A Thesis entitled Homology-based Structural Prediction of the Binding Interface Between the Tick-Borne Encephalitis Virus Restriction Factor TRIM79 and the Flavivirus Non-structural 5 Protein. by Heather Piehl Brown Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Master of Science Degree in Biomedical Science _________________________________________ R. Travis Taylor, PhD, Committee Chair _________________________________________ Xiche Hu, PhD, Committee Member _________________________________________ Robert M. Blumenthal, PhD, Committee Member _________________________________________ Amanda Bryant-Friedrich, PhD, Dean College of Graduate Studies The University of Toledo December 2016 Copyright 2016, Heather Piehl Brown This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Homology-based Structural Prediction of the Binding Interface Between the Tick-Borne Encephalitis Virus Restriction Factor TRIM79 and the Flavivirus Non-structural 5 Protein. by Heather P. Brown Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Master of Science Degree in Biomedical Sciences The University of Toledo December 2016 The innate immune system of the host is vital for determining the outcome of virulent virus infections. Successful immune responses depend on detecting the specific virus, through interactions of the proteins or genomic material of the virus and host factors. We previously identified a host antiviral protein of the tripartite motif (TRIM) family, TRIM79, which plays a critical role in the antiviral response to flaviviruses. The Flavivirus genus includes many arboviruses that are significant human pathogens, such as tick-borne encephalitis virus (TBEV) and West Nile virus (WNV). We found that TRIM79 directly interacts with the viral polymerase, nonstructural protein 5 (NS5), and leads to lysosomal degradation of NS5. This restriction is specific to TBEV, as the direct binding of TRIM79 and subsequent degradation of NS5 was not seen in the mosquito-borne flavivirus WNV, despite the TBEV and WNV NS5 proteins sharing a 59% identity. Thus, dissecting the TRIM79/NS5 interaction will provide an effective model of how antiviral proteins differentiate between similar viral proteins. To begin addressing how TRIM79 targets only TBEV NS5 the 3D structures of TBEV and WNV NS5 were compared and modeled the TBEV NS5/TRIM79 interaction complex to identify critical residues for this interaction. Because the structures of TRIM79 and TBEV NS5 are unsolved, homology-based protein modeling was used to create preliminary structures for both proteins. These structures iii were then used to predict the binding interface for TRIM79 monomers and dimers. From the predicted binding interfaces, residues important for binding were identified that were unique to TBEV NS5 that could then be mutated to disrupt the interaction, rendering TBEV NS5 resistant to TRIM79 restriction. iv To my husband, TJ, who believed in me even when I did not believe in myself. I would never have gotten to here without your love and support. Acknowledgements This thesis would not have been possible without the support of many individuals. A special thanks to my committee members Dr. Bob Blumenthal and Dr. Xiche Hu, both of whom contributed lots of support with learning new techniques and methods, and by posing thought provoking questions that helped guide the research and kept it from being an impossibly large question to answer. I would like to thank all the past and present members of the Taylor Lab, but especially John Presloid, Adaeze Izuogu, and Brian Youseff, who have provided insight, guidance, and encouragement that helped me develop into a better scientist. I owe a special debt of gratitude to Dr. Travis Taylor, who was kind enough to invite a budding bioinformatician into a virology lab. Travis’s ability to get on the same level and explain things in a way that made daunting concepts seem easy was invaluable. His endless guidance and patience mean more to me than I can ever express. This endeavor would never have been possible without the support of my family. My parents have provided a guiding light for me to follow and never tried to quell my relentless desire to learn more about the world around me. My brothers and my sisters have all provided unconditional emotional support and let me ramble on about my project. My grandmother Paula provided not only emotional and financial support of this endeavor, but was also a valuable source with which to discuss my project. vi Table of Contents Abstract iii Acknowledgements vi Table of Contents vii List of Tables ix List of Figures x I. Introduction 1 A. Flaviviruses 1 B. Flavivirus Entry 2 C. Flavivirus Replication 3 