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ABSTRACT SCHUCHMAN, RYAN MICHAEL. on New Host Cell ABSTRACT SCHUCHMAN, RYAN MICHAEL. On New Host Cell Proteins Involved in Alphavirus Replication (Under the direction of Dr. Dennis T. Brown). These studies redefined the function of vacuolar-ATPase and identified new host cell proteins involved in alphavirus replication. Traditionally, the vacuolar-ATPase has been thought to induce conformational changes in the structural proteins by decreasing the pH in endomembrane compartments during infection through receptor-mediated endocytosis. The conformational changes in the structural proteins of the virus would allow for fusion of the virus particle and endomembrane compartment to release the viral genome into the cytoplasm. However, using the heat-resistant strain of Sindbis virus, we have generated a bafilomycin- resistant mutant of Sindbis virus (BRSV). Using BRSV, and other mutants that were generated by reverse genetics, in combination with electron microscopy, quantitative polymerase chain reaction, and plaque assay, we showed that the vacuolar-ATPase helps facilitate direct entry at the plasma membrane, promotes synthesis of viral RNA, and functions in efficient assembly of mature virus particles. Identifying host cell proteins that can be used as targets of antiviral therapeutic strategies is vastly important in controlling the impact of viruses on the human species. To that end, we have identified and validated sorting nexin 5 as host cell protein that has a critical function during the infection cycle of alphaviruses. The identification of host cell proteins in this study was made by purifying mature Sindbis virus particles from various cell backgrounds and interrogating the complement of proteins associated with the virus particles with liquid chromatography coupled with tandem mass spectrometry. Significantly, we also identified non- structural protein 2, the viral protease, as a component of the mature virus. The function of nsP2 outside of the role of the viral protease during replication is unknown, however it is possible that it may play some role in the initial phases of infection. On New Host Cell Proteins Involved in Alphavirus Replication by Ryan Michael Schuchman A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Biochemistry Raleigh, North Carolina 2018 APPROVED BY: _______________________________ _______________________________ Dennis T. Brown Cynthia L. Hemenway Committee Chair _______________________________ _______________________________ Raquel Hernandez Frank Scholle DEDICATION This work is dedicated to anyone who has ever dared to dream. ii BIOGRAPHY On 7 January 1988, Ryan Michael Schuchman was born at Cape Fear Valley Medical Center in Fayetteville, North Carolina to Wendy and Michael. He would find himself graduating from Douglas Byrd Senior High School and Fayetteville State University for his undergraduate studies. Though he always loved the sciences, it wasn't until much introspection that he would decide that a doctoral degree in biochemistry was in his journey. He would eventually find himself completing his PhD at North Carolina State University under the guidance of Dr. Dennis T. Brown. iii ACKNOWLEDGMENTS Firstly, I want to thank Drs. Dennis Brown and Raquel Hernandez. I will never forget the generosity, advice, and friendship that you have extended to me through the last few years—it means the world to me. I also want to thank you for always allowing me to explore my curiosities. To the rest of my committee members, both present and past, Drs. Cindy Hemenway, Stu Maxwell, and Frank Scholle, thank you for your guidance, encouragement, and advice through my education—I couldn’t have picked a better committee to advocate for me. Cindy, I also want to thank you for offering your beakers to me, but I'm glad that I never had to use them. Next, I want to thank Michael—I know having stuck by my side almost every day of this adventure has been challenging. I am so very grateful for your patience, support, and especially for taking care of Watson and Newton when I couldn't be there because of late nights in lab. I am incredibly indebted to my family; your encouragement, love, and support through the last 30 years have been paramount to my success. RueAnne, I'm going to need to sell a kidney on the black market to even come close to repaying you for all of the conversations, diversions, and late night car rides. Drs. Finley Bryan, Faren Wolter, and Paul Swartz, you three will have to split the money that I get from my other kidney. The three of you have been instrumental in the maintenance of my sanity more times than I care to count. To the best work wife a guy could ask for, Dr. Alex Breuer, I will always appreciate all of our conversations about science and life. Next, in no particular order, I'd like to thank all of my dear friends that I met in Polk Hall: Dmitry Grinevich, Dr. Davis Ferreira, Dr. Laura Herring, Dr. Annette Bodenheimer, Sophia Yang, Matt Coco, Colin Woolard, Kate Emmerich, Hannah Conley, Kevin Blackburn, Amanda iv Piper, Jonatan Isacsson, Gabby O’Neill, Dr. Tara Nash, Jigar Desai, Dipti Paudel, Dr. Joe Maciag, Bryan Rogers, Kaylin Prestage, Dr. Melvin Thomas, Dr. Bob Grinshpon, and Dr. Brad O’Dell. Thank you all for being part of my journey. Lastly, I would be remiss if I did not mention my gratitude for the Cavanagh Lab: thank you all for your individual contributions to my education—my experiences with you are unforgettable. v TABLE OF CONTENTS LIST OF FIGURES ..................................................................................................................... vii Chapter 1: Divergence from Cannon: an Additional Opportunity for Alphavirus Entry ... 1 1.1: Abstract ...................................................................................................................... 2 1.2: Introduction:Alphaviruses, Their Superstructure, and Genomic Organization .......... 3 1.3: Alphavirus Structural Proteins ................................................................................... 5 1.4: The Study of Virus Entry ........................................................................................... 8 1.5: A Brief Overview of Inhibitors used to Study Virus Entry ........................................ 9 1.6: Biological Complications in Studying Virus Entry ................................................. 11 1.7: Evidence for Receptor-Mediated Endocytosis ......................................................... 13 1.8: Evidence for pH-Independent Entry......................................................................... 16 1.9: Summary .................................................................................................................. 20 Chapter 2: Role of the Vacuolar ATPase in the Alphavirus Replication Cycle ................... 21 2.1: Abstract .................................................................................................................... 22 2.2: Introduction .............................................................................................................. 22 2.3: Results ...................................................................................................................... 26 2.4: Discussion ................................................................................................................ 44 2.5: Methods .................................................................................................................... 52 2.6: Acknowledgements .................................................................................................. 56 Chapter 3: Comparative Characterization of the Sindbis Virus Proteome from Mammalian and Invertebrate Hosts Identifies nsP2 as a Component of the Virus Nucleocapsid and Sorting Nexin 5 as a Significant Host Factor for Alphavirus Replication ................................... 57 3.1: Abstract .................................................................................................................... 58 3.2: Importance................................................................................................................ 59 3.3: Introduction .............................................................................................................. 60 3.4: Results ...................................................................................................................... 66 3.5: Discussion and Conclusions ..................................................................................... 80 3.6: Materials and Method .............................................................................................. 88 3.7: Acknowledgements .................................................................................................. 94 Chapter 4: Summary and Future Perspectives ...................................................................... 95 REFERENCES ............................................................................................................................ 99 APPENDIX ............................................................................................................................... 124 vi LIST OF FIGURES Figure 1.1 An EM reconstruction of Sindbis Virus. In this reconstruction, the E1/E2 glycoprotein coat is seen in yellow, the lipid bilayer is seen in red, and the nucleocapsid core is seen in blue. Though not seen in this image, the E2 protein crosses the membrane and interacts with the capsid
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