Abstract Hepatitis C virus (HCV) is a leading cause of chronic liver disease. Identification of key host cellular factors involved in the HCV replication cycle is important for understanding disease pathogenesis and identifying novel antiviral therapeutic targets. The objective of this study was to identify novel host factors with important roles in the HCV replication cycle. Two approaches were used to select factors to test as potential HCV cofactors. The first approach investigated factors previously shown to facilitate the replication of other RNA viruses. This included Rab GTPases and the RNA binding proteins, Staufen, TIAL1 and TIA1. The second approach investigated factors observed to be upregulated during HCV infection following microarray expression profiling of HCV (JFH-1 clone) infected Huh7 hepatoma cells. Host genes controlling innate immunity, proliferation, lipid and protein metabolism and intracellular transport were observed to increase in expression, whilst genes controlling oxidative stress and cytoskeletal regulation decreased in expression. The expression patterns observed also indicated that HCV associated pathogenesis may be caused by HCV-induced gene expression changes. Further investigation into the upregulation of lipid synthesis genes observed during HCV infection demonstrated that intracellular lipid abundance also increased significantly, and may contribute to the development of hepatic steatosis in chronically infected patients. Host gene silencing by siRNA knockdown was used to investigate the potential cofactor role of the selected candidate host factors. Factors shown to be essential for effective HCV replication and secretion included those involved in lipid metabolism (TXNIP, CYP1A1 and CIDEC), intracellular transport (RAB2B, RAB4A, RAB11B, RAB27A/B, RAB33B and ABLIM3), and mRNA regulation (Staufen1). It was also shown that TXNIP may control lipid metabolite abundance during HCV replication, and Staufen1 may colocalise with HCV replication complexes to control HCV genome translation, replication or trafficking. Further investigation into the potential HCV cofactors identified should now be performed to improve understanding of the HCV replication cycle and enable identification of novel factors for antiviral targeting. 3 4 Table of Contents Abstract .................................................................................................................. 3 Table of Contents ................................................................................................... 5 Table of Figures ................................................................................................... 11 Table of Tables ..................................................................................................... 14 Abbreviations ........................................................................................................ 15 Publications and Presentations ............................................................................ 22 Acknowledgements .............................................................................................. 23 Declaration ........................................................................................................... 24 Chapter 1: General Introduction ........................................................................... 25 1.1 The pathogenesis, treatment and classification of Hepatitis C virus ............. 27 1.1.1 Pathogenesis of HCV infection .............................................................. 27 1.1.2 Treatment of HCV infection .................................................................... 27 1.1.3 Classification of HCV infection ............................................................... 29 1.2 The HCV RNA genome ................................................................................ 30 1.3 The HCV replication cycle ............................................................................ 31 1.3.1 HCV entry .............................................................................................. 31 1.3.2 Genome translation and replication ....................................................... 33 1.3.3 Virus particle assembly and secretion .................................................... 34 1.4 The role of virus proteins in the HCV replication cycle ................................. 40 1.4.1 The structural HCV proteins ................................................................... 40 1.4.2 The non-structural HCV proteins ............................................................ 42 1.5 The role of cellular host factors in the HCV replication cycle ........................ 47 1.5.1 Host factors with a role in genome translation and replication ............... 47 1.5.2 Host factors with a role in assembly and secretion ................................ 50 1.5.3 Host factors involved in the production of HCV-essential metabolites ... 53 1.5.4 The role of the cellular cytoskeleton in the HCV replication cycle .......... 54 5 1.6 In vitro and in vivo systems for studying the HCV replication cycle .............. 54 1.6.1 In vitro systems for studying the HCV replication cycle .......................... 54 1.6.2 In vivo systems for studying the HCV replication cycle .......................... 58 1.7 Experimental approaches used to study the HCV replication cycle .............. 59 1.7.1 Protein capture assays ........................................................................... 59 1.7.2 Genomic studies of HCV infection .......................................................... 60 1.7.3 Proteomic studies of HCV infection ........................................................ 63 1.7.4 Host factors involved in the replication of other related RNA viruses ..... 65 1.8 Hypothesis and Project Aims ........................................................................ 77 Chapter 2: Materials and Methods ........................................................................ 79 2.1 Cell culture .................................................................................................... 81 2.2 Production of infectious JFH-1 virus stocks .................................................. 81 2.3 Affymetrix microarray profiling of host expression during JFH-1 infection..... 85 2.4 Quantitative PCR analysis of host expression during JFH-1 infection .......... 86 2.5 Generation of genotype 2a (GT2a) replicon expressing cell lines ................. 91 2.6 Generation of JFH-1 core expressing Huh7 cells ......................................... 93 2.7 Western blot analysis of host proteins during JFH-1 infection ...................... 96 2.8 Triglyceride quantification assay ................................................................... 99 2.9 Cholesterol quantification assay ................................................................. 101 2.10 Lipid droplet staining and quantification .................................................... 102 2.11 Glucose quantification assay .................................................................... 104 2.12 siRNA transfection of Huh7 and Huh7.5 cells ........................................... 104 2.13 Alamar blue cell viability assay ................................................................. 110 2.14 Quantitative PCR expression analysis of interferon stimulated genes ...... 111 2.15 Immunofluorescent staining of host proteins ............................................. 112 2.16 Statistical Analysis of Results ................................................................... 113 6 Chapter 3: The Effect of HCV Infection on Host Gene Expression ..................... 115 Chapter 3: Introduction ....................................................................................... 117 Chapter 3: Results .............................................................................................. 119 3.1 The JFH-1 cell culture system .................................................................... 119 3.1.1 Production of infectious JFH-1 virus..................................................... 119 3.1.2 A high-titre production line for JFH-1 virus ........................................... 119 3.1.3 JFH-1 infectivity in Huh7 and Huh7.5 cells ........................................... 120 3.2 Why study the effect of HCV infection on host gene expression? .............. 121 3.3 Expression profiling of factors involved in the replication of other viruses .. 121 3.4 Identification of host factors upregulated during HCV infection .................. 123 3.4.1 Microarray gene expression profiling of JFH-1 infected Huh7 cells ..... 123 3.4.2 Intracellular JFH-1 RNA abundance in the JFH-1 infected Huh7 cells . 124 3.4.3 Gene ontology analysis of the microarray expression data .................. 124 3.4.4 JFH-1 infection alters the expression of cellular defence genes .......... 126 3.4.5 JFH-1 infection alters the expression of metabolism and transport genes ............................................................................................................ 127 3.4.6 The biological significance of HCV induced gene expression patterns 129 3.4.7 Validation of the microarray expression data by quantitative PCR ....... 131 3.4.8 Comparing host cell response to infection: Huh7 versus Huh7.5 cells . 134 3.5 Gene expression profiling of cells expressing a subgenomic replicon ........ 137 3.5.1 Generating cells that express a genotype 2a subgenomic replicon ..... 137 3.5.2 Quantitative PCR analysis of subgenomic