D. Flavivirus Immune Evasion 5 E. Host Cell TRIM Restriction Factors 7 F. TRIM79 10 G. Protein Structural Modeling 11 II. Methods 16 A. Homology Modeling 16 B. Predicting the Interaction 16 C. Selecting Key Residues 17 D. Calculating Interaction Energies 18 E. In silico Mutagenesis 19 F. Eukaryotic Linear Motif 20 III. Results 21 A. Structural Modeling 21 vii B. Interaction Complex Modeling 23 C. Modeling Effects of Mutation 24 D. Summary 25 IV. Discussion 27 References 60 viii List of Tables Table 1 Excerpt from a contact map output file. .........................................................48 Table 2 Preliminary list of TBEV mutagenesis candidates with the corresponding amino acid mutations. ....................................................................................50 Table 3 List of TBEV mutagenesis candidates with the corresponding amino acid mutations from TRIM79 monomer cross referenced with the alignment of TRIM79 and TRIM30. ....................................................................................51 Table 4 Final list of TBEV mutagenesis candidates with the corresponding amino acid mutations from docking the TRIM79 dimer and cross referenced with the alignment of TRIM79 and TRIM30. .........................................................52 Table 5 Energy calculations for all interaction complexes. ........................................54 Table 6 Energy calculations for the impact of point mutations on complex 19. ........56 Table 7 Energy calculations for the impact of the mutation P95K on other interaction complexes. .....................................................................................................59 ix List of Figures Figure 1 Code used in TK Console to generate the contact map. .................................28 Figure 2 Sequence alignment of JEV NS5 and TBEV NS5. .........................................29 Figure 3 Homology modeled structure of TBEV NS5 utilizing the solved crystal structure of JEV NS5. .....................................................................................30 Figure 4 Structural comparison of TBEV NS5 and WNV NS5 modeled on the TBEV NS5 structure. .................................................................................................35 Figure 5 Multiple sequence alignment (MSA) of WNV NS5, LGTV NS5, and TBEV NS5. ................................................................................................................39 Figure 6 Structure of TBEV NS5 with all non-identical and non-similar residues marked. ............................................................................................................41 Figure 7 Sequence alignment of TRIM5α and TRIM79 ................................................42 Figure 8 Homology modeled partial structure of mouse TRIM79 utilizing the solved partial crystal structure of rhesus macaque TRIM5α .......................................43 Figure 9 Sequence alignment of mouse TRIM79 and mouse TRIM30. .......................47 Figure 10 Top four predicted interaction complexes. .....................................................49 Figure 11 Structure of TBEV NS5 with preliminary candidate residues marked. ..........53 Figure 12 Model of interaction complex 19. ...................................................................55 Figure 13 Graph of ΔE impact of point mutations. .........................................................57 Figure 14 Graph of ΔΔE impact of point mutation. ........................................................58 x Chapter One Introduction The Flaviviruses. Mankind has always battled against foes that were invisible to the naked eye. Bacteria and viruses predate the human species and, though antibiotics have been developed to treat many of the bacteria that can cause us harm, viruses have proved to be more difficult to treat. Among the myriad of pathogenic viruses one genus has been of interest to the Taylor Laboratory, the flaviviruses due to the large incidences of disease caused by them. Members of this genus are geographically widespread, and can be found on every continent that has a dense human population. There are currently over 70 members of this genus, of which forty species are able to cause disease in humans. In this genus are several well known and emerging human pathogens including Dengue virus (DENV), Yellow Fever virus (YFV), Tick-borne Encephalitis virus (TBEV), Japanese Encephalitis virus (JEV), St. Louis Encephalitis virus (SLEV), West Nile virus (WNV), Zika virus (ZIKV), and Powassan virus (POWV). These viruses are responsible for hundreds of thousands of cases reported each year, and often lead to hospitalization or in extreme cases,