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Inhibition of Polyomavirus DNA Replication by Nucleotide Analogs

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Inhibition of Polyomavirus DNA Replication by Nucleotide Analogs Provided by the author(s) and NUI Galway in accordance with publisher policies. Please cite the published version when available. Title Inhibition of polyomavirus DNA replication by nucleotide analogs Author(s) Onwubiko, Nichodemus Okechukwu Publication Date 2017-01-31 Item record http://hdl.handle.net/10379/6277 Downloaded 2021-09-27T20:27:15Z Some rights reserved. For more information, please see the item record link above. Inhibition of Polyomavirus DNA Replication by Nucleotide Analogs Nichodemus Okechukwu Onwubiko Supervised by Professor Heinz-Peter Nasheuer School of Natural Sciences, Biochemistry National University of Ireland, Galway A thesis submitted to the National University of Ireland, Galway for a degree of Doctor of Philosophy January, 2017 Table of Contents Table of contents List of Abbreviation…………………………………………………………….…….…....iv Acknowledgment…………………………………………………………………………..vii Abstract...….………………………………………………………………………………viii 1.0 Introduction.........……………………………………………………………………….1 1.1 Polyomaviruses.............…………………………………………………….………....2 1.1.1 Discovery, Classification and Disease Association of Human Polyomaviruses....2 1.1.1.1 JC Virus..….............……………………………………………………………3 1.1.1.1.1 Reactivation of JCV in Humans.......………..........…….....……………...4 1.1.1.2 BK Virus...……….............…………………………………...……………..….7 1.1.1.2.1 Disease Association of BKV...…………....……............………………...8 1.1.1.2.1.1 Polyomavirus-Associated Nephropathy...…………….................……..8 1.1.1.2.1.2 Polyomavirus-Assoiated Hemorrhagic Cystitis...……………..............10 1.1.1.2.1.3 Ureteric Stenosis and other BKV-Associated Diseases..…....………...10 1.1.1.3 Trichodysplasia Spinulosa Virus (TSV)……….......……………..…………...11 1.1.1.3.1 TSV-Associated Diseases………….............………….……..………….12 1.1.1.4 Merkel Cell Carcinoma Virus..……...........……………………………….12 1.1.1.4.1 Disease Association of MCV..………….…….....…..... ….…………….13 1.1.1.5 Other Human Polyomaviruses...……..………… …………………………..13 1.1.1.6 Representation of Selected Human Polyomaviruses………………………17 1.1.2 Structure, Genome Architecture, Transcriptome and Proteome of Polyomaviruses....…...….……………………………………………………...18 1.1.2.1 Polyomavirus Structure..………….…………….…………………………….18 1.1.2.2 Polyomavirus Genome Architecture, Transcriptome and Proteome.......18 1.1.2.2.1 The Large Tumor Antigen.…………..…....…..….…….…..…………...20 1.1.2.2.2 Additional Gene Product Encoded by the Early Region.………….....…23 1.1.2.3 Non Coding Control Region…..….……………………….…………....24 1.1.2.3.1 The Replication Origin..…………....…….……………….…………….25 1.2 Polyomavirus DNA Replication: A Model for Eukaryotic DNA Replication........27 1.2.1 Replication Protein A..……..……………………………….…………………..30 1.2.2 DNA Polymerase α-primase........….……………………………….…………...33 1.2.3 DNA Polymerase δ.....…………………………………………………………..35 1.2.4 Proliferating Cell Nuclear Antigen.....…………………………………………..36 1.2.5 Human Topoisomerase I...………..…………………………….……………….37 1.3 Host Immune Responses and Control of Polyomavirus Infection….…......….…..38 1.4 Pharmaceutical Agents for the Treatment of Polyomavirus Infections….……..39 1.4.1 Acyclovir......…………………………………….……………………………...40 1.4.2 Cidofovir and Derivatives: Application, Metabolism and Mechanism of Action ...…………………………………………………………………………….......40 1.4.2.1 Use of Cidofovir for the Management of Polyomavirus-Associated Symptoms ..………………………………………………………………………….…....42 1.5 Aims and Objectives..…………………………………………….……………….…44 2.0 Materials and Methods...……………………………………………………….……..45 2.1 Materials.....…....……………………………………………………..…………...….46 i Table of Contents 2.1.1 Equipment and Suppliers............………………………………….…………….46 2.1.2 Chemicals, Kits and Solutions....……………………………………….……….47 2.1.3 Buffers and Solutions………………………………………….………………..47 2.1.4 Services.………………………………………………………………………....48 2.1.5 Primers.………………………………………….……………………………....48 2.1.6 Recombinant Plasmid DNA and Baculoviruses......……………….…………....49 2.1.7 Bacterial Strains and Eukaryotic Cell Lines.……..…….………………….…....50 2.1.8 Analogs and Chemical Agents Studied.....………….…………………………..50 2.2 Methods…..….……………………………………………………………………….51 2.2.1 Culture Techmiques.………...………………………………………………...51 2.2.1.1 Bacterial Culture…………………………………………………………...….51 2.2.1.2 Insect Cultures………………………………………………………….….….51 2.2.1.3 HeLa Cell Culture.…………………………………………….…….…….… .51 2.2.1.4 Heat Inactivation of Fetal Bovine Serum (FBS)………………………………51 2.2.2 Molecular Biology Techniques.……………….… ……..…………………….52 2.2.2.1 Transformation of Competent Cells…….………….………………………....52 2.2.2.2 Plasmid Purification and Analyses….………………………………………...52 2.2.2.3 Horizontal DNA Gel Electrophoresis….………….… ……………………….52 2.2.2.4 Protein Analysis Gels………………………………………….