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Dissertation Role of Endogenous Retrovirus In DISSERTATION ROLE OF ENDOGENOUS RETROVIRUS IN CONTROL OF FELINE LEUKEMIA VIRUS INFECTION AND IMPLICATIONS FOR CROSS SPECIES TRANSMISSION Submitted by Elliott S. Chiu Department of Microbiology, Immunology, and Pathology In partial fulfillment of the requirements For the Degree of Doctor of Philosophy Colorado State University Fort Collins, Colorado Summer 2019 Doctoral Committee: Advisor: Sue VandeWoude Edward A. Hoover Gregory D. Ebel W. Chris Funk Copyright by Elliott Sinming Chiu 2019 All Rights Reserved ABSTRACT ROLE OF ENDOGENOUS RETROVIRUS IN CONTROL OF FELINE LEUKEMIA VIRUS INFECTION AND IMPLICATIONS FOR CROSS SPECIES TRANSMISSION Endogenous retroviruses (ERV) are markers of ancient retroviral infections, though evolutionary forces have limited the capacity for ERV replication and virulence. While they are seldom considered infectious alone, they maintain the ability to interact with their exogenous retroviral (XRV) progenitors. In Chapter One, we review the interactions that exist between ERV and XRV dyads. One such couplet includes feline leukemia virus (FeLV), a common domestic cat pathogen. In Chapter Two, we review FeLV subgroup taxonomy and the methods used from which they were originally characterized. Though the domestic cat is regarded as the natural host for the virus, recent reports have documented FeLV infections in wild felids with pathogenic consequences. Chapter Three examines the root of a contemporary FeLV outbreak in Florida panthers (Puma concolor coryi), a species that lacks endogenous FeLV. Our phylogenetic analysis of the contemporary FeLV outbreak has further implicated domestic cats (Felis catus) as the origin of FeLV infections in wild felids. Furthermore, we detected a recombinant oncogenic variant in Florida panthers that is believed to be non-horizontally transmissible. These field studies have prompted us to examine the cellular basis of infection and intrinsic resistance to the virus. In Chapter Four, we interrogate the cellular basis of FeLV infections between puma (P. concolor) and domestic cat cells using in vitro approaches. We demonstrated that puma cells support greater infection and replication. Additionally, we documented enFeLV long terminal repeats (LTR) in domestic cats are negatively correlated to FeLV infection outcomes in vitro. Natural FeLV infections in both Florida panther and domestic cat tissues offered us the opportunity to examine end stage disease dynamics, which demonstrate that Florida panthers have the ability to produce more virus despite having lower proviral loads than domestic cats. The results of both in vivo and in vitro experiments prompted us to further ii investigate enFeLV-LTRs and their role in FeLV infection. Chapter Five took advantage of the publicly available data in the NCBI Sequence Read Archive (SRA) to evaluate enFeLV-LTR basal transcription levels. Data-mining the domestic cat transcriptome showed that lymphoid cells, which are relatively resistant to in vitro FeLV infection, transcribe more enFeLV elements than relatively susceptible cells (i.e., fibroblasts). We also identified microRNA transcripts are produced that have the potential ability to down-regulate FeLV RNA transcripts. In Chapter Six, we innovated a new methodology to characterize the enFeLV-LTR integration sites across the entire genome of 20 related and unrelated domestic cats in an attempt to uncover genes that may be influenced by LTR enhancement of gene expression. We found one LTR integration site in a limited number of cats that is within 1MB of APOBEC1, an antiviral gene, and that the most common gene found in close proximity to LTR integration sites are zinc fingers, a broad-acting class of regulatory proteins. Collectively, this groundwork provides future directions to uncover direct and indirect mechanisms of enFeLV-mediated restriction of FeLV infection. We conclude that because wild felids lack enFeLV, they may be more vulnerable to FeLV infection. As urbanization forces niche overlap and contact between wild and domestic felids, the risk of infection of these species is likely to increase, and thus it will be important to consider contacts between FeLV-infected domestic cats and wild felid populations during development of conservation action plans. iii ACKNOWLEDGEMENTS This work could not have been completed without the intellectual contributions and emotional support from an ever-growing cadre of collaborators. They have helped me immeasurably by providing samples, developing protocols in addition to reviewing and providing constructive criticism to my experimental approaches and writing. Below is a list of collaborators who contributed to my work separated by chapter. Chapter 1 – Roderick Gagne, Sue VandeWoude Chapter 2 – Edward