https://theses.gla.ac.uk/ Theses Digitisation: https://www.gla.ac.uk/myglasgow/research/enlighten/theses/digitisation/ This is a digitised version of the original print thesis. Copyright and moral rights for this work are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This work cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Enlighten: Theses https://theses.gla.ac.uk/ research-enlighten@glasgow.ac.uk Characterisation of the translation efficiency and quasispecies composition of the 5’untranslated region of Hepatitis C Virus in genotype 1 and 3 infected patientsbyMorteza Motazakker, M.D.A thesis presented for the degree of Doctor of PhilosophyinThe Faculty of Biomedical and Life Sciencesat theUniversity of GlasgowInstitute of Virology University of Glasgow Church St GlasgowG il 5JR May 2005ProQuest Number: 10800562All rights reservedINFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted.In the unlikely event that the author did not send a com p le te manuscript and there are missing pages, these will be noted. Also, if material had to be removed,a note will indicate the deletion.uestProQuest 10800562Published by ProQuest LLC(2018). Copyright of the Dissertation is held by the Author.All rights reserved.This work is protected against unauthorized copying under Title 17, United States C odeMicroform Edition © ProQuest LLC.ProQuest LLC.789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346f GLASGOW I UNIVERSITY I u b m a r y .AcknowledgementsFirstly, I would like to thank Professor Richard Elliott for providing research facilitie in the Institute of Virology.I would like to thank my supervisor, Dr Liz McCruden for her valuable comments ard encouragement throughout this project and especially for her critical reading of this thesis so promptly.I am grateful to Dr Frazer Rixon, my second supervisor for his helpful advice and support.I am grateful to Prof Richard Elliot for providing me pRL vector and some other reagents used in this project. I also wish to thank Dr John McLauchlan for providing the core product and anticore monoclonal antibody, and for his helpful discussions. I am grateful to Dr. Karin Oien for providing me with the liver biopsy scoring data. Many thanks to Gwen Gunning at the Regional Virus Laboratory for her help in quantitation of viral load using the COBAS system. Thanks Dr Rory Bowden, for his helpful comments on work presented in Chapter 6.I wish to thank all past and present members of lab 311/301 for both technical assistance and for providing a friendly environment in which to work. Many thanks tc Dr Petra Preiskchat for providing sequencing data and PCR products. Special thanks 10 Carol-Anne Smith for looking after the sera and for teaching me how to use the real time PCR system. Thanks Selma Erwa, for carrying out part of work described in chapter 7. Thanks Victoria McKechnie for providing me the HVR clones used in chapter 7 .1 also wish to thank the washroom and media staff who supplied the communal glassware and solutions used in this projectMany thanks to all my friends in the Institute and elsewhere in the university, especially Babak Sioofy and Rahim Rezaee who have made the bad days seem not so bad and the good days seem even better.I have to thank my parents, for their continuous strength and support throughout the course of my study. A big thank you to all o f the members of our family in Iran. My wife is the last in this list but deserves the biggest thanks for all her love, encouragement and making the most of our life together in Glasgow. My daughter, Denise for giving me the most enjoyable moments in my life.The author was the recipient of Islamic republic of Iran student scholarship. I thank ths ministry of Health and Medical Education for generous financial support. Unless otherwise, all work presented in this thesis was carried out by the author.Morteza Motazakker May 2005Table of contentsContentsA ck n ow led gem en ts L ist o f F igures and T ables A bbreviations Sum m aryCHAPTER 1 .......................................................................................................................................... 1INTRODUCTION................................................................................................................................... 11.1. Historical background........................................................................................................................................ 11.1.1. Non-A, non-B hepatitis............................................................................................................................ 11.1.2. Cloning of the NANB hepatitis infectious agent................................................................................... 21.1.3. Virus morphology.................................................................................................................................... 31.1.4. Classification of HCV..............................................................................................................................41.2. The HCV genome and its gene products........................................................................................................ 41.2.1. Processing of the HCV polyprotein........................................................................................................41.2.2. Core.......................................................................................................................................................... 51.2.3. El and E2..................................................................................................................................................81.2.4. p7 .............................................................................................................................................................. 91.2.5. NS2......................................................................................................................................................... 101.2.6. NS3......................................................................................................................................................... 101.2.7. NS4A and NS4B.................................................................................................................................... 111.2.8. NS5A...................................................................................................................................................... 121.2.9. NS5B...................................................................................................................................................... 141.3. HCV untranslated regions.............................................................................................................................. 141.3.1. 5’UTR..................................................................................................................................................... 141.3.2. 3TJTR..................................................................................................................................................... 171.4. Translation......................................................................................................................................................... 181.4.1. Cap-dependent translation.....................................................................................................................181.4.2. IRES-mediated translation.....................................................................................................................191.4.3. IRES-mediated translation in picomaviruses........................................................................................191.5. Initiation of translation by the HCV IRES..................................................................................................201.5.1. Cellular factors involved in HCV Cap-independent translation......................................................... 221.5.2. Assembly of the translation complex....................................................................................................241.5.3. Mapping the HCV internal ribosome entry site................................................................................... 251.5.3.1. The 5’ border of HCV IRES..........................................................................................................251.5.3.2. The 3’ border of HCV IRES..........................................................................................................261.5.4. The impact of the 3’ UTR in HCV translation..................................................................................... 271.6. Replication of HCV........................................................................................................................................... 281.6.1. Subgenomic replicon system............................................................................................................. 301.7. Immune response to HCV............................................................................................................................... 31Table of contents1.8. Genetic variability of the HCV genome...................................................................................................331.8.1. Genotypes...............................................................................................................................................331.8.2. Variability in the HCV genome............................................................................................................ 341.8.3. Viral quasispecies..................................................................................................................................351.9. Natural history of HCV infection............................................................................................................. 361.9.1. Epidemiology and transmission............................................................................................................ 361.9.1.1. Parenteral Transmission of HCV...................................................................................................371.9.1.2. Non-parenteral Transmission of HCV.......................................................................................... 381.9.2. Prevention of HCV infection................................................................................................................ 381.9.3. Clinical features of HCV infection.......................................................................................................391.9.3.1. Acute hepatitis C.............................................................................................................................391.9.3.2. Chronic hepatitis C .........................................................................................................................401.9.3.3. Extrahepatic manifestations of HCV infection.............................................................................401.9.4. Genotypes and natural history of HCV.................................................................................................411.10. Treatment of HCV.................................................................................................................................... 411.10.1. Antiviral therapy..................................................................................................................................411.10.2. Predicting response to treatment.........................................................................................................421.11. The background to the work in this thesis............................................................................................451.12. Aims and objectives..................................................................................................................................47CHAPTER 2 ........................................................................................................................................ 48MATERIALS AND METHODS........................................................................................................... 482.1. M aterials.......................................................................................................................................................482.1.1. Bacterial Strains..................................................................................................................................... 482.1.2. Vectors....................................................................................................................................................482.1.3. Synthetic oligonucleotides.................................................................................................................... 482.1.4. Kits and enzymes for RNA/DNA modification................................................................................... 482.1.5. Mammalian cell lines and culture media............................................................................................. 492.1.6. Human sera............................................................................................................................................ 492.1.7. Luciferase assay..................................................................................................................................... 502.1.8. Antibodies...............................................................................................................................................502.1.9. Chemicals...............................................................................................................................................502.1.10. SSCP.....................................................................................................................................................502.1.11. Commonly used solutions................................................................................................................... 