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A Dissertation Submitted to the Faculty of the Graduate School of Arts And CHARACTERIZATION OF GII NOROVIRUS VPG NUCLEOTIDYLYLATION AND NUCLEOSIDE TRIPHOSPHATE BINDING A Dissertation submitted to the Faculty of the Graduate School of Arts and Sciences of Georgetown University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology By Alexei Medvedev, B.S. Washington, DC December 6, 2016 Copyright 2016 by Alexei Medvedev All Rights Reserved ii CHARACTERIZATION OF GII NOROVIRUS VPG NUCLEOTIDYLYLATION AND NUCLEOSIDE TRIPHOSPHATE BINDING Alexei Medvedev, B.S. Thesis Advisor: Dr. Brent Korba, Ph.D. ABSTRACT Human Norovirus (hNoV) is a single-stranded positive-sense RNA virus of Caliciviridae family. It is the major cause of gastroenteritis outbreaks in the United States. The VPg protein of hNoV is a multi-functional protein essential for virus replication. Sequential and single-point alanine substitutions revealed that several positively charged amino acids in the N-terminal region of hNoV VPg regulate its nucleotidylylation. I provide evidence that hNoV VPg directly binds nucleoside triphosphates (NTPs), that inhibition of binding inhibits nucleotidylylation, and that the NTP binding appears to involve the first 13 amino acids of the protein. Substitution of multiple positively charged amino acids within the first 12 amino acids of the N-terminal region reduces nucleotidylylation without affecting binding. Substitution of only Lys20 abolishes nucleotidylylation, but not NTP binding. These studies indicate that positively charged amino acids in the first 20 amino acids of hNoV VPg regulate its nucleotidylylation through several potential mechanisms. Additionally, I demonstrate that the un-cleaved viral precursor protein, ProPol (NS5-6) is 100-fold more efficient in catalyzing VPg nucleotidylylation than the mature polymerase (Pol, NS6), suggesting a specific role for ProPol during the hNoV infection. iii The research and writing of this dissertation is a monument to everyone who helped me along the way. First and foremost, I would like to thank my amazing mentor, Dr. Brent Korba. It is hard to imagine that I could have done my thesis work anywhere else, but your lab. You instilled in me a special kind of appreciation for the scientific process, one that is not always straightforward, having a distinct set of guidelines, but, rather, one that requires creative thinking, ability to adjust on the fly, and just a tad of luck. Thank you for letting me run wild with my ideas and for always finding positives in every outcome, good or bad. Most importantly, thank you for being a great fatherly figure, caring and understanding, especially in the last (and very difficult) year of my thesis work. I can proudly admit that I never had to “work” in the lab under your guidance. Thank you, my lab-mates and co-mentors, Drs. Jared May and Prasanth Viswanathan. Your friendship and scientific advice were invaluable in my growth as a scientist. My sanity is relatively intact, all thanks to the fun and collaborative environment you helped me create in our lab. I also would like to acknowledge Dr. Brittany Griffin, who was very instrumental in my development as a scientist in the early stages of my PhD education. Thank you for always fighting for my right to do science. I think the scientific community suffered a tremendous loss when you hung up your lab coat, and I hope you can find your way back one day. Dear members of my Thesis Committee, Drs. John Casey, William Fonzi, Kim Green, and Radhakrishnan Padmanabhan, thank you for guiding me along on this journey. Thank you for pushing me forward when I needed it and for acknowledging my progress when I did something worthy. You helped me create high standards for my scientific work and were instrumental in making sure that I do not fall short of expectations. Big thanks to my friends Vanya Kamyshko, Zo Noor, and Roma Dogadin. I will never be able to thank you enough for your support in my work, but, even more importantly, in my life outside the lab. You have come through for me on more than one occasion and helped me keep a clear and calm mind in the darkest, most turbulent times. With you, I never feel alone. Thank you, Katerina Satanovsky and the rest of the Satanovsky clan for helping me start this journey and for supporting me along the way. Most importantly, thank you, Anya Stolpe, for helping me complete that journey. Your unprecedented support and believe in my abilities have given me strength and confidence to finish my work. You make me a better man. My family. Where would I be without you? Great many thanks to my mother, Tamara Medvedeva, for allowing her little boy to leave for America, an impossible decision for any parent. Thank you, my grandparents, Viktor and Valentina Palanevich, as well as Tamara and Gennady Medvedev for stoking fires of curiosity and instilling great human values in me from an early age. The rest of my Belarussian, Canadian, German, and American relatives, thank you very much for always keeping me in your thoughts and checking up on me from time to time. My mom-away-from-mom, Natasha Medvedev, thank you for taking care of me like your own son. My little siblings, Maxim, Nikita, and Ksenya, thank you for showing me how to be cool and how to have fun. My lovely sister Anastasia, I am in awe of your superhuman abilities to do everything you do. Thank you for inspiring me to work harder, and a special thank you for bringing Milena, a constant beacon of light, into this life. Finally, thank you, my father, Vladimir Medvedev. None of this would be possible without you. Period. Your relentless scientific and academic pursuits opened up a path to America for me and the rest of our family. You are the most brilliant, driven, and yet humble man I have ever encountered. I have looked up to you my whole life and I would be extremely happy someday to be even half the man and scientist you are. Thank you for your continued support in my work and in my life, and thank you for being the best father a son could ever ask for. I dedicate this work to you. Many thanks, ALEXEI MEDVEDEV iv TABLE OF CONTENTS I. Introduction ................................................................................................................................ 1 II. Background ................................................................................................................................ 5 A. Picornavirus VPg Biology ................................................................................................... 5 B. Potyvirus VPg Biology ........................................................................................................ 7 C. Calicivirus VPg Biology ...................................................................................................... 11 III. Materials and Methods ............................................................................................................... 14 A. Cloning and Mutagenesis .................................................................................................... 14 B. Protein Expression ............................................................................................................... 16 C. hNoV Polymerase (Pol) Assay ............................................................................................ 18 D. hNoV VPg Nucleotidylylation Assay ................................................................................. 19 E. hNoV VPg NTP Binding Assay .......................................................................................... 21 F. Structure Modeling .............................................................................................................. 21 IV. Characterization of Human Norovirus VPg ............................................................................... 23 A. hNoV VPg Nucleotidylylation Occurs Through Covalent Linkage to the α-Phosphate of the Incoming Nucleotide ................................................................................................. 24 B. Nucleotidylylation of Native hNoV VPg by hNoV ProPol Is Significantly Greater Than Nucleotidylylation Catalyzed by hNoV Pol ........................................................................ 26 C. GMP and UMP Are Preferentially Added to hNoV VPg .................................................... 28 D. Positively-Charged Amino Acids in the N-Terminus of hNoV VPg Regulate Its Nucleotidylylation by hNoV ProPol ................................................................................... 28 E. The Central Core Region of hNoV VPg Does Not Contain Amino Acids Required for the Stability of the Nucleotidylylation Reaction ............................................................ 31 F. hNoV VPg Binds NTPs in the Absence of Polymerase ...................................................... 33 v V. Future Work ............................................................................................................................... 35 A. Plasmon Resonance Required to Study NTP Binding by hNoV VPg ................................. 35 B. hNoV VPg Is a Protein Primer for Initiation of hNoV Genomic RNA Synthesis .............. 36 C. Protease Domain of Intact hNoV ProPol Is Required for Interaction with hNoV VPg and Its Subsequent Nucleotidylylation ................................................................................ 39 VI. Conclusions ................................................................................................................................ 41 Appendices .............................................................................................................................................
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