee (Drs. Peter Krell, George Harauz, and Alicia Viloria-Petit), with the following exceptions: Jacob van Vloten conducted the resazurin assays and titrated the MG1-eGFP, AdGFP and VACV-GFP viruses in Chapter 2. Lisa Santry produced and titrated NDV-GFP for Chapter 2. Laura van Lieshout helped with the mouse delivery and produced AAV-FT-Hyal2 virus for Chapters 3 and 4. Jakob Domm also produced the AAV-FT-Hyal2 virus for Chapters 3 and 4. ix TABLE OF CONTENTS ABSTRACT ......................................................................................................................ii DEDICATION .................................................................................................................. v ACKNOWLEDGEMENTS ...............................................................................................vi LIST OF CONTRIBUTIONS .......................................................................................... viii LIST OF FIGURES ........................................................................................................ xiv LIST OF TABLES .......................................................................................................... xvi LIST OF ABBREVIATIONS .......................................................................................... xvii Chapter 1: Literature Review ........................................................................................... 1 Introduction .................................................................................................................. 1 Biology of Betaretroviruses .......................................................................................... 2 Betaretroviral Life Cycle ............................................................................................... 9 Pathogenesis of Betaretroviruses .............................................................................. 14 Ovine Pulmonary Adenocarcinoma and Enzootic Nasal Adenocarcinoma Pathology 15 JSRV and ENTV Tissue Selectivity ............................................................................ 17 The Long Terminal Repeats and their Role in Tropism .......................................... 17 The Env Protein and its Role in Tissue Specificity ..................................................... 19 Statement of Rationale .................................................................................................. 24 Hypotheses ................................................................................................................... 26 Experimental Approach ................................................................................................. 27 x Chapter 2: Use of precision-cut lung slices as an ex vivo tool for evaluating viruses and viral vectors for gene and oncolytic therapy .................................................................. 30 Abstract ...................................................................................................................... 31 Background ................................................................................................................ 32 Reagent Setup ........................................................................................................... 37 Equipment Setup ........................................................................................................... 37 Experimental Procedure ................................................................................................ 39 1. Preparation of low melting point agarose (30 min) (Day 0) .............................. 39 2. Preparation of lung tissue for sectioning with a vibratome (2 h) (Day 0) .......... 39 3. Sectioning with the Vibratome (1 – 4 h) (Day 0) ............................................... 41 4. Acclimation Period (Day 0 – 3) ......................................................................... 42 5. Viability Measurement (Day 3 and Day 28) ...................................................... 42 6. Transduction and infection of lung slices (15 min) (Day 3-4) ........................... 43 7. Detection of Viral Transduction/Infection (Day 5-7) .......................................... 44 8. Immunohistochemistry (4 days) (Week 3-4) ..................................................... 44 Timeline ........................................................................................................................
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