502.1.12. Virus stocks.......................................................................................................................................... 512.1.13. Other materials and apparatus............................................................................................................. 522.2. Manipulation of DNA and RNA............................................................................................................... 522.2.1. Small scale preparation of plasmid DNA (Minipreps).........................................................................522.2.1.1. Using QIAprep Spin Miniprep Kit.................................................................................................522.2.1.2. “In-house” Miniprep method......................................................................................................... 532.2.2. Large scale preparation of plasmid DNA (Midipreps).........................................................................532.2.3. Quantification of plasmid DNA and oligonucleotides.........................................................................542.2.4. Restriction enzyme digestion of DNA..................................................................................................542.2.5. Dephosphorylation of linearised plasmid DNA................................................................................... 552.2.6. Extraction of RNA from human sera....................................................................................................552.2.7. Reverse transcription of RNA............................................................................................................... 562.2.7.1. Omniscript reverse transcriptase................................................................................................. 56Table of contents22.1.2. Superscript reverse transcriptase....................................................................................................562.2.8. Polymerase chain reaction (PCR)......................................................................................................... 562.2.8.1. Amplification of 5'UTR................................................................................................................. 562.2.8.2. Amplification of the 5'UTR and core.............................................................................................572.2.8.3. Amplification of cDNA for quantitation of viral load................................................................. 582.2.9. Electrophoretic separation and isolation of DNA................................................................................ 582.2.9.1. Agarose gel electrophoresis........................................................................................................... 582.2.9.2. Purification of DNA from agarose gels........................................................................................ 592.2.9.3. Phenol/Chloroform extraction....................................................................................................... 592.2.9.4. Ethanol precipitation...................................................................................................................... 602.2.10. Ligation of DNA fragments.................................................................................................................602.2.10.1. Ligation of DNA fragment into pRL vector (Collier et al., 1998).............................................602.2.10.2. Ligation of DNA fragment into pCRII vector (TA cloning)......................................................602.2.11. Single Stranded Conformation Polymorphism (SSCP) analysis....................................................... 602.2.11.1. Generation of double stranded DNA........................................................................................... 602.2.11.2. Purification of double stranded DNA......................................................................................... 612.2.11.3. SSCP of PCR product.................................................................................................................. 612.2.11.4. Silver staining method................................................................................................................. 612.2.11.5. Extraction of DNA from silver stained gel................................................................................. 622.2.12. Automated DNA sequencing.............................................................................................................. 622.3. Transformation of competent E.coli cells................................................................................................622.4. Maintenance of mammalian cells............................................................................................................. 632.4.1. BHK cells................................................................................................................................................632.4.2. BHKsinT7 cells..................................................................................................................................... 632.4.3. HuH7 cells............................................................................................................................................. 632.4.4. CV-1 cells.............................................................................................................................................. 642.5. Transfection of mammalian cells.............................................................................................................. 642.5.1. Transfection of pRL constructs into BHKsinT7 cells..........................................................................642.5.2. Transfection of pRL constructs using vTF7.3 vaccinia virus............................................................. 642.5.3. Growth and purification of vTF7.3.......................................................................................................652.5.4. Titration of vTF7.3 stock virus............................................................................................................. 652.6. Dual-Luciferase reporter assay................................................................................................................ 662.6.1. Cell extract preparation..........................................................................................................................662.6.2. Luciferase quantification assay............................................................................................................. 672.6.3. Validation and normalisation of luciferase assay results.....................................................................672.7. Protein analysis by SDS-PAGE................................................................................................................ 682.7.1. Electroblotting to nitrocellulose membrane......................................................................................... 682.7.2. Immunodetection...................................................................................................................................692.7.3. Stripping membrane for reprobing........................................................................................................692.8. Computer software..................................................................................................................................... 692.9. Statistical analysis....................................................................................................................................... 70CHAPTER 3 ........................................................................................................................................ 71COMPARISON OF TRANSLATION EFFICIENCY OF 5’UTR DERIVED FROM GENOTYPE 1 AND 3 INFECTED PATIENTS........................................................................................................... 71Table of contents3.1. Introduction....................................................................................................................................................... 713.2. Study patients and samples.............................................................................................................................743.3. Patients’ majority sequences...........................................................................................................................743.3.1. Majority sequence of GT 1 samples.....................................................................................................753.4. Amplification of 5’UTR region for study of relative translational activities of different sequences ....................................................................................................................................................................................... 763.5. Construction of plasmids containing 5'UTR (pRLN)............................................................................... 763.5.1. Reconstruction ofpRLN clones............................................................................................................ 773.6. The effect of the first 17 nucleotides of the 5’UTR on IRES activity.................................................... 783.7. Relative translation activities in BHKsinT7 cells....................................................................................... 783.8. Relative translation activities in HuH7 cells................................................................................................793.9. Is the IRES of GT 1 is more efficient than that of GT 3?.........................................................................803.10. Comparison of relative translation efficiency in BHKsinT7 and BHK cells..................................... 813.11. Discussion......................................................................................................................................................... 82CHAPTER 4 ........................................................................................................................................ 88COMPARISON OF IRES ACTIVITY OF 5'UTR PLUS CORE REGIONS FROM GT 1 AND 3 INFECTED PATIENTS........................................................................................................................884.1. Introduction....................................................................................................................................................... 884.2. Amplification of 5’UTR plus core regions................................................................................................... 904.3. Sequencing of core coding sequences from patients..................................................................................914.4. Construction of plasmids containing 5'UTR plus core (pRLNC)...........................................................914.5. Relative translation efficiency of 5’UTR plus Core constructs (pRLNC) to equivalent 5’UTR constructs (pRLN)....................................................................................................................................................924.6. IRES activity in GT1 and GT3 pRLNC constructs...................................................................................934.7. The effect of core on IRES activity................................................................................................................944.7.1 Glycosylation of firefly luciferase protein.............................................................................................954.8. Discussion........................................................................................................................................................... 96CHAPTER 5 ...................................................................................................................................... 100STUDYING THE RELATION BETWEEN VIRAL LOAD, LIVER HISTOLOGY AND TRANSLATION EFFICIENCY OF HCV IN PATIENTS.................................................................. 100Table of contents5.1. Introduction..................................................................................................................................................... 1005.2. Clinical sam ples...............................................................................................................................................1035.3. HCV RNA quantification.............................................................................................................................. 1045.3.1. Calibration of the assay........................................................................................................................ 1055.3.2. Comparison of TaqMan real time PCR and COB AS Amplicor Monitor Assay for the quantitation of HCV PCR....................................................................................................................................................... 1065.3.3. Quantitation of viral load in serum samples.......................................................................................1085.4. Liver histology scoring...................................................................................................................................1085.5. Analysis of the correlation between IRES activity, viral load and histological features................ 