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SDSU Template, Version 11.1 UC San Diego UC San Diego Electronic Theses and Dissertations Title Use of retroviral peptide libraries for the identification of novel cellular targets of HIV-1 and the discovery of novel inhibitors Permalink https://escholarship.org/uc/item/60q2v5fq Authors Stotland, Aleksandr Borisovich Stotland, Aleksandr Borisovich Publication Date 2012 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA, SAN DIEGO SAN DIEGO STATE UNIVERSITY Use of Retroviral Peptide Libraries for the Identification of Novel Cellular Targets of HIV-1 and the Discovery of Novel Inhibitors A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Biology by Aleksandr Borisovich Stotland Committee in charge: University of California, San Diego Professor Michael David Professor Deborah H. Spector Professor Celsa A. Spina San Diego State University Professor Roland Wolkowicz, Chair Professor Ralph Feuer Professor Christopher C. Glembotski 2012 Copyright © 2012 by Aleksandr Borisovich Stotland All Rights Reserved The Dissertation of Aleksandr Borisovich Stotland is approved, and it is acceptable in quality and form for publication on microfilm and electronically: _______________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ Chair University Of California, San Diego San Diego State University 2012 iii DEDICATION I would like to dedicate this thesis to all of my family, friends and lab mates without whose support none of my work would be possible. iv TABLE OF CONTENTS Signature Page……………………………………………………………………………iii Dedication………………………………………………………………………………...iv Table of Contents………………………………………………………………………….v List of Figures……………………………………………………………………………vii List of Tables……………………………………………………………………………...x Acknowledgements……………………………………………………………………….xi Vita………………………………………………………………………………………xii Abstract………………………………………………………………………………….xiv Introduction to Dissertation……………………………………………………………….1 References…………………………………………………………………………6 Chapter I: Purification of the COP9 Signalosome Complex and Binding Partners from Human T-cells in the presence of HIV-1 Proteins ……………………………………......9 Introduction………………………………………………………………………10 Materials and Methods…………………………………………………………...14 Results…………………………………………………………............................18 Discussion………………………………………………………………………..28 References………………………………………………………………………..33 Appendix…………………………………………………………………………38 Chapter II: Development of Random Retroviral Nuclear-Targeted Peptide Libraries as Tools for Discovery of Novel HIV-1 Inhibitors…………………………………………59 Introduction………………………………………………………………………60 Materials and Methods…………………………………………………………...62 v Results…………………………………………………………............................68 Discussion………………………………………………………………………..81 References………………………………………………………………………..84 Chapter III: Further Applications of Retroviral Peptide Libraries for Targeting HIV-1 Gag-Pol and Envelope Processing……………………………………………………….87 Introduction………………………………………………………………………88 Materials and Methods…………………………………………………………...91 Results…………………………………………………………............................94 Discussion………………………………………………………………………102 References………………………………………………………………………105 vi LIST OF FIGURES Figure 1-1: Establishment of an SBP-CSN1 expressing cell line. (A) Representation of pBMN.SBP-CSN1.i.mCherry, the pBMN.i.mCherry retroviral vector harboring the SBP sequence fused to the N′ terminus of CSN subunit 1. (B) Retroviral particles produced from pBMN.SBP-CSN1.i.mCherry were used to infect naive SupT1 cells. ……………19 Figure 1-2: Schematic representation of the SBP-based pulldown technique for the purification of the CSN and binding partners. ………………………………………….20 Figure 1-3: Purification of the CSN from T cells. (A) Coomassie stain of the eluted fractions isolated from SBP-Citrine control and SBP-CSN1 cell line lysates. (B) Confirmation by Western blots (B) and LC-MS (Scaffold3 Software) (C) of the presence of all subunits of the CSN in the eluted fractions from the SBP-CSN1 lysate…………..20 Figure 1-4: Western blot analysis of SBP-Citrine and SBP-CSN1 cell lysates to confirm CSN binding partners. (A) Cell line eluates were probed with anti-Nedd8 antibody to show the interaction of the CSN with neddylated proteins (three arrows) in the SBP- CSN1 lysate but not in the SBP-Citrine control. Anti-Cullin 4A ……………………..25 Figure 1-5: 14-3-3 interacts with the subunits of the CSN in the presence of HIV-1 proteins. Scaffold3 readout of the LC-MS data demonstrating the presence of the 14-3-3 Zeta/Delta in the SBP-CSN1 eluate from pCMV ΔR8.2 cell lysate (A). Western blot of a pulldown