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Characterization and Targeting the Dynamic Recognition of Trp37-G During the Interaction of Ncp7 Protein of HIV-1 with Nucleic Acids Rajhans Sharma

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Characterization and Targeting the Dynamic Recognition of Trp37-G During the Interaction of Ncp7 Protein of HIV-1 with Nucleic Acids Rajhans Sharma Characterization and targeting the dynamic recognition of Trp37-G during the interaction of NCp7 protein of HIV-1 with nucleic acids Rajhans Sharma To cite this version: Rajhans Sharma. Characterization and targeting the dynamic recognition of Trp37-G during the interaction of NCp7 protein of HIV-1 with nucleic acids. Biophysics. Université de Strasbourg, 2018. English. NNT : 2018STRAJ014. tel-02918150 HAL Id: tel-02918150 https://tel.archives-ouvertes.fr/tel-02918150 Submitted on 20 Aug 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. UNIVERSITÉ DE STRASBOURG ÉCOLE DOCTORALE DES SCIENCES DE LA VIE ET DE LA SANTE UMR CNRS 7021 Laboratoire de Bioimagerie et Pathologies THÈSE présentée par : Rajhans SHARMA soutenue le : 10th Avril 2018 pour obtenir le grade de : Docteur de l’université de Strasbourg Discipline/ Spécialité : Sciences du vivant/Biophysique Caractérisation et ciblage de la reconnaissance dynamique de Trp37-G lors de l’interaction de la protéine NCp7 de HIV-1 avec des acides nucléiques THÈSE dirigée par : M. MELY Yves Professeur, Université de Strasbourg RAPPORTEURS : Mme. MAHUTEAU-BETZER Florence Chargée de recherche, Institut Curie,Paris Mme. GATTO Barbara Associate Professor, University of Padova AUTRES MEMBRES DU JURY : M. LEONARD Jérémie Chargé de recherche, Universitié de Strasbourg UNIVERSITY OF STRASBOURG ÉCOLE DOCTORALE DES SCIENCES DE LA VIE ET DE LA SANTÉ UMR CNRS 7021 Laboratory of Bioimaging and Pathologies THESIS presented by : Rajhans SHARMA Defense on : 10th April, 2018 For obtaining the degree of : Doctorate of University of Strasbourg Discipline/ Specialization : Life sciences/Biophysics Characterization and targeting the dynamic recognition of Trp37-G during the interaction of NCp7 protein of HIV-1 with nucleic acids THESIS directed by : Mr. MELY Yves Professor, University of Strasbourg REPORTERS : Mrs. MAHUTEAU-BETZER Florence Chargée de recherche, Institut Curie, Paris Mrs. GATTO Barbara Associate Professor, University of Padova OTHER MEMBERS OF JURY : Mr. LEONARD Jérémie Chargé de recherche, University of Strasbourg 3 Table of Contents Acknowledgement ......................................................................................................... 6 List of Abbreviations ..................................................................................................... 8 1. Bibliographic Review ........................................................................................ 10 1.1. Human Immunodeficiency Virus Type 1 (HIV-1) ..................................... 11 1.1.1. Overview ................................................................................................. 11 1.1.2. Origin Timeline ....................................................................................... 11 1.1.3. Classification and Transmission.............................................................. 11 1.1.4. Structural Organization ........................................................................... 12 1.1.5. Genomic Organization ............................................................................ 13 1.1.6. Viral Proteins........................................................................................... 18 1.1.7. Replication Cycle of HIV-1 .................................................................... 23 1.1.8. Antiretroviral Therapy (ART) ................................................................. 28 1.2. Structure-Function Relationship of Nucleocapsid Protein ......................... 31 1.2.1. Introduction ............................................................................................. 31 1.2.2. Nucleic Acid Chaperoning of NC ........................................................... 32 1.2.3. Role of NC During HIV-1 Reverse Transcription .................................. 34 1.2.4. Role of NC Domain of Gag in Viral Assembly ...................................... 38 1.3. Fluorescence and Nucleic Acids................................................................. 41 1.3.1. Introduction to Fluorescence ................................................................... 41 1.3.2. Introduction to Nucleic Acids ................................................................. 45 1.3.3. Fluorescence Nucleobase Analogues ...................................................... 52 1.3.4. Emerging Applications of Fluorescent Nucleobase Analogues .............. 59 2. Research Objectives ........................................................................................... 62 3. Materials and Methods ....................................................................................... 66 3.1. Fluorescent Nucleobase Analogues ............................................................ 67 3.2. Peptides ....................................................................................................... 68 3.3. Oligonucleotides ......................................................................................... 69 3.4. UV/Vis Absorption and Fluorescence Spectroscopy. ................................ 71 3.5. Time Correlated Single Photon Counting .................................................. 74 3.6. Kinetic Measurements ................................................................................ 76 3.7. Isothermal Titration Calorimetry (ITC) ...................................................... 78 4 3.8. Molecular Dynamics (MD) ........................................................................ 79 4. Results and Discussions ..................................................................................... 80 4.1. CHAPTER 1: Photophysical Investigation of Fluorescent Nucleobase Analogues. 81 4.1.1. Publication 1: Tautomers Of A Fluorescent G Surrogate and Their Distinct Photophysics Provide Additional Information Channels. ................................................ 81 4.1.2. Identifying the Fluorescence Lifetimes of thG Tautomers ...................... 84 4.1.3. Photostability of thG as substituted in (–)PBS ......................................... 85 4.1.4. Publication 2: Conquering 2‑Aminopurine’s Deficiencies: Highly Emissive Isomorphic Guanosine Surrogate Faithfully Monitors Guanosine Conformation and Dynamics in DNA ..................................................................................................... 90 4.2. CHAPTER 2: Fluorescent Nucleobase Analogues as a Tool for Characterizing Dynamics of Nucleocapsid Protein Promoted Annealing of (–)/(+)PBS. ... 92 4.2.1. Publication 3: Dynamic Interconversion of Nucleic Acid Structures Surveyed by Environment-Sensitive Fluorescent Nucleoside Analogues. ...................... 92 4.3. CHAPTER 3: Investigating the Role of Sugar Deoxyribose in Binding Polarity of HIV-1 Nucleocapsid Protein. ............................................................................. 94 4.3.1. Introduction ............................................................................................. 94 4.3.2. Results and Discussions ........................................................................ 101 4.3.3. Conclusions ........................................................................................... 128 4.4. CHAPTER 4: Screening the Inhibitors of Nucleocapsid Protein ............. 131 4.4.1. Introduction ........................................................................................... 131 4.4.2. THINPAD ............................................................................................. 132 4.4.3. NCinh assay........................................................................................... 134 4.4.4. Site Specific Binding Assay .................................................................. 135 4.4.5. Conclusions ........................................................................................... 139 5. Conclusions ...................................................................................................... 140 6. Perspectives...................................................................................................... 146 7. Résume en Français ......................................................................................... 148 8. Appendix .......................................................................................................... 156 9. References ........................................................................................................ 168 5 Acknowledgement I am immensely grateful to Prof. Yves Mély for giving me this opportunity to pursue the PhD research work under his supervision. Thank you for your guidance, discussions, sharing knowledge, pushing me to higher limits, improving my skills, encouragements and help me grow as a researcher. I admire your positivity, dynamism, sense of humor and winning attitude. Your ‘perfectionism’ has always inspired me to excel my inputs. I am grateful to all the members of jury, Dr. Florence Mahuteau-Betzer, Dr. Barbara Gatto and Dr. Jerémié Leonard for accepting the proposal to evaluate this thesis work. I am grateful for
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