1095.6. Discussion..........................................................................................................................................................I l lCHAPTER 6 ...................................................................................................................................... 114QUASISPECIES COMPOSITION OF 5’UTR IN SERUM AND LIVER......................................... 1146.1. Introduction..................................................................................................................................................... 1146.2. Study patients and samples........................................................................................................................... 1166.3. Amplification of 5’UTR/core from patients and comparison of resulting “majority” sequence.. 1166.4. Cloning of 5’UTR/core into pCR 2.1 vector.............................................................................................. 1176.5. Sequencing of clones....................................................................................................................................... 1186.6. Comparison of liver and serum quasispecies composition in patients................................................ 1186.7. Artefactual sources of variation...................................................................................................................1196.8. Characteristics of observed 5' UTR changes.............................................................................................1216.9. Comparison of heterogeneity of sequences in serum and liver............................................................. 1216.10. Comparison of variation between paired and unpaired regions........................................................1226.11. Discussion........................................................................................................................................................122CHAPTER 7 ...................................................................................................................................... 127SSCP ANALYSIS OF QUASISPECIES COMPOSITION OF 5’UTR IN SERUM AND LIVER.... 1277.1. Introduction..................................................................................................................................................... 1277.2. Optimisation of SSCP..................................................................................................................................... 1297.2.1. Sensitivity of SSCP when two variants present.................................................................................. 1307.2.2. Sensitivity of SSCP analysis using Silver staining............................................................................ 1317.3. Study patients................................................................................................................................................... 131Table of contents7.4. Amplification of 5'UTR region for SSCP analysis...................................................................... - .......... 1327.4.1. Control clones:..................................................................................................................................... 1327.4.2. Patients samples:.................................................................................................................................. 1327.5. SSCP profile of representative clones............................................................................................. - ......... 1337.6. Effect of nucleotide 107 on SSCP pattern...................................................................................... - ......... 1347.7. SSCP analysis of 5’UTR from GT 1 patients................................................................................- ......... 1357.8. SSCP analysis of 5’UTR from GT 3 patients................................................................................ - ......... 1367.9. SSCP analysis of DNA extracted from silver stained polyacrylamide g e l......................................... 1367.10. SSCP analysis of PCR product derived from new cDNA........................................................ 1377.11. Discussion....................................................................................................................................................... 137CHAPTER 8 ......................................................................................................................................141GENERAL DISCUSSION................................................................................................................ 1418.1. Future studies................................................................................................................................................146AppendixReferencesList of Figures and TablesFigure Previous page number1.1 Schematic representation o f the HCV genome and the protein products of the polyprotein.31.2 A predicted model of HCV NS2 membrane topology. 91.3 Crystal structure of the HCV NS5B polymerase. 131.4 Sequence and proposed secondary structure o f the HCV IRES. 141.5 Predicted model of the secondary structure o f the HCV 3’UTR RNA. 161.6 Proposed mechanism of translation initiation by the cap-dependent mechanism.171.7 Schematic diagrams of picomavirus IRESs. 191.8 Proposed structure o f the HCV IRES RNA-40S subunit complex. 201.9 Two possible mechanisms of assembly o f the translation complex on the HCV 5’UTR.241.10 Putative HCV replication cycle. 271.11 Structure of the HCV genome and a typical subgenomic replicon. 291.12 Phylogenetic analysis o f the nucleotide sequences in part of the HCV NS5A region.322.1 Comparison of linear ranges of firefly and renilla luciferases. 673.1 Predicted secondary structure of HCV 5’UTR. 713.2 Differences between the 5’UTR majority sequences obtained from patients.753.3. Position of nucleotide differences on the predicted secondary structure of the 5’UTR.753.4 Structure of the bicistronic dual luciferase reporter plasmid containing 5 ’UTR and 15 nucleotides of core (pRLN).763.5 Reconstruction of pRLN clones of HCV isolates using fragments from two clones.773.6 The effect of the first 17 nucleotides of H77c on IRES activity. 783.7 Relative IRES activity o f GT1 and GT3 constructs in BHKsinT7 cells. 793.8 Relative IRES activity o f GT1 and GT3 constructs in HuH7 cells. 803.9 Comparison of IRES activity of GT1 and GT3 isolates. 803.10 Comparison of relative translation efficiencies in BHK and BHKsinT7 cells.814.1 Hydrophobicity profile of the core polypeptide and the three proposed domains of core.894.2 Alignment of the majority nucleotide sequence of the core region obtained from GT1 infected patients.914.3 Alignment of the majority nucleotide sequence of the core region obtained from GT3 infected patients.914.4 Alignment of the amino acid sequence of the core region. 914.5 Structure of the bicistronic dual luciferase reporter plasmid containing the 5’UTR and full length core coding sequence (pRLNC).924.6 Comparison o f relative IRES activity o f pRLNC clones containing 5’UTR and full length core from 6 patients.934.7 IRES activities of GT1 and GT3 pRLNC clones in BHKsinT7 cell. 934.8 Comparison o f mean IRES activity of GT1 and GT3 pRLNC constructs in BHKsinT7 cells.934.9 Analysis of the expression of firefly and core genes by western blot analysis.944.10 Amino acid sequence of firefly luciferase protein. 955.1 Schematic representation of TaqMan real time PCR. 1005.2 Schematic representation of the primers and probe used for quantitation of viral loads.1045.3 Standard curve plot of transcribed RNA. 1045.4 Representative results of quantitative real time RT-PCR assays. 1055.5 Comparison o f viral load quantitated by TaqMan and COB AS Amplicor. 1065.6 Quantitation o f the HCV RNA serum levels by TaqMan real time PCR assay.1086.1 Schematic representation of the 5’UTR/core RT PCR. 1156.2 Nucleotide differences between majority sequences of patients and reference sequences.1176.3 Schematic representation of pCR2.1-TOPO vector and insert. 1186.4 Nucleotide differences observed in clones derived from patients LA. 1196.5 Nucleotide differences observed in clones derived from patients BH. 1196.6 Nucleotide differences observed in clones derived from patients SA. 1196.7 Nucleotide differences observed in clones derived from patients ME. 1196.8 Nucleotide differences observed in clones derived from patients MO. 1196.9 Nucleotide differences observed in clones derived from patients RJ. 1196.10 Complexity of sequences obtained from serum and liver of six patients. 1226.11 The location o f nucleotide differences in sequences studied from 3 genotype 1 infected patients on the secondary structure o f the 5’UTR.1226.12 The location o f nucleotide differences in sequences studied from 3 genotype 3 infected patients on the secondary structure o f the 5’UTR.122III7.1 SSCP analysis of HVR clones. 1297.2 Sensitivity of SSCP when two variants present. 1307.3 SSCP analysis of clones matched with majority sequences from patients. 1337.4 The effect of nucleotide differences at position 107 on SSCP. 1347.5 Secondary structure model of the 3’ end of HCV genotype la negative strand RNA.1347.6 “Mfold” analysis of sense and antisense strands o f the 5’UTR DNA. 1357.7 “Mfold” analysis of sense and antisense strands of the 5’UTR RNA. 1357.8 SSCP analysis of HCV 5’UTR sequences amplified from serum and liver from GT1 patients.1357.9 SSCP analysis of HCV 5’UTR sequences amplified from GT3 infected patients.1367.10 SSCP analysis of DNA extracted from a silver stained gel. 1377.11 SSCP analysis of PCR product derived from new cDNA. 137A-l Alignment of the majority nucleotide sequence of the 5’UTR and 5’ end of the core region from genotype 1 infected patients.147A-2 Alignment of the majority nucleotide sequence of 5’UTR and 5’ end of core.147Table Previous page number3.1 Oligonucleotide primers for RT-PCR of GT1 and 3 5’UTR region. 743.2 Firefly and renilla luciferase light outputs in BHKsinT7 cells. 793.3 Firefly and renilla luciferase light outputs in HuH7 cells. 804.1 Oligonucleotide primers for amplification and sequencing of core region.904.2 Firefly and renilla luciferase light outputs in BHKsinT7 cells. 935.1 Oligonucleotide primers for quantitation of RT-PCR. 1035.2 Ishak and steatosis scores in liver biopsies from patients. 1085.3 Patient’s data used for statistical analysis. 1096.1 Amplification of 5’UTR and core region and primers used. 1176.2 Frequency of nucleotide substitutions in 5’UTR region in patients. 1206.3 Sequence complexity o f the 5’UTR in liver and serum quasispecies. 1217.1 Oligonucleotide primers for PCR o f HVR1 region from clones. 1297.2.A Oligunucleotide primers for PCR o f the 5’UTR. 1327.2.B PCR condition used for amplification of 5’UTR region from clones and patient samples.132Abbreviations5’UTR 5’ untranslated region3’UTR 3’ untranslated region% percentage°C degree Centigradepg microgram(s)pi microlitre(s)A adenosineaa amino acid(s)ALT alanine aminotransferaseAST aspartate aminotransferaseAPS Ammonium persulphateARFP alternate reading frame proteinas antisenseASGP-R asialoglycoprotein receptorATP (2'-deoxy)adenosine 5'-triphosphateBHK Baby hamster kidney cellsBHKsinT7 BHK cells expressing T7 RNA polymerasebp base pairBSA Bovine serum albuminBVDV bovine viral diarrhoea virusC cytosineCAT Chloramphenicol Acetyl TransferaseC-terminal Carboxy-terminalCaCb Calcium chloridecDNA Complementary DNACIP Calf intestinal phosphatasecm centimetreCMV cytomegalovirusCO2 Carbon dioxideCOS-1 African Green Monkey kidney cellsCt threshold cycleCTL cytotoxic T lymphocyte(d)CTP (2'-deoxy)cytidine 5'-triphosphatedH20 Distilled waterDMEM Dulbecco’s modified Eagles MediumDNA Deoxyribonucleic acidDNase DeoxyribonucleaseDTT dithiothreitolEl envelope glycoprotein 1E2 envelope glycoprotein 2EBV Epstein Barr virusE.coli Escherichia coliECL Enhanced chemiluminescenceEDTA ethlenediaminetetra-acetic acid (disodium salt)elF Eukariotic initiation factorEMCV encephalomyocarditis virusER Endoplasmic reticulumEtBr ethidium bromideFCS Foetal calf serumFLuc firefly luciferaseFMDV Foot-and-mouth disease virusGAPDH glyceraldehyde-3-phosphate dehydrogenaseGBV-B GB virus strain BGMEM Glasgow minimal Eagles MediumGST glutathione S-transferaseGT genotype(d)GTP (2'-deoxy)guanosine 5'-triphosphateG guanosineHAV hepatitis A virusHBV hepatitis B virusHCC hepatocellular carcinomaH20 WaterHC1 Hydrochloric acidhGH human growth hormonehnRNPk heteronuclear ribonucleoprotein kinaseHRP Horseradish peroxidaseHoCV hog cholera virusHVR hypervariable regionHuH7 Human hepatocellular carcinoma cell lineIFN InterferonIgG Immunoglobulin GIL interleukinIRES internal ribosome entry siteIRF-3 IFN regulatory factor-3ISDR IFN sensitivity determining regionIU International unitsIVDU intravenous drug userkDa kilodaltonkb kilobase pairKC1 Potassium chlorideKH2PO4 Potassium dihydrogen orthophosphateKOAc Potassium acetateKOH Potassium hydroxideLa Lupus antigen proteinL-broth Luria Bertani mediumLDL Low density lipoproteinLT-PR lymphotoxin-P receptorM molarmAb monoclonal antibodymg miligramMgCl2 Magnesium chlorideMg(OAc)2 Magnesium acetateM gS04 Magnesium sulphateMHC major histocompatibility complexmin minute(s)ml millilitre(s)mMmmMolt4mRNAN-terminalNaClNa2H P04NANBHNaOHNBCS(NH4)2S 0 4ngnmNMRNSnt(s)(d)NTPODORFPABPPAGEPBMCPBSPCRPCR 1PCR 2PEGp.f.u.PKRpmolesPnpRLpRLNpRLNCPTBRlucRdRpRFLPRLURNARNaseRNasinr.p.mrRNARTRT-PCRsSDSSDS-PAGEmillimolarmillimetre(s)human T lymphoblastic leukaemia cell messenger RNA Amino-terminal Sodium chloridedi-sodium hydrogen orthophosphate anhydrousnon-A, non-B hepatitisSodium hydroxideNew bom calf serumAmmonium sulphatenanogram(s)nanometre(s)Nuclear