Hoover, Sandra Quackenbush, Sue VandeWoude Chapter 3 –Simona Kraberger, Mark Cunningham, Lara Cusack, Dave Onorato, Melody Roelke, Sue VandeWoude Chapter 4 – Ryan Troyer, Ryan Mackie, Lisa Wolfe, Karen Fox, Mark Fisher, Ivy Levan, Jennifer Malmberg, Gary Mason, Alex Byas, Laura Hoon-Hanks, Lauren Harris, Devin von Stade, Esther Musselman, Mary Nehring, Craig Miller, Melody Roelke, Sue VandeWoude Chapter 5 – Matthew Moxcey, Mark Stenglein, Justin Lee, Sue VandeWoude Chapter 6 – Roderick Gagne, Matthew Moxcey, Mark Stenglein, Justin Lee, Susan Bailey, Miles McKenna, David Maranon, Sue VandeWoude Finally, I would like to thank my friends, family, lab-mates, and DVM/PhD colleagues whose encouragement I have leaned on every step of the way. I would not be at this point in my professional and personal development if it were not for them and am privileged to have them in my life. iv TABLE OF CONTENTS ABSTRACT ………………………………………………………………………………………………. ii ACKNOWLEDGEMENTS ……………………………………………………………………………… iv CHAPTER ONE: Interactions Between Endogenous Retroviruses and Their Exogenous Counterparts … 1 Figures ……………………………………………………………………………………………….. 19 LITERATURE CITED…………………………………………………………………………………… 22 CHAPTER TWO: A Retrospective Examination of Feline Leukemia Subgroup Characterization: Viral Interference Assays to Deep Sequencing ………………………………………………………………... 31 Figures ………………………………………………………………………………………………. 42 LITERATURE CITED…………………………………………………………………………………… 45 CHAPTER THREE: Multiple Introductions of Domestic Cat (Felis catus) Feline Leukemia Virus in Endangered Florida Panthers (Puma concolor coryi)……………………………………………………. 52 Introduction ………………………………………………………………………………………….. 52 Methods ……………………………………………………………………………………………… 54 Results ……………………………………………………………………………………………….. 57 Discussion …………………………………………………………………………………………... 59 Figures ………………………………………………………………………………………………. 63 Tables ……………………………………………………………………………………………….. 70 LITERATURE CITED…………………………………………………………………………………… 73 CHAPTER FOUR: Comparisons of feline leukemia virus (FeLV) Susceptibility of puma (Puma concolor) and domestic cat (Felis catus) cells in relation to endogenous FeLV elements ……………… 76 Introduction ………………………………………………………………………………………….. 76 Methods ……………………………………………………………………………………………… 78 Results ……………………………………………………………………………………………….. 82 Discussion …………………………………………………………………………………………… 84 Figures ………………………………………………………………………………………………. 89 Tables ………………………………………………………………………………………………... 95 LITERATURE CITED…………………………………………………………………………………… 97 v CHAPTER FIVE: Endogenous Feline Leukemia Virus (enFeLV) siRNA May Drive Direct Interference with FeLV Infection ……………………………………………………………………………………. 101 Introduction ………………………………………………………………………………………… 101 Methods ……………………………………………………………………………………………. 103 Results ……………………………………………………………………………………………… 104 Discussion …………………………………………………………………………………………. 106 Figures ……………………………………………………………………………………………… 111 Tables ………………………………………………………………………………………………. 117 LITERATURE CITED………………………………………………………………………………….. 119 CHAPTER SIX: Characterization of Endogenous Feline Leukemia Virus (enFeLV) Long Terminal Repeat (LTR) Integration Site Diversity ……………………………………………………………….. 121 Introduction ………………………………………………………………………………………… 121 Methods …………………………………………………………………………………………….. 122 Results ……………………………………………………………………………………………… 126 Discussion …………………………………………………………………………………………. 128 Figures ……………………………………………………………………………………………… 132 Tables ………………………………………………………………………………………………. 139 LITERATURE CITED………………………………………………………………………………….. 216 CONCLUSION ………………………………………………………………………………………… 218 vi CHAPTER ONE Interactions Between Endogenous Retroviruses and Their Exogenous Counterparts INTRODUCTION Animal genomes are riddled with evolutionary fossils left over from ancient viral infections; notable among these are remnants of retroviruses. Retroviruses require conversion of RNA genomes into DNA that stably integrate into host DNA (Coffin, 1992). Following integration, retroviruses hijack host cellular machinery for replication. In normal somatic cell infections, this process allows for the establishment of life long infections, as integrated viral genomes are not easily excised. In the special case of retroviral germline infections, integrated viral genomes may be passed along to progeny by vertical transmission (Figure 1). This results in permanent viral penetrance into the animal’s cellular genome, resulting in an endogenous retrovirus (ERV), in contrast to horizontally transmitted counterparts, referred to as exogenous retroviruses (XRV) (Coffin, 2004). Early on in infection, ERVs are identical to their XRV progenitors. At this stage, they may be transcribed and can be infectious. After generations, ERV mutations accumulate, mitigating deleterious impacts of fully functional viruses. Defective ERVs
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