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University of Southampton Research Repository Copyright © and Moral Rights for this thesis and, where applicable, any accompanying data are retained by the author and/or other copyright owners. A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. This thesis and the accompanying data cannot be reproduced or quoted extensively from without first obtaining permission in writing from the copyright holder/s. The content of the thesis and accompanying research data (where applicable) must not be changed in any way or sold commercially in any format or medium without the formal permission of the copyright holder/s. When referring to this thesis and any accompanying data, full bibliographic details must be given, e.g. Thesis: Author (Year of Submission) "Full thesis title", University of Southampton, name of the University Faculty or School or Department, PhD Thesis, pagination. Academic Thesis: Declaration Of Authorship I, Rafael Gonçalves Barbosa Gomes declare that this thesis and the work presented in it are my own and has been generated by me as the result of my own original research. Exploring genetic and physical aspects of Hepatitis C Virus non-structural proteins involved in the formation of the replication complex I confirm that: 1. This work was done wholly or mainly while in candidature for a research degree at this University; 2. Where any part of this thesis has previously been submitted for a degree or any other qualification at this University or any other institution, this has been clearly stated; 3. Where I have consulted the published work of others, this is always clearly attributed; 4. Where I have quoted from the work of others, the source is always given. With the exception of such quotations, this thesis is entirely my own work; 5. I have acknowledged all main sources of help; 6. Where the thesis is based on work done by myself jointly with others, I have made clear exactly what was done by others and what I have contributed myself; 7. Either none of this work has been published before submission, or parts of this work have been published as: Gomes RG, Isken O, Tautz N, McLauchlan J, McCormick CJ. Polyprotein-Driven Formation of Two Interdependent Sets of Complexes Supporting Hepatitis C Virus Genome Replication. J Virol. 2015;90(6):2868-83. Signed: ………………………………………………………………………… Date: ………………………………………………………………………… UNIVERSITY OF SOUTHAMPTON FACULTY OF MEDICINE Clinical and Experimental Science Exploring genetic and physical aspects of Hepatitis C Virus non-structural proteins involved in the formation of the replication complex by Rafael Gonçalves Barbosa Gomes Thesis for the degree of Doctor of Philosophy September 2017 UNIVERSITY OF SOUTHAMPTON ABSTRACT FACULTY OF MEDICINE INFECTION AND IMMUNITY Thesis for the degree of Doctor of Philosophy EXPLORING GENETIC AND PHYSICAL ASPECTS OF HEPATITIS C VIRUS NON-STRUCTURAL PROTEINS INVOLVED IN THE FORMATION OF THE REPLICATION COMPLEX Rafael Gonçalves Barbosa Gomes Hepatitis C Virus (HCV) is the causative agent of Hepatitis C, a chronic liver disease that is responsible for significant morbidity and mortality throughout the