magnetic resonancenon-structuralnucleotide(s)(deoxy)nucleotide triphosphate Optical density open reading frame polyadenylate binding protein Polyacrylamide gel electrophoresis peripheral blood mononuclear cells Phosphate-buffered saline Polymerase Chain Reaction First round PCR Second round PCR polyethylene glycol plaque forming units RNA-dependent protein kinase picomolesthe ratios o f number of polymorphic sites tonumber o f nucleotides sequencedempty bicistronic vector (Collier et al., 1998)pRL vector containing 5’UTR regionpRL vector containing 5’UTR plus core regionPolyptrimidine tract binding proteinrenilla luciferaseRNA dependent RNA polymeraseRestriction fragment length polymorphismRelative light unitRibonucleic acidRibonucleaseRibonuclease inhibitorrevolutions per minuteribosomal RNAReverse transcriptionReverse transcription PCRsensesodium dodecyl sulphate SDS-polyacrylamide gel electrophoresisSGB Stacking gel bufferSPP signal peptic peptidasess single stranded DNASSCP single stranded conformation polymorphismSL stem loopSV40 simian 40 virusTAH transfusion associated hepatitisTBE Tris-boric acid-EDTA bufferTEMED N,N,NTSl-tetramethylethylenediamineT thymidineTMB Tetramethyl benzidineTMD transmembrane domainTNF-R1 tumour necrosis factor-receptor 1TP broth Tryptose phosphate brothtRNA transfer RNA(d)TTP (2'-deoxy)thymidine 5-triphosphateU unitUTR untranslated regionUV ultra violetu uracilV voltsVLP virus like particlev/v volume per volumeWHO World Health Organisationw/v weight per volumeReference HCV isolates:Genotype la H77 Accession no: AF011751Genotype 3a NZL1 Accession no: D 17763All patients studied in this thesis were infected with either genotype la or 3a but for simplicity are usually referred to as genotype 1 or 3 (GT1 or GT3).One and three letter abbreviations for amino acidsAmino acid Three letter code One letterAlanine Ala AArginine Arg RAsparagine Asn NAspartic acid Asp DCysteine Cys CGlutamic acid Glu EGlutamine Gin QGlycine Gly GHistidine His HIsoleucine lie ILeucine Leu LLysine Lys KMethionine Met MPhenylalanine Phe FProline Pro PSerine Ser SSummaryHepatitis C virus (HCV) infects over 170 million people worldwide. Chronic infection occurs in 50-80% of cases and eventually leads to cirrhosis and hepatocellular carcinoma. HCV can be classified into six genotypes. Genotypes 1, 2 and 3 have a world-wide distribution but their prevalence differs from one geographical area to another. In Scotland there is an approximate 50/50 split between patients infected with HCV genotype 1 and genotype 3. One difference which has been consistently demonstrated is the better response o f patients infected with genotypes 2 and 3 to interferon treatment than those infected with genotype 1.The HCV lifecycle is only partly understood owing to the lack of a productive cell culture system. There is no vaccine to prevent infection by HCV. Given the predicted future impact of the disease, there is a great need to understand the molecular basis of the HCV life cycle. Protein translation is one of the important processes in HCV replication. It involves an internal ribosome entry site (IRES) in the 5’untranslated region (5’UTR). Comparison of the sequence and function o f the 5’UTR from different genotypes might differentiate features essential to the virus life cycle in all genotypes from those relevant only to individual genotypes. In this study, the 5’UTR region of genotype 3 was compared with that o f genotype 1 with respect to translation initiation and quasispecies composition. The association between translation efficiencies, serum viral loads and the histology of the liver was also investigated.The work presented in chapters 3 and 4 was undertaken to compare the translation efficiencies o f genotypes 1 and 3 IRESs obtained from a number of infected patients. There are conflicting reports with respect to the translation efficiency of the 5’UTR in genotypes 1 and 3 (Buratti et al., 1997; Collier et al., 1998). These studies used only a single sequence as representative o f its genotype. In this study, the system developed by Collier et al. (1998) in this Institute was used to measure the translation efficiency of a number o f 5’UTR sequences matched with the relevant majority sequence fromgenotype land 3 infected patients. In this system, the upstream reporter (renilla luciferase) is driven by the T7 promoter sequence and the IRES sequences are inserted before the downstream firefly luciferase reporter gene. Sixteen constructs (8 genotype 1 and 8 genotype 3) containing different 5'UTR sequences from our patient cohort were constructed and their activities were measured in two different cell lines, BHKsinT7 and HuH7. Differences in the 5'UTR nucleotide sequence resulted in variations in IRES activity. Translation efficiencies differed in the two cell lines tested. Mean translational activities of genotype 1 isolates were statistically significantly higher than genotype 3 in BHKsinT7 cells in 3 out o f 5 experiments but further consideration o f the variation in the raw data led us to the conclusion that the differences may not be biologically relevant. We proceeded to repeat the experiments in HuH7 cells in which the differences in mean translational levels between genotypes were shown not to be significant.To compare the translation efficiencies o f the 5’UTR combined with the putative core encoding region between genotype 1 and 3 patients, an attempt was made to construct clones matched with the majority sequence obtained from patients. Most previous reports suggested that the presence of core resulted in downregulation of HCV IRES activity. Unfortunately, no completely matched clone was identified. Therefore, the translation efficiency o f three random clones from each of 6 patients was measured. The results suggested that the presence of core region did not downregulate the translation efficiency o f 5’UTR as suggested previously. It was also shown that IRES activities of genotypes 1 and 3 constructs did not differ significantly.The work described in chapter 5 was carried out to investigate the association between IRES activity, serum viral load and the histopathological appearance of liver biopsies from the patients. Serum viral loads were measured using real-time PCR (Taqman) and relevant liver biopsies scored using the Ishak scoring system. The viral loads ranged from 104 to 107IU/ml. Statistical analyses o f these data found no association between translation efficiency, viral load and liver histology. However, a strong correlation was observed between the presence of steatosis in liver biopsies taken from genotype 3 infected patients compared to genotype 1.The work presented in chapters 6 and 7 was undertaken in order to determine the quasispecies composition of 5'UTR sequences in serum and matched liver samples from 6 HCV infected patients using 2 different methods including cloning and single stranded conformation polymorphism (SSCP). Previous work carried out by P. Preikschat in our laboratory showed the presence of an identical majority sequence in paired serum and liver samples from 26 patients (11 genotype 1 and 15 genotype 3). It was hypothesized that a minor population of virus with low translational activity could replicate in liver cells in order to escape from immune surveillance and result in virus persistence. The majority sequence data did not either support or refute this hypothesis. Therefore, amplified 5'UTR products from matched serum and liver samples were cloned and sequenced in both directions (chapter 6). It was shown that the majority o f clones in serum and liver were identical to the majority sequence obtained from each patient. In addition, single, distinct variants were observed suggesting the presence of a quasispecies in 5'UTR. However, there is a limit to the number of clones which can be sequenced and nucleotide misincorporation due to Taq polymerase and reverse transcription errors will occur. In order to address whether any of the clones detected by the above method were present as more than 2.5% of the quasispecies, SSCP analysis was performed (chapter 7). An identical SSCP profile was observed in serum and liver samples from each patient. Taken together, the results obtained from cloning and SSCP analysis o f 5’UTR region from matched serum and liver samples did not support the hypothesis that a minor subpopulation of virus in liver cells exists which translates the polyprotein with a lower efficiency.Chapter 1Chapter 1 Introduction1.1. Historical background1.1.1. Non-A, non-B hepatitisThe discovery of the hepatitis B surface antigen (HBsAg) in the mid 1960’s led to the introduction of serological tests to screen blood donors for hepatitis B infection. Hepatitis B virus (HBV) was the major cause of post-transfusion hepatitis prior to introduction of screening for HBsAg. The introduction of screening tests for HBV in 1970 reduced transfusion associated hepatitis (TAH) by approximately 80%. Despite the removal of all HBsAg-positive blood donations from the supply, transfusion associated hepatitis persisted. Later, Feinstone et a l (1975) discovered the hepatitis A virus (HAV) using immunoelectron microscopy on faecal samples from volunteers infected with this virus. Tests to detect IgM and IgG antibodies were then introduced and it became possible to screen non-HBV cases for HAV. It became apparent that another blood-borne agent was responsible and these cases were defined as having non-A, non-B hepatitis (NANBH)(Alter et al., 1975).In 1978, two studies by Alter et a l (1978) and Tabor et a l (1978) reported the transmission of the NANBH agent to chimpanzees. Alter et a l administered plasma obtained from patients with NANBH to chimpanzees intravenously. The chimpanzees1Chapter 1developed hepatitis but importantly, were negative for HAV, HBV or other hepatotropic viruses such as cytomegalovirus (CMV) and Epstein Barr virus (EBV). Ultrastructural changes, not reported in humans, were observed in the hepatocytes of the infected chimpanzees. The appearance of either cytoplasmic membranous tubules or intracellular particles of 20-27 nm or both were reported. Feinstone et al. (1983) extracted samples of human plasma which contained infectious doses of NANBH virus with chloroform which is a potent lipid solvent, and showed that the extract was non-infectious when tested in chimpanzees. This suggested that the agent might be a virus with a lipid envelope.Subsequently, He et a l (1987) determined the approximate size of one strain (H) of the NANBH viral agent by filtering it through polycarbonate membranes. The study indicated that strain H was 30 to 60 nm in diameter.1.1.2. Cloning of the NANB hepatitis infectious agentAfter more than a decade, attempts at identifying the NANBH agent by conventional immunological methods failed, possibly because of insufficient concentrations of viral antigen. Houghton et al. using their previous experience in cloning of the genome of hepatitis D virus, used molecular biological approaches to clone and express the genome (Choo et al., 1989) and then to develop immunoassays to detect antibody to the protein products of these clones (Kuo et al., 1989). In order to obtain infectious material with a high concentration of agent, they pooled large volumes of plasmapheresis samples from a chronically infected chimpanzee which had been shown to have an unusually high titre of the presumed virus. The plasma was centrifuged until a pellet was obtained. Because it was not known whether the virus had a DNA or a RNA genome, random primers were used to synthesise cDNA, which was cloned into the cloning vector A.gtl 1 and expressed in Escherichia coli (E.coli). This allowed the amplification of the cDNA and the proteins to be expressed. After the lysis of the bacteria, expressed proteins were screened with serum from a patient who had NANBH as a presumed source of antibodies and then with radiolabeled antiglobulin. After screening approximately 106 recombinants, a single positive clone, 5-1-1, was identified. To investigate the origin of clone 5-1-1, a larger2Chapter 1clone, clone 81, was isolated from the same cDNA library using clone 5-1-1 as a hybridisation probe. Clone 81 did not hybridise to control human DNA or RNA derived from non-NANBH infected chimpanzees, however it did hybridise to RNA extracted from the liver tissue of an NANBH infected chimpanzee. This activity was abolished after treatment with ribonuclease but not deoxyribonuclease. The investigators concluded that the clones 5-1-1 and 81 were derived from an exogenous RNA molecule associated with NANB hepatitis infection. Further analysis showed that this was a positive sense singlestranded RNA molecule, approximately 10 kb in length containing one continuous translational open reading frame (ORF) presumed to be the genome of the virus. The virus responsible for NANB agent was renamed hepatitis C virus (HCV). The cloning of the HCV led to the development of immunoassays for detection of HCV antibodies (Kuo et al., 1989). Using the original clone 5-1-1 as a probe, three overlapping clones were isolated and ligated together to build clone cl 00 consisting of part of NS4 region of the HCV genome. This clone was expressed in yeast and the resultant polypeptide was used to capture HCV antibodies from serum samples.1.1.3. Virus morphologyBefore the identification of HCV in 1989, Feinstone et a l (1983) reported inactivation of the non-A, non-B hepatitis agent when infectious material was treated with chloroform suggesting that the particles were surrounded by a lipid envelope. Microfiltration experiments demonstrated that this agent had a diameter of less than 80 nm (Bradley et al., 1990). Further filtration experiments by He et a l (1987) suggested a diameter of 30-60 nm. After identification of HCV, immunogold electron microscopy studies have shown that HCV is a spherical particle with a lipid membrane envelope containing projections of 6 nm. These particles were found in 1.14 to 1.16 g/ml fractions after sucrose density gradient centrifugation (Kaito et al., 1994). Similarly, Shimizu et a l (1996) detected viruslike particles with a diameter of approximately 50 nm in the liver of an infected chimpanzee. The buoyant density of HCV virions is heterogeneous, possibly due to association with immunoglobulins or low-density lipoproteins. In highly infectious sera, HCV was detected in fractions of low buoyant density (<1.06 g/ml), equivalent to that of3Figure 1.1. A schematic representation of the HCV genome and the individual protein products of the polyprotein.