UC Merced UC Merced Electronic Theses and Dissertations Title INVESTIGATION OF THE MECHANISM OF GRIFFITHSIN (GRFT): A POTENT HIV ENTRY INHIBITOR Permalink https://escholarship.org/uc/item/5176w2wz Author Xue, Jie Publication Date 2014 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA, MERCED INVESTIGATION OF THE MECHANISM OF GRIFFITHSIN (GRFT): A POTENT HIV ENTRY INHIBITOR A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Quantitative and Systems Biology by Jie Xue Committee in charge: Professor Andy LiWang, Chair Professor David Ojcius Professor Jinah Choi Professor Patricia J. LiWang, Supervisor 2014 Copyright Jie Xue, 2014 All rights reserved The dissertation of Jie Xue, titled Investigation of the Mechanism of Griffithsin (GRFT), a Potent HIV entry inhibitor, is approved, and it is acceptable in quality and form for publication on microfilm and electronically: Date Professor David Ojcius Date Professor Jinah Choi Supervisor Date Professor Patricia LiWang Chair Date Professor Andy LiWang University of California, Merced 2014 iii To My Family, Thank you for all of your love, support, and sacrifice throughout my life. iv Table of Contents Abstract .............................................................................................................. viii List of Figures .......................................................................................................ix List of Tables ....................................................................................................... xii List of Abbreviations ........................................................................................... xiii Acknowledgements ............................................................................................ xiv Chapter 1 ............................................................................................................. 1 Introduction ........................................................................................................... 1 1.1 HIV pandemic around the world .................................................................. 1 1.1.1 History and pandemic of HIV ................................................................ 1 1.1.2 HIV transmission ................................................................................... 4 1.1.3 HIV clades (subtypes) and geographic distribution ............................... 6 1.2 HIV entry process ........................................................................................ 7 1.3 Trimeric envelope spikes (env) on the HIV surface ..................................... 8 1.3.1 Env trimers form clusters on HIV surface .............................................. 8 1.3.2 HIV envelope protein gp120 and gp41 .................................................. 9 1.3.4 Gp120 undergoes significant conformational change upon receptor and coreceptor binding ....................................................................................... 12 1.3.5 Structure mechanism of env activation ............................................... 14 1.3.6 Glycan sites on the gp120 surface ...................................................... 14 1.4 Structure of the HIV coreceptor ................................................................. 16 1.4.1 Structure of CCR5 ............................................................................... 16 1.4.2 Model of chemokine-CCR5 interaction: ............................................... 17 1.4.3 Structure of HIV coreceptor CXCR4.................................................... 18 1.5 HIV entry inhibitors .................................................................................... 19 1.5.1 Inhibitors that bind glycoprotein gp120 ................................................ 19 1.5.2 Griffithsin (Grft): A protein lectin binds the carbohydrates on gp120 ... 20 1.5.3 Inhibitors that bind coreceptor CCR5: RANTES and its derivatives .... 21 1.5.4 Inhibitor binds both coreceptor CCR5 and CXCR4: vMIP-II ................ 22 1.5.5 Inhibitors that bind gp41: Fusion peptides ........................................... 23 1.6 Multivalent studies of anti-HIV lectins and antibodies ................................ 25 1.7 Microbicides .............................................................................................. 27 v Chapter 2 ........................................................................................................... 28 The Role of Individual Carbohydrate-Binding Sites in the Function of the Potent Anti-HIV Lectin Griffithsin ................................................................................... 28 2.1 Abstract ..................................................................................................... 28 2.2 Introduction ............................................................................................... 29 2.3 Material and Methods ................................................................................ 31 2.3.1 DNA construction ................................................................................ 31 2.3.2 Protein production and purification ...................................................... 31 2.3.3 GRFT binding to D-mannose-agarose column .................................... 32 2.3.4 ELISA studies of GRFT-gp120 interactions ........................................ 32 2.3.5 Virus isolates ....................................................................................... 32 2.3.6 Single-round infection assays ............................................................. 33 2.3.7 Anti-HIV assays in CEM cell cultures .................................................. 33 2.3.8 Cocultivation assay ............................................................................. 33 2.3.9 NMR spectroscopy .............................................................................. 33 2.3.10 Surface plasmon resonance (SPR) analysis ..................................... 34 2.3.11 Analytical Ultracentrifugation ............................................................. 35 2.4 Results ...................................................................................................... 36 2.4.1 NMR chemical shift assignments of wild-type GRFT. ......................... 36 2.4.1 NMR of GRFT with mannose. ............................................................. 37 2.4.3 Mutations of carbohydrate-binding sites in GRFT. .............................. 38 2.4.4 Mutations in the CBS of GRFT reduce the ability of GRFT to bind mannose and gp120. ................................................................................... 42 2.4.5 Mutation of the individual CBS markedly reduces the anti-HIV activity of GRFT. .......................................................................................................... 48 2.5 Discussion ................................................................................................. 51 2.6 Conclusion ................................................................................................ 54 Chapter 3 ........................................................................................................... 55 The Griffithsin Dimer Is Required for High-Potency Inhibition of HIV-1: Evidence for Manipulation of the Structure of gp120 as Part of the Griffithsin Dimer Mechanism ......................................................................................................... 55 3.1 Abstract ..................................................................................................... 55 3.2 Introduction ............................................................................................... 56 3.3 Material and Methods ................................................................................ 59 3.3.1 DNA construction ................................................................................ 59 vi 3.3.2 Protein purification .............................................................................. 59 3.3.3 NMR spectroscopy .............................................................................. 59 3.3.4 Analytical Ultracentrifugation ............................................................... 60 3.3.5 ELISA studies of Grft obligate dimer interaction with HIV gp120 ........ 60 3.3.6 Surface plasmon resonance (SPR) analysis ....................................... 61 3.3.7 Viral Reagents .................................................................................... 61 3.3.8 Virus capture ELISA ............................................................................ 62 3.3.9 Single round infection assays ............................................................. 62 3.3.10 Anti-HIV Assays in CEM Cell Cultures .............................................. 62 3.3.11 Grft-induced CD4 binding site exposure ........................................... 63 3.3.12 Cell surface gp120 shedding ............................................................. 63 3.3.13 Grft cross-linking separate gp120 subunits ....................................... 63 3.4 Results ...................................................................................................... 64 3.4.1 Design of the obligate Griffithsin dimer. .............................................. 64 3.4.2 Obligate Grft dimer binding to gp120. ................................................. 67 3.4.3