experiment with lysates transfected with pCMV ΔR8.2 and 3XFLAG-Vpr probed with pan 14-3-3 antibody (B)…………………………………………………..27 Figure 2-1: Schematic representation of scaffolds used in library construction………...69 Figure 2-2: Expression of scaffold constructs (A) Fluorescent micrographs of 293T cells transiently transfected with the GNF scaffold and SupT1 cells infected with MLV produced with the GNF as the transfer vector. (B) Coomassie stain of the eluted fraction of a pulldown from 293T cells transfected with SBP-GNF……………………………70 Figure 2-3: Analysis of library complexity and ligation efficiency (A) Restriction digest analysis (BamHI and XhoI) of the bacterial colonies produced by the ligation of the 3XF(GGS)3Blast library. (B, C, D) Peptide sequences produced as a result of the 3XF(GGS)3Blast ligation, GNF ligation and SBP-GNF ligation………………………72 Figure 2-4: Sub-cellular localization of the 3XF(GGS)3 NLS RPL and 3XFmCherry NLS RPL (A) Immunofluorecent staining for FLAG expression of 293T cells transfected with either the 3XF(GGS)3Blast scaffold or the 3XF(GGS)3 NLS RPL. (B)…………………74 vii Figure 2-5: Establishment of NLS RPL-expressing SupT1 cells. (A) The retroviral library is transfected along with the VSVg envelope into Phoenix-GP cells, for the production of (B) non-replicative MLV virions carrying the peptide sequences, which are used to infect (C) naïve SupT1 cells…………………………………………………………………….76 Figure 2-6: Screening of the 3XF(GGS)3 NLS RPL library for inhibitors of HIV-1 Graphical representation of the screening process (A). Infection rates of mCherry and 3XF(GGS)3 NLS RPL SupT1 cells after the second round of screening (B) compared to infection rates after the twelfth round of screening (C)………………………………….77 Figure 2-7: Screening of clonal populations derived from the 3XF(GGS)3 NLS RPL library. Infection of selected clones with sinHIV-GFP (A) compared to infection with sinMLV-GFP (B)………………………………………………………………………...78 Figure 2-8: Recovery and Identification of Sequences from the 3XF(GGS)3 clones Amplification of the clone sequences from genomic PCR of 3XF(GGS)3 clones 2 and 6 (A) Full peptide sequences rescued by genomic PCR from Clones 2, 6, and 8 (B)……..79 Figure 3-1: Schematic Overview of the T-cell-based PR Assay. The assay is based on a conditional (Tet-On) expression of the Gal4 transcription factor driving the expression of GFP from the UAS promoter (A, B). The autocatalytic activity of PR, inserted between the DBD and TAD domains inhibits GFP expression…………………………………...95 Figure 3-2: Expression of GFP in Assay Cells SupT1 cells expressing either Gal4 (A), Gal4 in fusion with a mutant PR (B), or Gal4 in fusion with wild-type PR (C) activated with 1 µg/ml of Dox in the absence or presence of 10 µM Indinavir (IDV)…………….95 Figure 3-3: Random Cytosolic Retroviral Peptide Library The library scaffold in the context of the MoMLV retroviral transfer vector pmGIB MG*XhoI (Nolan Lab, Stanford, CA) (A) and the detailed DNA sequence of the SKVILFE dimerization scaffold (B)………………………………………………………………………………………..96 Figure 3-4: Assay Cells Transduced with the RPL Flow cytometry profiles of SupT1 cells expressing the PR assay transduced at an MOI of 1 with the RPL virions and activated with 1 µg/ml Dox (A). Clonal cell populations 5 and 6 expanded from the screen and activated with Dox (Forward Scatter Area on the X-axis, FITC on the Y-axis) (B)…….97 Figure 3-5: Clonal Populations Isolated from The Screen Flow cytometry profiles of the 18 clonal cell lines established from the screen (Forward Scatter Area on the X-axis, FITC on the Y-axis)………………………………………………………………….…..98 Figure 3-6: Amplification of the Peptide Sequence from the Genomic DNA of Selected Clones Peptide sequences amplified by PCR from the genomic DNA of Clones 1, 10, 11, and 12 recovered by the screen…………………………………………………………..99 viii Figure 3-7: Random peptide library targeted to the ER (A) Schematic representation of the retroviral scaffold for ER localization. (B) Design of the RPL insert oligo. (C) Confocal microgarphs of 293T cells transfected with the ER scaffold and stained for FLAG-FITC and ER resident protein GRP 78 to confirm ER localization…………….101 ix LIST OF TABLES Table 1-1: Peptides of the CSN Subunits Identified by LC-MS…………………………21 Table 1-2: Peptides of Proteins Binding to the CSN Identified by LC-MS……………...24 Table 1-3: Putative CSN-Interacting Proteins Identified With an 80% or Greater Probability………………………………………………………………………………..38 Table 1-4: All Putative CSN-Interacting Proteins Identified by LC-MS………………..42
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