(modified from Pawlotsky, 2004).co'5bCJ02OGGOZUHDCOC /3C02i-i . i« 4. t 4! PuX' O 3' direction until it identifies the initiator codon AUG. Scanning is assisted by the factors elFl and elFl A. Stable binding of the 43 S preinitiation complex to the AUG codon forms the 48S initiation complex. Subsequent joining of the large (60S) ribosomal subunit results in the formation of the 80S initiation complex. Both AUG recognition and joining of the large ribosomal subunit induces GTP18Chapter 1hydrolysis of eIF2 and eIF5B, respectively. Subsequently, the 80S complex catalyses the formation of the first peptide bond. (Gebauer and Hentze, 2004).1.4.2. IRES-mediated translationThe initiation of translation in this system does not require a 5’ cap structure but requires a RNA segment upstream of the initiation codon which directs the 40S ribosome subunit to the site of translation initiation. This segment of highly structured RNA has been referred to as the “internal ribosome entry site” (IRES).In 1988, it was discovered that uncapped picomaviral mRNAs were translated by an mechanism distinct from scanning (Jang et al., 1988). The development of bicistronic expression vectors containing the poliovirus 5’UTR (Pelletier and Sonenberg, 1988) or the encephalemyocarditis virus (EMCV) 5’UTR (Jang et al., 1988) located in the intercistronic region showed that this region (5’UTR) could confer internal initiation of translation on RNA. Translation of the downstream cistron occurred even when translation of the upstream cistron was abolished.So far, all picomaviral RNAs have been found to contain IRES elements. The RNA genome of many other RNA viruses including HCV, classical swine fever virus, murine leukemia vims, simian immunodeficiency and cricket paralysis viruses use IRES- mediated translation. In addition, some cellular mRNAs, including translation initiation factors, transcription factors, oncogenes and growth factors, contain IRES elements in their 5’UTRs (Hellen and Samow, 2001).1.4.3. IRES-mediated translation in picornavirusesPicomaviruses have several characteristics that preclude their use of the cap-dependent scanning mechanism for initiation of translation. The RNA genomes of picomaviruses do not contain a cap structure but have a covalently linked protein called VPg at the 5’ end. A19Figure 1.7. Schematic diagrams of the picornavirus IRESs. (A) Type 1 IRES, (B) Type 2 IRES and (C) Type 3 IRES, (taken from Le., 1998)U0 _] L -----UCQUfflpdfloXaaooflsO*P73flflS.-W0>-s<*Hom1 CHON Sfi 8 S wif-a&aChapter 1scenarios, subsequent binding of the 60S subunit results in the assembly of a functional 80S ribosome, which starts to translate the HCV polyprotein. It should be noted that the precise order of these events remains uncertain.1.5.3. Mapping the HCV internal ribosome entry siteSeveral approaches have been used to identify the minimal sequence required for HCV IRES dependent translation and to map the 5’ and 3’ borders of the HCV IRES elements. The commonly used technique has been the analysis of bicistronic RNAs in which two reporter protein coding sequences are separated by an IRES sequence. Translation of the upstream gene occurs in a 5’cap-dependent mechanism, while translation of the downstream reading frame is driven by the IRES element. Quantifiable reporter proteins like luciferase (Tsukiyama-Kohara et al., 1992; Wang et al., 1993) or chloramphenicol acetyl transferase (CAT) (Rijnbrand et al., 1995) have been used by several groups. However, the HCV core protein coding region (Fukushi et al., 1994; Tsukiyama-Kohara et al., 1992) or the complete HCV open reading frame have been used by other groups as the reporter sequence in order to study the translation efficiency in a more natural condition (Honda et al., 1996b).1.53.1. The 5’ border of HCV IRESAn early study by Tsukiyama-Kohara et a l (1992) reported the 5' border to be located between nts 100 and 156. Later, Fukushi et a l (1994) reported that the entire 5'UTR was needed for IRES activity. Despite this report, most studies have shown the 5' border of the IRES to be at or near the 5' end of domain II (nt 44) (Honda et al., 1996b; Kamoshita et al., 1997; Reynolds et al., 1996). Honda et a l (1999c) showed that deletion of nts 32-37 did not have any effect on IRES activity, while substitutions at nts 45-46 had a strong, negative effect on translation (Honda et al., 1999). They suggested that the 5' border of the HCV IRES was between nts 38 and 46, i.e., just upstream of stem-loop II. As can be seen, most studies agreed that stem-loop I is not required for IRES activity. In fact, some studies have shown that removal of stem loop I enhanced the translation efficiency of the25Chapter 1IRES (Honda et al., 1996b; Rijnbrand et al., 1995). In addition, the data presented by Kamoshita et a l (1997) suggested that the inhibitory effect of stem-loop I on IRES activity may be cell-type specific. They showed that RNA transcripts lacking nts 1-22 were translated more efficiently in a HeLa cell lysate, but less efficiently in African green monkey kidney cells, suggesting that host cell-specific factors may interact with stem-loop I. More recently, Friebe et a l (2001) observed that deletions of stem loop I or the spacer sequence (nts 20-44) decrease RNA translation by two- to threefold in a bicistronic system in HuH7 cells. Luo et a l (2003) also reported that deletion and nucleotide substitutions in the 5’ proximal stem-loop structure resulted in reduction of translation of a reporter gene (CAT) by two- to five fold, suggesting that the 5’ proximal stem-loop RNA element also modulates HCV RNA translation.I.5.3.2. The 3’ border of HCV IRESThe 3' boundary of the IRES is less certain. The IRES extends in a 3’ direction as far as the initiator AUG codon. It has been shown that the RNA pseudoknot involving stem-loop Illf was essential for translation of HCV (Wang et al., 1995). In early studies, efficient IRES activity was observed in dicistronic constructs containing between 3 (initiator AUG codon) and 8 nucleotides of the core-coding sequence (Rijnbrand et al., 1995; Tsukiyama- Kohara et al., 1992). In contrast, Reynolds et al (1995) reported a significant increase in IRES activity if 12 to 30 nts of core coding sequence were included in the RNA transcript. The importance of the first 24 nucleotides of the core-coding sequence in IRES-dependent translation was supported by the observation that chimeric polioviruses containing the HCV IRES required at least 24 nts of core-coding sequence for viral replication (Lu and Wimmer, 1996). A chimeric virus containing 370 nts of the core protein coding region showed the most efficient replication.Two different explanations have been proposed for the discrepancy between these observations. First, Reynolds et a l suggested that a sequence homology existing between the firefly luciferase and HCV core nt sequences may be responsible for the IRES activity observed by Wang et a l (Tsukiyama-Kohara et al., 1992; Wang et al., 1993) in constructs26Chapter 1containing the luciferase sequence fused directly to the HCV initiator AUG. Another explanation was that the fusion of some reporter gene sequences to the HCV 5'UTR may result in an RNA structure which may have an inhibitory effect on translation initiation (Honda et al., 1996a). It has been shown that the 40S ribosome interacted with the RNA directly at the site of the initiator AUG codon and structures around the AUG reduced IRES activity (Pestova et al., 1998). So it may be that the inclusion of the 5’ core coding sequence simply ensures the absence of an unfavourable base pairing structure around the AUG initiation codon. Taken together, the findings suggested that the minimal sequence for IRES function includes stem loops II and III as far as the initiator AUG codon and extends into the core encoding region which makes up part of the domain IV structure.1.5.4. The impact of the 3’ UTR on HCV translationThe RNA of HCV, GBV-B and pestiviruses differs from other RNAs containing IRESs by the lack of a poly (A) tail at the 3’ end of the RNA. Instead there is a variable poly (U-C) stretch plus a conserved X region at the 3’ end (Kolykhalov et al., 1996). The X region seemed to form athree-stem-loop structure and bound to polypyrimidine tract-binding protein (PTB) (Tsuchihara et al., 1997). PTB has been reported to bind to the IRES region of HCV, and regulate its translation (Ali and Siddiqui, 1995). There are controversial reports regarding the effect of the 3’UTR on translation of HCV. One study published by Ito et a l (1998) suggested that the X region at the 3’ end of the HCV genome enhanced IRES-dependent translation weakly. They showed that HCV RNA containing the X region was translated three- to five fold more than the corresponding RNAs without this region. Mutations that abolished PTB binding in the X region reduced, but did not completely abolish, the enhancement of translation. However, they did not use the entire HCV 3'UTR. In contrast, Murakami et a l (2001) observed that the complete 3'UTR downregulated HCV translation in vitro. This inhibition was removed when the poly (U/UC) or stem-loop III (SL3) regions of the 3'UTR were deleted. The absence of regulation of HCV IRES activity by the 3'UTR has been reported by other studies. Fang & Moyer (2000) demonstrated that the presence or absence of the 3'UTR sequence did not affect translation efficiency in an in vitro system. Recently, the impact on translation27EntryEndosome?UncoatingTranslation NS3/4A N%4B NS5BNS2 'LJLA-nssaNucleusReleaseExocytosisCytoplasmReceplor(s)? , Coreceptor(s)?LDL-R? Scavenger-R? DC-SIGN (CD209)7 L-SIGN (CD209L)? CD81?Progenygenom esV/N/N\ / \ / NFigure 1.10. Putative hepatitis C virus (HCV) replication cycle.ER=endoplasmic reticulum, (taken from Racanelli & Rehermann, 2003).Chapter 1efficiency of a series of mutations on the 3'UTR was analysed. The results showed that complete deletion of the variable region, the poly (U/UC) tract or the 3'X region did not modify HCV IRES activity (Friebe and Bartenschlager, 2002). Moreover, Kong & Samow(2002) have shown that the HCV 3'UTR modulates neither the translation nor the stability of a chimeric mRNA. These results were supported by the data presented by Imbert et al.(2003) suggesting that the HCV 3'UTR does not affect IRES-dependent translation efficiency, even in the presence of HCV structural or non-structural proteins in hepatic and non-hepatic cell lines tested.1.6. Replication of HCVLittle is known about the mechanism of replication of HCV due to lack of an efficient culture system for its study. Because of the lack of convenient animal models and cell culture systems, the proposed model of replication has been suggested based on the relationship of HCV with the other members of Flaviviridea family and characterisation of recombinant HCV proteins (Figure 1.10). The detection of HCV non-structural proteins and viral RNA in the livers of infected patients (Blight et al., 1993) or experimentally infected chimpanzees showed that the liver is a main site of HCV replication. It has been reported that HCV can also replicate in peripheral blood mononuclear cells (PBMCs) (Cribier et al., 1995) and experimentally infected B and T cell lines (Mizutani et al., 1996).How HCV enters the cell is unknown, but it may be by interaction with a receptor. Several putative receptors have been identified. The first to be identified was CD81(Pileri et al., 1998). CD81 is a 25 kDa protein and a member of the tetraspanin superfamily of cell surface proteins. Binding of E2 was mapped to the major extracellular loop of CD81. This is the most variable part of the molecule but it is highly conserved in humans and chimpanzees, the only species permissive for HCV infection (Pileri et al., 1998). Analysis of inhibition of the E2-CD81 interaction by using a panel of anti-E2 monoclonal antibodies has shown that amino acids 412-417 of E2 are involved in this interaction28Chapter 1(Owsianka et al., 2001). A more recent report suggested that CD81 is an entry coreceptor and that other cellular factors are involved during binding and entry (Cormier et al., 2004). The LDL receptor has also been proposed as a candidate for the HCV receptor. Agnello et al. (1999) showed a direct correlation between cell surface expression of LDL-R and the number of infected cells positive for the presence of HCV RNA using different cell lines. It has also been observed that HCV is not able to bind to COS-7 cells unless they express the LDL receptor (Monazahian et al., 1999). However, endocytosis of HCV has been reported in LDL-deficient fibroblasts (Agnello et al., 1999).Other putative receptors that have been reported are human scavenger receptor class B type I (SR-BI) (Scarselli et al., 2002), asialoglycoprotein receptor (ASGP-R) (Saunier et al., 2003) and liver/lymph node-specific intercellular adhesion molecule-3-grabbing integrin (L-SIGN) which is a calcium-dependent lectin expressed on endothelial cells of liver and lymph nodes (Gardner et al., 2003).Coprecipitation experiments have indicated that the non-structural proteins probably form a replicase complex on the cytoplasmic side of the ER membrane (Ishido et al., 1998) which is similar to other flaviviruses and pestiviruses. The NS5B protein is responsible for the synthesis of both plus- and minus-strand RNA, although the individual steps involved in this process are unknown. Numerous attempts have been made at propagating HCV in cell culture, through infection of either primary cells or established cell lines, or cultivation of primary cells from chronically infected patients (Ito et al., 1996; Lanford et al., 1994; Rumin et al., 1999). These systems suffer from both poor efficiency and poor reproducibility and none permitted detailed analysis of the replication cycle. The development of subgenomic HCV RNA replicons has allowed the study of RNA replication in the human hepatoma cell line (HuH7) (Lohmann et al., 1999; Pietschmann et al., 2002). This system will be covered in more detail in section 1.6.1.The chimpanzee is the only known animal to support HCV replication but its use is limited by its scarcity, high maintenance costs and for ethical reasons. In the absence of a successful cell culture system, the chimpanzee has proved invaluable for the study of293o«acjuoa« 0 X3CA"3o' a’Va3VEo3a>ox>UXJ=OX . 