… …………..53 2.2.2.5 Protein Transfer and Western Blotting.……………………………………….53 2.2.2.6 Coomassie Brilliant Blue Staining of Protein Gels…………………………...54 2.2.3 Preparation of HeLa S100 Extract...………………………………………….54 2.2.4 Protein Expression, Purifications and Characterization...………….………55 2.2.4.1 Purification of SV40 Larger Tumor Antigen.….………...……….…………..55 2.2.4.2 Purification of JCV Larger Tumor Antigen.………...……………….……….56 2.2.4.3 Purification of Wild-type BKV Large Tumor Antigen.……………...……....57 2.2.4.4 Purification of FLAG-tagged BKV Large Tumor Antigen…..……..……......58 2.2.4.5 DNA Polymerase α-Primase Expression and Purification…………….……..58 2.2.4.6 His-Tagged Topoisomerase I Expression and Purification…………….…….60 2.2.4.7 Expresssion and Purification of Human Replication Protein A……………...61 2.2.4.8 Purification of Proliferating Cell Nuclear Antigen and Pol δ………………...62 2.2.5 Biochemical Assays....………………………………………….……………....63 2.2.5.1 In vitro DNA Replication Assay..………………………………………….….63 2.2.5.2 Monopolymerase Asssay.…………….……………………………………….63 2.2.5.3 DNA Synthesis Assay….……………………………………….…………….64 2.2.5.4 DNA Helicase Assay for Polyomavirus T Antigens.…………….……….......65 2.2.5.5 Preparation of DNA Substrates for Helicase Activity and DNA Synthesis Assay..………………………………………………………………………....65 2.2.5.6 ATPase and UTPase Assays for Polyomavirus T Antigens....…………….…66 2.2.5.7 Electrophoretic Mobility Shift Assay (EMSA)....………….………………...67 2.2.5.8 DNA Relaxation Assay……………………………………………………….67 2.2.5.9 Data Analysis Programs and Methods………………………………………...67 3.0 Analyses and Characterization of Purified Proteins..…....………………….….…...69 3.1 Polyomavirus Large Tumor Antigens.………..….……………………………….…..70 3.2 Human Topoisomerase I....………………………………………………….…….….71 3.3 Human DNA Polymerase -Primase......….……………………………………….…73 ii Table of Contents 3.4 Replication Protein A..........…………………………………………….………….....74 3.5 Proliferating Cell Nuclear Antigen....….……………………………….…………….75 4.0 Nucleotide Analogs in In Vitro Replication of Polyomaviruses and Species Specifcity.....………………………………………………………………………..…..77 4.1 In Vitro Replication Of SV40 DNA Is Inhibited by Fatty-Ester Linked Nucleotide Analogs...……………………………………………………………………………...78 4.2 Inhibitory Effects of the Analogs on Polyomavirus DNA Replication: Species-Specific Implications …………………………………………………………………………..81 4.3 BKV DNA Replication Is Inhibited by Fatty-Ester Conjugated Analogs …...............84 5.0 Monopolymerase Replication of Polyomavirus DNA, the Effetcs of Nucleotide Analogs………………………………………………………………………………....86 5.1 Monopolymerase Replication of SV4O DNA Is Sensitized to Fatty-Ester Derivatives of Cidofovir...…………………………………….……………………………..…….87 5.2 Effects of Analogs on Monopolymerase Replication of JCV DNA..……………........89 5.3 Effects of Analogs on Monopolymerase Replication of BKV DNA..……………........90 6.0 Studying Human DNA Polymerases as Cellular Targets of Nucleotide Analogs…........................................................................................................................92 6.1 Determination of the Enzyme Activity of Human DNA Polymerase α-Primase…......93 6.2 Effects of Analogs on Synthesis of DNA by Human DNA Polymerase α–Primase ...……...……………………………………………………………………………….94 6.3 Effects of Analogs in the Presence of Different Concentrations of Deoxynucleotides ………………………………………………………………………………………...95 6.3.1 Effects of Fatty-Ester Conjugated Analogs on Human DNA Polymerase α-Primase DNA Synthesis with Respect to dCTP………………………………..................95 6.3.2 Effects of Fatty-Ester Conjugated Analogs on Human DNA Polymerase α-Primase DNA Synthesis with Respect to dGTP, dTTP and dATP....................................101 6.4 Understanding the Mechanism of Inhibition of dCTP Utilization by HDP-CDV and Its Enatiomer CMX029………………………………………………………………....111 6.5 The Effects, Kinetics and Mechanism of Inhbition of Human DNA Polymerase α – primase by CMX029....…...…………………………………………………………115 6.6 Effects of Analogs on Synthesis of DNA by Human DNA Polymerase δ.…..……...119 7.0 Polyomavirus-Encoded TAgs as Candidate Targets of Nucleotide Analogs..……120 7.1 Optimization of Helicase Assay Conditions….…………………..…………..….…121 7.1.1 Nucleotide Analogs and the Helicase Activity of JCV Polyomavirus ...….……122 7.2 Effects of Fatty-Ester-Derived Cidofovir (HDP-CDV) on Primate Polyomavirus TAgs’ Helicase Activity ……………………………………………….….….……123 7.3 ATPase Activities of Primate Polyomaviruses TAgs and Nucleotide Analogs…….126 7.4 Effects of Nucleotide Analogs on the ATPase Activities of Primate Polyomaviruses. ......…..………………………………………………………………….…………..127 7.5 Effects of Nucleotide Analogs on Additional Host Replication Proteins .........…....131 8.0 Discussion, Conclusion AND Future Studies.….…………….……………………..134 References.........…..…………………….………………………………………………...141 Appendices………………………………………………………………………………..184
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