world. Replication of the HCV RNA genome relies on the expression of non-structural proteins (NS), produced initially as a single polyprotein. Often subgenomic viral transcripts that express the NS polyprotein are used to study RNA replication. Previous work in the laboratory led to the development of a novel ‘intragenomic’ replicon, a construct expressing two copies of the NS polyprotein, thus readily enabling genetic complementation studies to be undertaken. Using this system, it was shown that genetic defects in NS4B and NS5A could be experimentally separated on the basis of the minimum polyprotein required to rescue these defects; some mutations were dependent on NS3-NS4A-NS4B-NS5A (NS3-5A) but others required NS3-NS4A-NS4B-NS5A-NS5B (NS3-5B). In this project, we investigated if this categorization could be extended to mutations elsewhere in the NS polyprotein, focussing on the viral protease/helicase NS3, as well as its co-factor NS4A. Three mutations were identified whose rescue could be facilitated by NS3- 5A; one in the NS3 linker region (PP1220-1GG), another in the NS3 protease domain (L1157A), and a third in the NS4A acidic domain (Y1706A). In contrast, one mutation in the NS4A transmembrane domain (V1665G) could not be rescued by NS3-5A. Furthermore, this same mutation exhibited a dominant negative phenotype, but consistent with the model of polyprotein–dependent complex formation, only when expressed in the context of NS3- 5B, but not when expressed in the context of NS3-4A or NS3-5A. An investigation into the mechanisms governing this difference in polyprotein rescue found that many of the mutations typically dependent on NS3-5B for rescue could be rescued by NS3-5A if this latter polyprotein was linked to a downstream coding region. This suggested that nascent NS3-5A was making interactions critical to replication complex formation while tethered to the genome via partially translated NS5B. In a separate study looking at the role that NS proteins play in remodelling lipid bilayers, the interaction of NS4B with neutral and anionic lipids was assessed by static and magic angle spinning (MAS) 31P nuclear magnetic resonance (NMR) spectroscopy. Static NMR showed that NS4B caused a broadening of the powder pattern of POPC multilamellar vesicles (MLVs) and that, at high temperature (45°C), there is significant bilayer disturbance with loss of the axially symmetric pattern. The MAS NMR showed that in the presence of anionic lipids, NS4B leads to changes in the surface charge density. This suggests that NS4B interacts with anionic lipids and that this interaction changes the bilayer mobility. These changes were also confirmed by measuring the T1 relaxation time. Page 4 of 212 TABLE OF CONTENTS CHAPTER 1 INTRODUCTION 21 1.1 HEPATITIS C VIRUS 21 1.2 FLAVIVIRIDAE FAMILY 21 1.3 TRANSMISSION AND SYMPTOMS 24 1.4 DIAGNOSIS AND TREATMENT 25 1.5 VIRUS LIFE CYCLE 27 1.5.1 THE VIRUS PARTICLE 27 1.5.2 VIRUS ENTRY 28 1.5.3 TRANSLATION 29 1.5.4 POLYPROTEIN PROCESSING 32 1.5.5 GENOME REPLICATION 33 1.5.5.1 NS2 34 1.5.5.2 NS3/4A 34 1.5.5.3 NS4B 36 1.5.5.4 NS5A 39 1.5.5.5 NS5B 40 1.5.5.6 HOST CELL PROTEINS 41 1.5.5.7 RNA ELEMENTS 41 1.5.6 VIRAL ASSEMBLY AND RELEASE 