333. 3 52 standard deviations (SD) below the mean were considered to represent an inadequate transfection for that clone in that experiment.674 -3 -■ Firefly • Renilla2~300o.J 1 -0--1? -15 - 1 4 - 1 3Log (moles luciferase/reaction)Figure 2.1. Comparison of the linear ranges of firefly and renilla luciferase in the dual luciferase assay.As shown in this graph, the linear range of the firefly luciferase assay is seven orders of magnitude, providing detection sensitivity o f <1 femtogram (approximately 10 ~20 moles) o f experimental reporter enzyme. The renilla luciferase assay has a linear range o f greater than five orders o f magnitude and allows for the detection o f approximately 30 femtograms (3 x 1 0 ~19) of control reporter enzyme. Taken from manufacturer’s catalogue (Dual-Luciferase ® Reporter assay system, Promega).Chapter 2The firefly/renilla light output ratios obtained from matched 5’UTR and 5’UTR plus core clones of the H77c were taken arbitrarily as 100% where indicated. The mean firefly/renilla ratio obtained from each replicate was expressed as a percentage of the ratio from H77c.2.7. Protein analysis by SDS-PAGECells were washed with PBSA and lysed using lysis buffer. Lysates were centrifuged and boiled for 3 min after addition of SDS-PAGE denaturation buffer (sample loading buffer) (0.5ml) and allowed to cool on ice. Samples were subjected to SDS-PAGE using the Bio- Rad Miniprotein II apparatus. The apparatus and glass plates (10 x 8cm) were assembled according to the manufacturers instructions using 1.5mm wide spacers. A 10% acrylamide resolving gel mix was used containing 3.6ml of 30% acrylamide solution (consisting of acrylamide and A'.A’methylene bisacrylamide (ratio 37.5:1), 2.7ml of resolving gel buffer, 4.5ml of dfbO, 70pl of 10% ammonium persulphate (APS) and 5 pi of TEMED. Gels were poured and levelled using dfLO to leave a smooth interface after polymerisation. After polymerisation, the top surface of the resolving gel was washed with resolving gel buffer. The stacking gel was prepared containing 1ml of 30% acrylamide solution, 1.5ml of stacking gel buffer, 3.5ml dfLO, 50pl 10% APS and 5jnl TEMED. This was poured on top of the set resolving gel surface and a 1.5mm wide comb inserted to form loading wells for the protein samples. Following polymerisation, the comb was removed and gels were immersed in lx running gel buffer and samples loaded alongside “rainbow protein marker” (Amersham Bioscience) (7 pl/lane). Gels were run at 100 V for 3 hour or until the dye front ran off the gel.2.7.1. Electrobiotting to nitrocellulose membraneProteins were resolved by SDS-PAGE and were then transferred to a nitrocellulose membrane in a Bio-Rad mini transblot apparatus, as described by Towbin et al. (1979). Briefly, a blotting sandwich was set up where the gel and nitocellulose membrane were placed in contact with each other, between two pieces of Whatman 3mm paper which in68Chapter 2turn were placed between two fibre pads. All materials were soaked in Towbin buffer prior to assembly of the sandwich and the sandwich was then transferred to the transblot apparatus and the reservoir filled with Towbin buffer. Electrotransfer was carried out at 100mA for 1.5 hours at 4°C.2.7.2. ImmunodetectionFollowing transfer, membranes were incubated in PBS (A) containing 5% skimmed milk (Marvel) overnight to block non-specific binding of antibody. Membranes were washed in PBS A containing 0.05% (v/v) Tween-20, 6 times for 10 min and probed with the primary antibody diluted in 1% BSA in PBST for 3 hours at room temperature. Membranes were washed as previously and incubated with secondary antibody conjugated to HRP for 2h at room temperature. The membranes were washed for a final six times and the proteins detected using the Amersham enhanced chemiluminescence (ECL) system. The two supplied reagents, I and II in equal ratio, were mixed and placed on the membranes for 2 min with agitation. The membranes were then placed between two sheets of mellanine and exposed to Kodak XS-1 film for 10-60 seconds.2.7.3. Stripping membrane for reprobingBound primary and secondary antibodies were removed by incubating the membrane in stripping buffer (0.28 mM P-Mercaptoethanol, 2% v/v SDS, 62.5mM Tris-Cl pH 6.7) at 50°C for 30 min with shaking followed by 3 x 10 min washes in PBS-T. Membranes could then be blocked and reprobed as previously described.2.8. Computer softwareSequences were initially analysed and edited using the “Sequence Navigator” program (Applied Biosystems). Alignments and final editing of DNA and protein sequences were carried out using the programs PILEUP, PRETTY, GELASSEMBLE AND SEQED in the Genetic Computer Group sequence analysis package version 10.2. The Sequence69Chapter 2Detector Software from Perkin-Elmer was used for detecting PCR product produced in the Perkin Elmer Applied Biosystems 5700 sequence detection system. EndNote 7.0 was used for creating a bibliography.2.9. Statistical analysisStatistical analysis was carried out in chapters 3 and 4 using both the Student t and Mann- Whitney tests. The Student t test is appropriate to test for the significance of observed differences between the means of 2 groups of measurements which fall into a normal distribution. One of the requirements for a valid t-test is that the standard deviations of the two groups being analysed should not be significantly different. If standard deviations do differ significantly, the Mann-Whitney test, a non-parametric test, can be used instead. It does not require the data to have similar standard deviations, nor to be normally distributed. In both tests, p value <0.05 was considered as significant but it should be noted that means have to differ by more to reach significance in the Mann-Whitney test.The Spearman correlation coefficient test was used to analyse the correlations in chapter 5. This test is a non-parametric test based on ranking the data to measuring the strength of the relationship between pairs of variables. The correlation coefficient is restricted to values within the range -1.0 to +1.0 with +1.0 showing a perfect positive correlation.Statistical calculations were performed using SPSS version 11.0 (SPSS, Inc., Chicago IL).70Chapter 3Chapter 3 Comparison of translation efficiency of 5’UTRs derived from genotype 1 and 3 infected patients3.1. IntroductionThe 5’ untranslated region (5’UTR) of the HCV genome is highly conserved and forms a stable secondary and tertiary structure (Brown et al., 1992; Honda et al., 1996a). It has been shown that the 5’UTR of HCV is able to direct translation of the open reading frame (ORF) by a cap independent internal ribosome entry mechanism mediated by an internal ribosome entry site (IRES) situated within the 5’UTR (Tsukiyama-Kohara et al., 1992; Wang et al., 1993).Four major structural domains can be distinguished in the HCV 5’UTR (Figure 3.1). The short stem loop I is formed by nucleotides 5 to 20. Some reports suggested that it is not required for IRES activity and may have an inhibitory effect on translation (Honda et al., 1996b; Rijnbrand et al., 1995). In contrast, others have shown that deletion of the 5’ end resulted in a reduction (two to threefold) in IRES activity (Friebe et al., 2001; Luo et al., 2003). It has been reported that stem loop II (nt 44-118) enhanced translation (Fukushi et al., 1994; Honda et al., 1996b; Reynolds et al., 1996). Lafuente et a l (2002), using transcripts that contained the sequences of the HCV IRES domains, showed specific RNA-RNA interactions between domains II and IV, and between domains II and Illabcd demonstrating the importance of domain II for IRES activity. Stem loop III forms the core of the IRES and showed weak translational initiation activity even in the absence of domain II (Tsukiyama-Kohara et al., 1992). Stem loop IV, which includes the initiator AUG codon, may play an important role in regulation of the initiation of translation on the viral RNA. It has been shown that the stability of this domain correlated inversely with translation efficiency (Honda et al., 1996a).71,200UG,nib uuucUC Au uU ACCUA AGCGCGCC CGCAUG C a C AA A r U GCC GC AU CG CG 175, GAlib Dla'UG UA--225 AU -A U .CG UG GC CG 75— GU AUAAU CACCGG“ “ ij c G GUGGCC GGG CGC CCC G GIIIcIlau GVCGGA JOO CGAU „ r c g u c ° u gu c u u cACA , . A G G A U c A / G50 U GG C c u cUA CGGC CG150— GCUACG UA GC GUUG 250 GC AUUA AGGCCG UAGUG G CGGA AGCGC.. G C A UG,UU, niduCGCGA g GA\27525 c g J25 a ^ a\ CG / AU GGCCA^. GACACUCCACCAUGAAUCACUCC A C C C C C C C U C C C G G G GCCU A I H e ZZ G G A G G G C C C J G G G .UCG CG"18 CG CG CGu uG AI325U u 300G C C VGGAUCCGAAGACFigure 3.1. Predicted secondary structure of the HCV 5’UTR.The sequence and numbering shown is that o f GTla (H77c) and the structure is based on that proposed by Honda et al (1996).Chapter 3Differences in the efficiency with which the IRES of different genotypes of HCV direct translation from reporter genes have been reported. The first study (Tsukiyama-Kohara et al., 1992) investigated the relative efficiencies of 5’UTRs (lacking 49 nts at the 5’ end) from genotypes lb and 2b in directing translation using an in vitro system. They reported a higher level of IRES activity by genotype (GT) 2b than GT lb. Kamoshita et al (1997) used different cell lines to transfect bicistronic constructs containing the 5TJTR of genotypes lb and 2b, and found similar results to those described by Tsukiyama-Kohara. They also noticed that ratio of 2b IRES activity to that of lb was different in different cell lines. They suggested that this might be due to different distributions of host cellular factors between cell lines which interact with IRES elements. Honda et al. (1996b) reported that the full length 5'UTR sequence of a GT la strain was two fold more efficient in directing translation of the downstream core protein than GT lb (N strain) in vitro and in vivo. Later, the same group (Honda et al., 1999c) showed that differences were due to specific differences at nts 34-35 of the HCV genome. Substitution of the AG dinucleotide sequence at nts 34 and 35 of HCV-N with GA (present in HCV-H) restored its IRES activity to that of the la. Buratti et al. (1997) studied the IRES activity of representative sequences from genotypes lb, 2a and 3. They inserted full-length (nts 1- 354) and truncated 5'UTR (nts 45 -342) sequences into a bicistronic expression vector containing human growth hormone (hGH) driven by the SV40 promoter and the CAT gene controlled by the HCV 5'UTR. The hGH levels were used to normalise the amount of cellular lysate used for measurement of CAT activity. They reported that the IRES activity of GT 3 was 50% of that observed for genotypes 1 and 2 in COS-1 cells. Comparable IRES activity was found between genotypes lb and 2. Later, Collier et al. (1998) cloned 7 different 5’UTR sequences (nt 18 of the 5’UTR to nt 15 of the core coding sequence ) from six different HCV genotypes (la, lb, 2b, 3a, 4a, 5a and 6a) into a bicistronic, dual luciferase reporter system. In contrast to Buratti’s observation, similar IRES activity was found between genotypes la and 3a. They reported that the genotype 2b IRES had the highest activity in four cell lines (BHK-21, HeLa, HuH7 and HepG2) while GT 6a had the lowest activity.72Chapter 3Genotype is an established predictor of response to interferon (IFN) therapy. Numerous studies have shown that genotypes 2 and 3 are associated with a better response to treatment than GT 1. This is true whether IFN is used alone (Martinot-Peignoux et al., 1995) or in combination with ribavirin (McHutchinson et al., 1998) or if the pegylated form of IFN is used (Zeuzem et al., 2000). Correlation between translation efficiency and response to treatment is an interesting possibility for study. Saiz et al. (1999) studied the 5'UTR from 5 patients (a treatment responder and nonresponder from each of genotypes lb and 3a and a non-responder infected with GT 2). They concluded that the response to IFN therapy and the activity of the IRES are independent. It was shown that the IRES activity of GT 2 and 3a was higher than that of GT lb.Bicistronic constructs have commonly been used to identify and assess the ability of candidate IRES elements. In these constructs, translation of the 3' reporter is driven by the putative IRES whereas the translation of 5' reporter is supported by a cap dependent mechanism. One potential problem is that the 3' reporter might actually be translated not from an IRES but from a monocistronic mRNA produced by splicing as suggested by Kozak (2003). A study published by