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Retrocyclin Rc-101 Overcomes Cationic Mutations on the Heptad Repeat 2 of Hiv-1 Gp41

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Retrocyclin Rc-101 Overcomes Cationic Mutations on the Heptad Repeat 2 of Hiv-1 Gp41 University of Central Florida STARS Electronic Theses and Dissertations, 2004-2019 2007 Retrocyclin Rc-101 Overcomes Cationic Mutations On The Heptad Repeat 2 Of Hiv-1 Gp41 Christopher Fuhrman University of Central Florida Part of the Immune System Diseases Commons, Microbiology Commons, and the Molecular Biology Commons Find similar works at: https://stars.library.ucf.edu/etd University of Central Florida Libraries http://library.ucf.edu This Masters Thesis (Open Access) is brought to you for free and open access by STARS. It has been accepted for inclusion in Electronic Theses and Dissertations, 2004-2019 by an authorized administrator of STARS. For more information, please contact [email protected]. STARS Citation Fuhrman, Christopher, "Retrocyclin Rc-101 Overcomes Cationic Mutations On The Heptad Repeat 2 Of Hiv-1 Gp41" (2007). Electronic Theses and Dissertations, 2004-2019. 3166. https://stars.library.ucf.edu/etd/3166 RETROCYCLIN RC-101 OVERCOMES CATIONIC MUTATIONS ON THE HEPTAD REPEAT 2 OF HIV-1 GP41 by CHRISTOPHER “KIT” ALLEN FUHRMAN B.S. University of Central Florida, 2005 A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in the Department of Molecular Biology and Microbiology in the Burnett College of Biomedical Sciences at the University of Central Florida Orlando, Florida Summer Term 2007 Major Professor: Alexander M. Cole © 2007 Christopher Allen Fuhrman ii ABSTRACT Retrocyclin RC-101, a θ-defensin with lectin-like properties, potently inhibits infection by many HIV-1 subtypes by binding to the heptad repeat (HR)-2 region of gp41 and preventing six-helix bundle formation. In the present study, we used in silico computational exploration to identify residues of HR2 that interacted with RC-101 and then analyzed the HIV-1 Sequence Database at LANL for residue variations in the HR1 and HR2 segments that could plausibly impart in vivo resistance. Docking RC-101 to gp41 peptides in silico confirmed its strong preference for HR2 over HR1, and implicated residues crucial for its ability to bind HR2. We mutagenized these residues in pseudotyped HIV-1 JR.FL reporter viruses, and subjected them to single round replication assays in the presence of 1.25-10ug/ml RC-101. Except for one mutant that was partially resistant to RC-101, the other pseudotyped viruses with single-site cationic mutations in HR2 manifested absent or impaired infectivity or retained wild-type susceptibility to RC-101. Overall, these data suggest that most mutations capable of rendering HIV-1 resistant to RC-101 will also exert deleterious effects on the ability of HIV-1 to initiate infections - an interesting and novel property for a potential topical microbicide. iii ACKNOWLEDGEMENTS I would like to thank Martine Kline for his excellent technical assistance. I am also very grateful to Dr. Wilfred Li, Teri Simas and Chris Misleh at the NBCR for the use of the rocks cluster supercomputer. The NBCR also granted a scholarship to attend a weeklong work in San Diego on Autodock for which I am very grateful. I also thank my lab-mates: Julie Martellini, Anna Herasimtschuk, Nick Felts and Nitya Venkataraman. Thanks especially to Andrew Warren whose dedication help push this project along. Thanks to my committee members for the guidance in my work and in my scientific career. And finally, thank you Dr. Alex and Amy Cole for their support and tutelage. I appreciate all that I have learned from both of you throughout the last two years. iv TABLE OF CONTENTS LIST OF FIGURES ...................................................................................................................... vii LIST OF ACRONYMS/ABBREVIATIONS................................................................................ ix CHAPTER ONE: INTRODUCTION............................................................................................. 1 Overview..................................................................................................................................... 1 Innate Immunity.......................................................................................................................... 2 Defensins..................................................................................................................................... 3 Alpha- and Beta-Defensins......................................................................................................... 5 Theta-Defensins .......................................................................................................................... 5 HIV-1 Epidemic........................................................................................................................ 10 Mechanism of HIV Infection.................................................................................................... 11 The Structure of gp41 ............................................................................................................... 14 HIV-1 and Retrocyclin.............................................................................................................. 18 Molecular Docking ................................................................................................................... 20 CHAPTER TWO: MATERIALS AND METHODS ................................................................... 23 Computational Analysis of the Variation in HR1 and HR2 ..................................................... 23 Preparation of HR1, HR2 and RC-Structure Models................................................................ 23 Computational Modeling of RC-101 Binding .......................................................................... 24 Defining Hydrogen and Non-Hydrogen Bonds ........................................................................ 25 Preparation of Peptide............................................................................................................... 25 Cell Culture............................................................................................................................... 26 HIV-1 Plasmid Constructs and Viral Entry Assay ................................................................... 26 v CHAPTER THREE: RESULTS................................................................................................... 29 Amino Acid Variation of HR1 and HR2 .................................................................................. 29 RC-101’s Preference for Anionic Charge................................................................................. 32 Anionic-to-Cationic Mutations on HR2.................................................................................... 35 RC-101 Binds to the HR1-binding regions of HR2.................................................................. 39 RC-101 Binds Anionic, Polar, and Hydrophobic Residues of HR2......................................... 41 Cationic Mutations in the HR1-Binding Domain of HR2 ........................................................ 43 CHAPTER FOUR: DISCUSSION............................................................................................... 45 CHAPTER FIVE: CONCLUSION............................................................................................... 47 APPENDIX A: MANUSCRIPT SUBMISSION.......................................................................... 49 APPENDIX B: COMPUTER PROGRAM .................................................................................. 52 Docking Log Parser Command Line Interface (DLGCLI)....................................................... 53 CountBonds............................................................................................................................... 54 PrimerFind ................................................................................................................................ 55 APPENDIX C: SEQUENCE INFORMATION ........................................................................... 59 REFERENCES ............................................................................................................................. 63 vi LIST OF FIGURES Figure 1 Defensins of the Innate Immune System........................................................................................4 Figure 2 Theta-Defensins..............................................................................................................................8 Figure 3 RC-101 and RC-100.......................................................................................................................9 Figure 4 Six-Helix Bundle Formation Drives Viral-Host Membrane Fusion.............................................13 Figure 5 Heptamer Designation of Alpha Helix .........................................................................................15 Figure 6 Illustration of the Heptamer Functionality of the 6-Helix Bundle................................................16 Figure 7 Rendering of the 6-Helix Bundle per Heptamer Functionality.....................................................17 Figure 8 The Lamarckian Genetic Algorithm.............................................................................................22 Figure 9 Pseudotype Methodology .............................................................................................................28 Figure 10 Amino Acid Diversity of HR1 and HR2 ....................................................................................30 Figure 11 Isoelectric Point of HR1 vs.
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