42 1.5.7 HOST RESPONSE TO IMMUNE INFECTION AND HCV EVASION STRATEGIES 45 1.6 MODEL SYSTEMS TO STUDY HCV INFECTION 48 1.6.1 ANIMAL MODELS 48 1.6.2 CELL CULTURE AND REPLICON SYSTEM 51 1.7 GENETIC COMPLEMENTATION 52 1.8 MEMBRANE PROTEINS 54 1.8.1 MEMBRANE PROTEIN EXPRESSION 54 1.8.2 PROTEIN PURIFICATION CHALLENGES 55 1.9 CIRCULAR DICHROISM 58 1.10 NUCLEAR MAGNETIC RESONANCE 59 1.10.1 SOLUTION AND SOLID-STATE NMR 61 1.10.2 CHEMICAL SHIFT 63 1.10.3 MAGIC ANGLE SPINNING NUCLEAR MAGNETIC RESONANCE 64 1.10.4 SPIN-LATTICE AND SPIN-SPIN RELAXATION 67 1.10.5 31P NMR OF LIPIDS 67 1.11 AIMS 70 CHAPTER 2 MATERIALS AND METHODS 73 2.1 CELL LINES AND STRAINS 73 Page 5 of 212 2.1.1 MAMMALIAN CELLS 73 2.1.2 INSECT CELLS 73 2.2 VIRUS PRODUCTION USING SUSPENSION CELL CULTURE 73 2.3 SF9 CELLS PLAQUE ASSAY 73 2.4 PROTEIN EXPRESSION AND HARVESTING 74 2.5 PROTEIN PURIFICATION 74 2.6 LIPID RECONSTITUTION 76 2.7 PROTEIN DIALYSIS 77 2.8 NUCLEAR MAGNETIC RESONANCE 78 31 2.8.1 P NUCLEAR MAGNETIC RESONANCE 78 31 2.8.2 P NUCLEAR MAGNETIC RESONANCE T1 RELAXATION 78 2.9 BACTERIAL GROWTH MEDIA AND SOLUTIONS 78 2.10 LIQUID BACTERIAL CULTURE 79 2.11 PREPARATION OF COMPETENT ESCHERICHIA COLI CELLS 79 2.12 TRANSFORMATION OF COMPETENT E. COLI CELLS 79 2.13 ISOLATION OF PLASMID DNA 80 2.14 POLYMERASE CHAIN REACTION 80 2.14.1 REACTION CONDITIONS 80 2.14.2 PCR-BASED MUTAGENESIS 81 2.14.3 PCR SCREENING OF BACTERIAL COLONIES 81 2.14.4 PRIMER SEQUENCES 82 2.15 PHENOL/CHLOROFORM EXTRACTION AND ETHANOL PRECIPITATION 83 2.16 SODIUM DODECYL SULFATE-POLYACRYLAMIDE GEL ELECTROPHORESIS 83 2.17 TRANSFER OF PROTEINS FROM A POLYACRYLAMIDE GEL TO POLYVINYLIDENE DIFLUORIDE MEMBRANE 84 2.18 WESTERN BLOT 85 2.19 DNA AGAROSE GEL ELECTROPHORESIS 86 2.20 PURIFICATION OF DNA FRAGMENTS FROM AGAROSE GEL 86 2.21 RESTRICTION ENDONUCLEASE DIGESTS 86 2.22 DNA LIGATIONS 87 2.23 SYNTHESIS OF RNA FROM CDNA TEMPLATES 87 2.24 PURIFICATION OF RNA FROM T7 REACTIONS 87 Page 6 of 212 2.25 MOPS/FORMALDEHYDE GEL ELECTROPHORESIS 88 2.26 TRANSFECTION 88 CHAPTER 3 ASSESSING POLYPROTEIN DEPENDENT DELIVERY OF NS3 & NS34A FUNCTIONS INTO THE HCV REPLICATION COMPLEX 91 3.1 INTRODUCTION 91 3.2 SELECTION AND TESTING OF POINT MUTATIONS IN NS3 AND NS4A THAT BLOCK REPLICATION 91 3.3 INTRODUCTION OF NS34A MUTATIONS INTO A SUBGENOMIC REPLICON 92 3.4 ASSESSING REPLICATION OF SGR-JFH1LUC-FLAG REPLICON CARRYING THE NS34A MUTATIONS 95 3.5 HELPER REPLICON-DEPENDENT TRANS-COMPLEMENTATION OF SGR-JFH1-LUC CARRYING NS34A MUTATIONS 96 3.6 ASSESSING THE POLYPROTEIN REQUIREMENTS FOR THE RESCUE OF NS34A MUTATIONS USING AN INTRAGENOMIC REPLICON 99 3.6.1 GENERATION OF INTRAGENOMIC REPLICONS CARRYING LETHAL NS34A MUTATIONS IN THE ORF2 REPLICASE 99 3.6.2 REPLICATION OF THE INTRAGENOMIC REPLICONS WITH THE NS34A MUTATIONS 103 3.7 ESTABLISHING POLYPROTEIN REQUIREMENTS NEEDED FOR TRANS-COMPLEMENTATION OF NS34A MUTATIONS 104 3.8 USING THE INTRAGENOMIC REPLICON TO ANALYSE RESCUE OF THE CIS-ACTING HELICASE MUTATION T1299A 106 3.8.1 GENERATION OF INTRAGENOMIC REPLICONS CARRYING T1299A MUTATION IN THE ORF2 REPLICASE 107 3.8.2 ASSESSMENT OF ORF1 ABILITY IN REPLACING THE CIS-ACTING ROLE OF ORF2 107 3.9 ESTABLISHING A POSSIBLE DOMINANT NEGATIVE EFFECT OF OTHER NS3