Dumas et a l (2003) reported that the HCV 5'UTR DNA sequence contained a cryptic promoter starting at nucleotide 67 which was able to drive the expression of genes inserted downstream. This activity was not detected when the 5'UTR was replaced by the HCV 3'UTR or the poliovirus 5'UTR. They concluded that study of the translational activity of the HCV 5'UTR using bicistronic DNA constructs should be analysed at both translational and transcriptional levels. The presence of a strong promoter in the HCV 5'UTR cDNA could undermine the results reported by most studies using the bicistronic system to evaluate HCV IRES activity.All previous studies measured the efficiency of translation of a single sequence as a representative of a genotype. Also, little or no attempt had been made to ensure the sequences matched those actually found in patients. It is not clear whether the observed IRES activity was genotype or actually only isolate specific. In the present study, I compared the ability of a number of GT 1 and 3 5’UTR sequences derived from patient samples to initiate translation in cell culture using a dual luciferase reporter system73Chapter 3(Collier et al., 1998) to find out whether there is a consistent difference between the IRES activity of GT 1 and GT 3. Also, it was possible to study the effect of individual substitutions in 5’UTR sequences obtained from different patient on IRES activity. I also conducted an experiment to ensure the results I obtained could not be explained by the present of a cryptic promoter in the DNA encoding the HCV 5’UTR.The work presented in this and the following chapter was based on initial work carried out by P. Preikschat in our laboratory. She amplified the 5’UTR and core regions from serum and liver samples obtained from 26 patients. Then, all PCR products were sequenced and majority sequences obtained as will be discussed in following sections. This allowed me to select specific variants for the translation study. I also used patients’ PCR products made by her as templates for re-amplification of 5’UTR and 5’UTR plus core regions for translation studies (discussed in chapter 4).3.2. Study patients and samplesLiver biopsies and serum taken at the same time from 26 patients with chronic HCV infection, including 15 infected with GT la and 11 with GT 3a, were used in this study. All of these patients had been referred to the liver clinic at Gartnaval General Hospital, Glasgow for assessment and possible treatment of chronic hepatitis C. Informed written consent for taking the samples was obtained from the patients and the study was approved by West Glasgow Ethics Committee. None of the patients was on interferon based therapy at the time when samples were collected.3.3. Patients’ majority sequencesThe following steps were carried out by P. Preikschat. PCR products corresponding to the entire 5’UTR and the core coding region were amplified by RT-PCR from the serum and liver of 11 HCV GT la and 15 GT 3a infected patients using primers shown in Table 3.1 using conditions described in section 2.2.8.2. The PCR products were sequenced directly.I aligned the majority sequences of the 5’UTR and the 5’ end of core (nts 1-360) obtained74CO ! c oCNJ N O : o o 05 CO : CNJCO CO i CO CO05CO ! CO00 I 0005 ' 05 05 no• P40X1©S.PiHPr smam-oaC3esHO0&UOh1H£i-.©I/Or - "i *Q*a /’OoOJ3sfloOX) 3+ Mu o+ Mo ot t lo o> > t t lo o < < t t lo oo u+ Mo o+M3 3+ Mo oo ot t l< »-«-<* coW 5E—H3u n3CDo£■ 3CDm3CD C /3o 3 ~o ° c: 3XJ ^CD CD.C r " -M Is *.CD L .T3 JUfli ^2 Occ3 CDS -CD£CD3 . s o 40 *45 ' O - 3OD _ 0 0"E xCD _'O cic . 3 'c o (D o 3 c^ 3 a3'3 TD 3 Ca . 303 03« c/3- O ^£ ^ 3 CD3 ‘- 53 .5 cCDc / 3£ ^ o Qj « 3 _ C~o -r5cd - r> 3OD T3*3 £C /3 0 3CD CD CD 3 CD 3a- ^CD CDCD O O CDS 5 5 33 3 3 •*—>g ’ 3 “ cr Z^ CD CD <~C O c / 3 c / 3 B used for further cloning.8888 383335833C XI X )C Qc / 3Cf t <3 O Cu8 8 3 8 8 3 3 3 3 3 ' 8 8 8 8 3 8 3 8 8 3 8 3 3 8 3 , 8 8 3 8O D rrDmHi r , u= ja/ N ■ = Z- w ^ U- 'Oo a0/ «*— o3 r - +■» r~-3 K• S c / 3« > c >> o • _ — 3 ;t3"O C lO2 303 O« « ,< D• a“ C , .I sa »— T3<*> C 0 > ^s ^'— w 4 /= H§ Oa/ .a2 ooo aa/ Qa a+ - 0 ) X C/3 C/3 (13x 5-13 CXw «' o -^ ro - a - i 2 ^ 5 - °ST 0E oT 3 a a■ a x-i12 o ox ^X f ljs -aaaC/3aaC /3oCur ^ iHC/3o cxT 30 D ^. C XS-H— i-3-*-<0 33—-*->C/5rfcu u 3.2fhmo303 3S3 gX i=o 533>> crO 03S -h C OW> 03X °o« £ 0 3 0 3•-S ^ •O H3_o_ 23a.§303a>t;oCl03CL)co3C OO (DS - 3C O 303OO0a c0)33H01C /23L.,03M—io3Oog03 XHi—"03S—>o£ oCL —r -X C3o g 6 «OO o'C ^03 i—1 C O f rg £O n ^2^ cS *X 03 00 co 3 3• 5 ML- Xo JS03PCz03_>-i—»0303CLC /203s-co03XOX )* a03o <« Z ~ Q3030300 -'O 3 -303JO ,xX .3 "5 £HoxC O are indicated under the cChapter 3However, the orientation of the insert within the vector was determined by cleavage with Ncol/Hindlll. A single Ncol site was identified in the 5’UTR sequence. A unique Hindlll site was present at the 3’ end of the firefly luciferase reporter gene. In total, 275 individual clones were screened for the presence and orientation of the insert. DNAs from clones with the correct orientation of insert were extracted using a Miniprep Kit (Qiagen) as described in chapter 2.2.1.1. and sequenced in both direction using primers 5’luc (5’ GCAAGAA GATGCACC TGATG 3’) as sense and 3’luc (5’ GCGTATCTCTTCATAG CCTT 3’) as antisense primer which bind to renilla and firefly luciferase reporter genes respectively. Nucleotide sequences were analysed using the "Sequence Navigator" program (Applied Biosystems) and aligned using the "PILEUP" and “PRETTY” programs [Genetic Computer Group (GCG) Wisconsin Programme Package]. Individual clones from each sample were sequenced until one was found which matched exactly that obtained by direct sequencing of the PCR product from the same patient sample. Construction of a matched clone from patient ME (GT3) was not successful. In total, 65 individual clones were sequenced to identify 16 pRLN matched clones which could be used for translation studies.3.5.1. Reconstruction of pRLN clonesOne problem encountered during the construction of the pRLN matched clones was the occurrence of substitutions particularly at the “left hand” end of the 5’UTR, at the primer binding site, without any other mismatch with the majority sequence from the relevant patient. This would appear to have been due to random misincorporation of bases during primer synthesis. To try and resolve the problem without expensive sequencing of additional clones, a rebuilding strategy was carried out. As shown in Figure 3.5, clone 1 which matched the majority at the 5' Ncol restriction site, and clone 2, which matched sequence 3’ to the Ncol site, were digested with Ncol /Hindlll. This released a short fragment containing most of the 5’UTR and the firefly luciferase gene and a long fragment containing the first 90 nts of the 5’UTR and the remainder of the pRL vector except for the firefly luciferase gene. Following purification by agarose gel electrophoresis, the long fragment from the first clone was ligated to the short fragment77Chapter 3from the second clone. The resulting clone was then sequenced and found to match the majority sequence.3.6. The effect of the first 17 nt of the 5’UTR on IRES activityAt the start of this study, the bicistronic dual reporter vector (pRL) containing the 5'UTR from H77c was obtained from P. Preikschat. Preliminary results of luciferase assays indicated that the IRES activity of most of the samples was higher than the activity of the clone thought to contain the entire IRES of H77c (Figure 3.6.A.B). Later, sequencing of this clone revealed the absence of 17 nucleotides comprising most of domain I of the 5’UTR. In order to include these 17 nucleotides, pRLN H77c was reconstructed as described in section 3.5.1 using the existing pRLNH77c clone and a clone from patient PE (pRLNPE) and the resultant construct was sequenced and shown to match H77c. Both constructs were transfected into BHKsinT7 cells and translation activity was analysed.The relative translation efficiency of the construct lacking the first 17 nts was 40% of that of the full length 5’UTR (Figure 3.6.C.D). The construct pRLNH77c containing the full 5’UTR was used as the arbitrary 100% standard in all subsequent experiments.3.7. Relative translation activities in BHKsinT7 cells5’UTR isolates including H77c were tested for translation activity in BHKsinT7 cells. In this system, transcription of plasmid DNA is controlled by the T7 promoter. The mRNA for the renilla luciferase gene is translated by a cap-dependent mechanism and expression of firefly luciferase by the HCV IRES mechanism. Each sample was tested on three different occasions. The first experiment was carried out in triplicate and second, third, forth and fifth experiments in duplicate. During analysis of the data, it was noticed that the renilla and firefly luciferase activities obtained in the third experiment were noticeably lower than those achieved during other experiments (Table 3.2).Applying the criteria described in methods (section 2.6.3) for evaluation of results of luciferase assays, renilla luciferase light output obtained from both replicates of pRLN78Figure 3.6. The effect of the first 17 nucleotide of H77c on IRES activity in BHKsinT7 cells.A) Firefly and renilla luciferase light outputs (RLU) obtained from pRLN H77c (nt 18-356) and 5 isolates. Comparison of relative IRES activities of the 5 isolates with H77c lacking the first 17 nucleotides (B). C) Firefly and renilla luciferase light outputs obtained from constructs containing full length (nt 1-365) and truncated (nt 18-356) 5’UTR. D)Comparison of IRES activity obtained from clone pRLNH77c (ntl 8-356) which was arbitrarily assigned as 100% with full length H77c. Data are presented as means +/- standard deviation (SD).A BS a m p le G TF i r e f l yo u tp u t(R L U )R e n i l l ao u tp u t(R L U )F i r e f l y /R e n i l l ar a t i oH 7 7 c ( n t 1 8 - 3 5 6 ) 1 1446 358.3 4.0H 7 7 c ( n t 1 8 - 3 5 6 ) 1 1276 344.6 3.7H 7 7 c ( n t 1 8 - 3 5 6 ) 1 1315 359.3 3.7P E 1 5765 609.8 9.5P E 1 5145 528.3 9.7P E 1 3455 375.5 9.1B A 1 5025 597.0 8.4B A 1 3828 479.4 7.9B A 1 2966 372.9 8.0S A 1 6595 783.1 8.4S A 1 5161 568.6 9.0S A 1 4405 512.1 8.6S J 3 3894 450.6 8.6S J 3 3523 413.0 8.5S J 3 2267 358.9 6.3M O 3 3814 553.0 6.9M O 3 4058 546.9 7.4M O 3 3743 471.2 7.9MO SJ SA BA PEH77c nt 18-3560 50 100 150 200 250 300R atio o f firefly /ren illa luc iferase ac tiv itycS a m p le G TF i r e f l yo u t p u t(R L U )R e n i l l ao u t p u t(R L U )F i r e f l y /R e n i l l ar a t i oH 7 7 c ( n t 1 8 - 3 5 6 ) 1 6022 379.6 15.9H 7 7 c ( n t 1 8 - 3 5 6 ) 1 5037 341.6 14.8H 7 7 c ( n t 1 8 - 3 5 6 ) 1 4973 327.4 15.19H 7 7 c ( n t 1 - 3 5 6 ) 1 7494 187.9 39.87H 7 7 c ( n t 1 - 3 5 6 ) 1 6192 174.5 35.49H 7 7 c ( n t 1 - 3 5 6 ) 1 5715 163.2 35DH77c nt l -356H77c nt 18-3560 50 100 150 200 250 300R atio o f firefly /ren illa luc iferase ac tiv ityChapter 3H77c in the second experiment was more than 2 standard deviations from the mean renilla activity of all replicates in the experiment. These results were therefore considered invalid. The first two experiments did not include clones obtained from patients BH, RJ and JN. Therefore these clones were tested later along with the H77c clone. Primary data including firefly and renilla luciferase activities are shown in Table 3.2. Data omitted from further calculations because they did not meet the criteria set down in section 2.6.3. are shown in red.It was impossible to compare IRES activities between experiments because of the lack of a valid H77c reference result in experiment 2. It was decided to analyse the mean firefly/renilla luciferase ratios obtained from each sample without normalisation to H77c taking each experiment separately. Figure 3.7 summarises the results for all 5 experiments. It is notable that, among GT1 isolates, the lowest translational activity was observed in the construct derived from patient OS, which had one substitution of A204C in loop Illb. Similarly, clones obtained from patients RJ (C121U) and RI (G34A and C204U) had consistently lower activity than other GT3 samples.3.8. Relative translation activities in HuH7 cellsThe IRES activities of 8 GT 1 and 8 GT3 isolates were compared in HuH7 cells. The plasmids were transfected into cells previously infected with vTF7-3, a recombinant vaccinia virus which expresses T7 RNA polymerase to allow cytoplasmic transcription of the bicistronic mRNA. All samples were tested in 3 different experiments. The criteria for validation of results were met for all replicates in all experiments except those indicated by red in Table 3.3. The mean firefly/renilla luciferase ratios obtained from all isolates were normalised against H77c in each experiment.The relative IRES efficiency of the 8 GT3 isolates varied from 60% to 120% of that of H77c as illustrated in Figure 3.8. Relative IRES activities obtained from RJ (C121U) and RI (G34A and C204U) were consistently lower in comparison to other GT3 isolates in both cell lines tested. However, The 5’UTR derived from patient OS with a substitution79Table 3.2. Firefly and renilla luciferase light outputs in BHKsinT7 cells.Firefly and renilla luciferase light outputs (RLU) obtained from 5 experiments are shown. Results are shown in the order in which experiments were performed. pRLN constructs from patients BH, JN and RJ were tested separately along with H77c. Renilla values not meeting criteria for acceptance of results are shown in red. ND= not doneE xperim ent 1 Experim ent 2 Experiment 3Sample c;iFireflyoutput(RLU)Renillaoutput(RLU)Firefly/RenillaratioMeanofratiosFireflyoutput(RLU)Renillaoutput(RLU)Firefly/RenillaratioMeanofratiosFireflyoutput(RLU)Renillaoutput(RLU)Firefly/RenillaratioMeanofratiosH77c 1 7494.0 187.9 39.9 1251 45.8 ND 887.7 14.8 60.1H77c 1 6192.0 174.5 35.5 36.8 1174 45.5 ND ND 910.0 16.2 56.2 58.1H77c 1 5715.0 163.2 35.0BA 1 6320.0 190.2 33.2 2403 118.8 20.2 907.6 18.4 49.3BA 1 4661.0 169.2 27.6 29.8 2103 97.9 21.5 20.9 788.6 17.6 44.8 47.1BA 1 5221.0 181.6 28.8SA 1 9481.0 358.7 26.4 3084 126.8 24.3 1100.0 20.3 54.1SA 1 8756.0 318.2 27.5 26.7 2865 110.0 26.0 25.2 1043.0 21.2 49.3 51.7SA 1 7977.0 306.3 26.0PE 1 8218.0 233.9 35.1 3630 139.2 26.1 1418.0 22.4 63.3PE 1 6956.0 224.6 31.0 33.0 3192 130.4 24.5 25.3 1549.0 25.2 61.6 62.4PE 1 6982.0 212.8 32.8CD 1 9981.0 319.1 31.3 2249 95.1 23.7 332.1 13.3 25.1CD 1 8870.0 285.8 31.0 30.7 3457 122.4 28.3 26.0 365.2 15.7 23.2 24.2CD 1 8765.0 293.4 29.9LA 1 6758.0 220.4 30.4 2238 71.4 31.3 969.0 19.2 50.4LA 1 7094.0 205.8 34.5 32.2 3531 119.0 29.7 30.5 749.9 17.2 43.6 47.0LA 1 7200.0 227.5 31.6o s 1 2863.0 115.3 24.8 1615 131.0 12.3 110.3 5.8 NDOS 1 3448.0 124.0 27.8 25.8 1590 128.3 12.4 12.4 125.6 7.9 ND NDos 1 3246.0 131.3 24.7BH ND ND ND ND 1014.0 16.9 60.2BH ND ND ND ND 1045.0 18.0 58.0 59.1SJ 3 4699.0 157.7 29.8 2298 110.6 20.8 688.5 16.2 42.4SJ 3 4430.0 157.0 28.2 29.1 2520 117.5 21.5 21.1 508.6 13.3 38.2 40.3SJ 3 4174.0 143.2 29.2JA 3 4652.0 151.4 30.7 2445 93.1 26.3 919.3 15.6 58.8JA 3 5126.0 161.0 31.9 32.3 2663 91.3 29.2 27.7 894.6 15.8 56.8 57.8JA 3 5182.0 151.6 34.2RI 3 4592.0 215.7 21.3 2007 126.4 15.9 551.0 21.6 25.5RI 3 4840.0 229.0 21.1 20.4 2195 133.1 16.5 16.2 389.0 16.5 23.5 24.5RI 3 4291.0 227.3 18.9MO 3 6103.0 183.8 33.2 2865 124.8 23.0 716.9 19.5 36.7MO 3 5438.0 161.7 33.6 34.1 3431 124.3 27.6 25.3 478.7 14.7 32.7 34.7MO 3 5565.0 157.1 35.4FV 3 5465.0 230.5 23.7 1126 57.7 19.5 355.8 13.3 26.8FV 3 5192.0 224.0 23.2 24.2 1251 58.7 21.3 20.4 220.5 10.9 20.3 23.5FV 3 4782.0 185.3 25.8BC 3 2703.0 109.1 24.8 1821 77.6 23.5 605.9 18.1 33.5BC 3 2734.0 98.4 27.8 26.0 1753 83.2 21.1 22.3 446.3 14.6 30.7 32.1BC 3 2601.0 102.2 25.5RJ ND ND ND ND 196.5 16.0 12.3RJ ND ND ND ND 174.2 15.3 11.4 11.9JN ND ND ND ND 334.4 12.3 27.2JN ND ND ND ND 351.3 11.2 31.5 29.3Experiment 4 Experim ent 5H77c 1 2517.0 301.0 8.4 514.9 133.7 3.9H77c 1 2365.0 264.2 9.0 8.7 443.0 127.1 3.5 3.7BH 1 1495.0 183.5 8.1 735.9 186.4 3.9BH 1 1161.0 167.4 6.9 7.5 559.9 166.6 3.4 3.7RJ 3 903.8 247.0 3.7 311.0 165.5 1.9RJ 3 809.0 239.0 3.4 3.5 315.2 154.8 2.0 2.0JN 3 1166.0 151.9 7.7 344.5 143.0 3.8JN 3 1129.0 171.2 6.6 7.1 521.0 157.3 3.3 3.6Figure 3.7. Relative IRES activities of GT1 and GT3 constructs in BHKsinT7 cells.Firefly/renilla luciferase ratios obtained from each sample are shown separately in each experiment. GT1 and GT3 samples in each experiment are shown by white and hatched bars respectively. Results are shown in the order in which the experiments were performed. pRLN constructs from patients BH, JN and RJ were tested separately along with H77c. Data are presented as means +/- standard deviation (SD). Arrows indicate values missing because transfection levels were invalid as described (Table 3.2).03 C/D:z:1 1id oCDExperiment 3Is s n i s sExperiment 4I s n lCO DC§ - ■Experiment 5Cd0.05) by applying both student t and the Mann-Whitney tests (Figure 3.9.C).80Table 3.3. Firefly and renilla luciferase light outputs in HuH7 cells.Firefly and renilla luciferase light outputs (RLU) obtained from 3 experiments are shown. Results are shown in the order in which the experiments were performed. Values are shown in red were excluded. ND: not doneExperiment 1 A Experiment 2 Experiment 3Patient'sinitial GTFireflyactivityRenillaactivityFirefly/RenillaratioMeanofratiosPatient'sinitialFireflyactivityRenillaactivityFirefly/RenillaratioMeanofratiosFireflyactivityRenillaactivityFirefly / Renilla ratioMeanofratiosH77cH77c1 270.3 1615 0.167 H77c 415.5 6986 0.0591 371.0 1961 0.189 H77c 276.4 5086 0.054 209.2 7092 0.029H77c 1 288 1565 0.184 0.180 H77c 438.5 6936 0.062 0.060 219.2 6831 0.032 0.031BA 1 | 331.7 | 1954 0.170 BA 437.1 7727 0.057BA 1 | 437.8 2235 0.196 BA 621.7 9977 0.062 156.0 5668 0.028BA 1 I 398.0 I 2300 0.173 0.180 BA 573.5 10056 0.057 0.059 235.0 7637 0.031 0.03SA | 1 321.7 | 1655 0.189 SA 382.1 6333 0.06SA 1 424.4 i 2055 0.206 SA 499.3 7549 0.066 149.7 5820 0.026SA M i 534.0 ! 2508 0.213 0.203 SA 520.3 8812 0.059 0.062 277.4 8774 0.032 0.029PE | 1 | 403.5 I 2083 0.194 PE 190.4 4731 0.04 IPE 1 ! 400.4 1963 0.204 PE 293.9 6402 0.046 189.5 5734 j 0.033 |PE 1 463.8 ! 2408 0.193 0.197 PE 287.4 6081 0.047 0.044 237.7 7393 | 0.032 0.033CD ! 1 | 444.6 I 2142 0.208 CD 301.8 5997 0.05 i ICD ! 1 CD “ j 1468.2 i 2133 0.220 CD 102.5 2416 0.042 120.3 5108 I 0.024 |439.1 ! 2024 | 0.217 j 0.215 CD 285.2 5564 0.051 0.048 234.7 7207 0.033 0.029LA M | 374.2 j 1771 j 0.211 ! LA 307.9 6699 0.046 I ILALA1 S 373.9 | 1811 ! 0.207 I LA 543.1 9875 0.055 143.6 6155 0.023 j1 I 353.0 , 1817 i 0.194 j 0.204 LA 390.9 7482 0.052 [0.051 200.6 7141 | 0.028 0.026OSOS "1 | 348.1 1833 I 0.190 [ OS 144.5 2310 0.063 I1 | 353.4 I 2019 0.175 I OS 167.9 2559 0.066 288.6 8928 0.032 IOS M l 465.0 | 2141 0.217 0.194 OS 178.8 2860 0.063 0.064 333.6 9486 | 0.035 0.034I Experiment 1B BH 346.5 5850 0.059H77H771 I 235.7 1809 0.130 BH r678^9”1 9703 0.07 278.0 7975 0.0351 | 210.6 j 1683 0.120 BH 697.2 10309 0.068 0.066 283.8 8260 0.034 0.035H77 i l l 233.3 1778 0.131 |0.127 SJ 284.1 6078 0.047bhBH1 ! 279.0 1917 0.146 | SJ 412.4 7709 0.054 315.8 10081 0.031 j1 | 290.5 1904 0.153 SJ 462.9 8788 0.053 ” 0.051 269.4 8107 0.033 | 0.032BH 1 1 1 298.3 i 1989 0.150 |0.150 JA 168 2937 0.057SJ 3 172.7 ! 1691 0.102 | JA 289.6 4531 0.064 385.7 8607 0.045SJ | 3 | 234.2 | 2073 0.113 JA 303.3 4766 0.064 | 0.062 375.7 8731 0.043 0.044SJ i 3^ 251.8 ! 2280 0.110 0.108 RI 390.7 8279 0.047JA 3 223.7 1419 0.158 | RI 138.9 2840 0.049 267.0 10222 0.026JA 3 i 267.4 M 629 0.164 i RI 102.2 2048 0.05 0.049 152.8 6470 0.024 0.025JA I 3 254.1 1635 0.155 0.159 MO 113.4 1733 0.065R|___ 3 206.4 2115 0.098 | MO 139 1959 0.071 0.068 307.9 8099 0.038RI 3 | 169.2 1648 0.103 MO 84.9 1165 ND 239.0 6685 0.036 0.037RI 3 i 219.6 2140 0.103 0.101 FV 51.52 1047 NDMO 3 i 265.6 1885 0.140 FV 92.35 1677 0.055 97.9 4510 0.022MO 3 244.4 1565 0.156 FV 72.67 1397 0.052 0.054 165.4 6548 0.025 0.024MO 3 281.6 1860 0.151 0.149 BC 46.9 817.4 NDFV 3 182.1 I 1492 0.122 BC 99.89 1555 0.064 76.6 3076 0.025FV 3 250.5 I 1841 0.136 i BC 96.8 1467 0.066 0.065 126.0 4416 0.029 0.027FV 3 I 218.0 j 1791 0.122 |0.127 JN ND ! NDBC I 3 ! 297.7 BC i 3 ! 281.01900 | 0.157 JN 85.34 j 1531 0.056 196.0 5775 0.0331832 I 0.153 i JN I 97.4 j 1483 0.065 0.056 221.3 I 6521 0.034 0.034BC I 3 j 275.8 i 1844 | 0.150 10.153 RJ | 42.24 I 1164 ND . _ ! !JN 3 1 206.5 j 1252 0.165 I RJ 79.04 I 2502 0.032 104.3 I 6755 0.015 !JN ! 3 i 183.6 I 1117 j ND I RJ 99.79 j 2434 0.039 0.036 163.6 I 7582 0.022 | 0.019JN i 3 i 177.8 I 1226 | 0.145 ! 0.155 ... I............J | ! ---------RJ | 3 | 154.0 i 1768 RJ 3 12973 ”’ 1647 RJ 3 133.1 16230.0870.083j|j ....... ■; ......~ •I0.0790.082iii---------Figure 3.8. Relative IRES activities of GT1 and GT3 constructs in HuH7 cells.Mean relative firefly/renilla luciferase ratios obtained from each sample in 3 experiments are shown. GT1 and GT3 samples are shown by bold and hatched bars respectively. Results are shown in the order in which experiments were performed. Firefly/renilla luciferase ratios obtained from all samples were normalised against H77c. Data are presented as means +/- standard deviation (SD).exp= experimentFigure 3.9. Comparison of IRES activities of GT1 and GT3 constructs.A) Mean IRES activities of GT1 (white bars) and GT3 (hatched bars) isolates in BHKsinT7 were compared within each experiment using the Mann-Whitney test.B) Mean normalised IRES activities of GT1 and GT3 isolates in HuH7 cells were Compared within each experiment using the Mann-Whitney test.C) Mean relative IRES activities of combined data from three experimentswere compared in HuH7 cells. Data are presented as means +/- standard deviation. Exp= experimentBHKsinT7 cells£h -S3 >~> • —CJ~. ■ r 0.05 p=0.01HuH7 cellsP= 0.04 p> 0.05p> 0.05Exp1p>0.05□ GT1 0 G T 3Exp 3 p> 0.05cc:0.05 GT3Chapter 33.10. Comparison of relative translation efficiencies in BHKsinT7 and BHK cellsOne report suggested that the 5’UTR cDNA has a cryptic promoter activity that can behave as a eukaryotic promoter in cells (Dumas et al., 2003). To test whether the expression of firefly luciferase in our experiments could be explained by this mechanism, it was decided to test two constructs; pRLNH77c (GT1) and that obtained from patient FV (GT3), in both BHK and BHKsinT7 cells. Unlike BHKsinT7 cells, ordinary BHK cells do not express T7 RNA polymerase, so transcription of DNA to RNA would not be expected and as a result no translation of either reporter genes would occur. In the presence of sufficient cryptic promoter activity in the 5’UTR DNA sequence to invalidate the results of our previous experiments, it would be expected that there would be firefly luciferase activity in BHK cells at a similar level to that in BHKsinT7 cells in the absence of renilla luciferase activity.DNA constructs were transfected into both BHK and BHKsinT7 cells in duplicate wells and light outputs from firefly and renilla luciferases were obtained. In order to ensure that there was no promoter activity in the empty vector to drive the transcription of firefly luciferase, DNA from the pRL vector with no insert was also transfected. pGL3 was also transfected into both BHKsinT7 and BHK cells. In this vector, translation of firefly luciferase is controlled by strong eukaryotic SV40 promoter. As expected, no firefly activity was observed in either the BHKsinT7 or the BHK cell lines transfected with the pRL vector. Transfection of pGL3 resulted in high levels of firefly luciferase light output in both cell lines. pRLNH77c and pRLNFV transfected BHKsinT7 cells showed levels of renilla and firefly luciferase activities comparable to previous experiments. Unexpectedly, upon transfection of pRLNH77c and pRLNFV constructs into BHK cells, low levels of both renilla and firefly luciferase activities were observed (Figure 3.10.A). However, firefly luciferase activities were 80 times lower than the values seen in BHKsinT7 cells. This suggests that activity observed from constructs in BHKsinT7 cells throughout this project could not be explained simply by cryptic promoter activity.81Figure 3.10. Firefly and renilla luciferase activities in BHK and BHKsinT7 cells.A) Renilla and firefly luciferase light outputs (RLU) obtained from transfection of pRLN H77c (GT1), pRLN FV (GT3), pGL3 and the empty pRL vector into BHKsinT7 and BHK cells are shown . Lower level of firefly (1/80) and renilla luciferase (1/8) activities were detected in BHK cells in comparison to BHKsinT7 cells. pGL3 containing the firefly reporter gene driven by the SV40 promoter was used as control construct for comparison of transfection efficiency. B) Renilla and firefly luciferase light outputs obtained from pRLNH77c, pRLNFV and pGL3 in BHKsinT7 and BHK cells are shown, (logarithmic scale)ASample Cell lineFireflyactivity(RLU)Renillaactivity(RLU)Firefly/RenillaratiopRLNH77c BHKsinT7 320.20 74.83 4.27pRLNH77c BHKsinT7 392.40 81.24 4.83pRLNH77c BHK 4.76 8.43 0.56pRLNH77c BHK 4.65 9.34 0.49pRLN FV BHKsinT7 80.39 23.25 3.45pRLN FV BHKsinT7 96.70 31.37 3pRLN FV BHK 1.40 3.11 0.45pRLN FV BHK 1.83 3.62 0.5pGL3 BHK 1884.00 0pGL3 BHK 2111.00 0pGL3 BHKsinT7 2372.00 0.02 86933pGL3 BHKsinT7 2744.00 0.01 250471pRL BHKsinT7 0.00 58.57pRL BHK 0.00 9.84B10000 I 1000I 10001 10 —11COCDa .XCQO s l .a Renilla ■ FireflyChapter 33.11. DiscussionFor the study of HCV IRES activities in vivo, bicistronic constructs were used in which 5’UTRs amplified from infected patients were inserted between the two reporter genes. Both reporter enzymes were assayed in the same cell lysate preparation and the activities were determined by the same method. In the present study, the bicistronic reporter vector, pRL, constructed by Collier et al. (1998) was used which contains the renilla luciferase gene act as the 5' reporter and the firefly luciferase gene as the 3’ reporter. The ratio of firefly luciferase to renilla luciferase activity was used as a measure of IRES activity. In the cell cytoplasm, the T7 promoter drives transcription of the bicistronic mRNA containing the renilla luciferase, the HCV 5’UTR and the firefly luciferase.The constructs were transfected into 2 cell lines. BHK sinT7 cells (Agapov et al., 1998) are able to express T7 RNA polymerase continuously from a noncytopathic replicon from sindbis virus (SINrepl9/T7pol). One problem with using the noncytopathic replicon to support the production of T7 RNA polymerase is the limitation on cell lines which can be used. The other method used was to supply T7 polymerase by co-infecting with the recombinant vaccinia virus, vTF7-3, to allow cytoplasmic transcription of the mRNA. The advantage of this is that different cell lines can then used for the assays. However, the cytopathic effects of vaccinia infection may interfere with cellular functions. These include the induction of early cell rounding, damage to the host genome and RNA, inhibition of host protein synthesis, and death of the infected cells (Tsung et al., 1996).In BHKsinT7 cells, the renilla luciferase light output was 20-30 times lower than that of firefly luciferase. It is possible that efficiency of translation using the sindbis replicon system was lower than that directed by the HCV 5’UTR due to inefficient capping of transcribed RNA. Unlike BHKsinT7 cells, in vaccinia infected cells higher levels of renilla luciferase activity in comparison to firefly luciferase activity was observed suggesting that transcription of T7 polymerase by vaccinia virus is more efficient than that achieved by the sindbis constructs.82Chapter 3We have compared the IRES activity of a number of isolates from GT 1 and 3 infected patients. It was important to ensure there were no artefactual base changes introduced by polymerase errors into the individual clones that were to be used to measure translation efficiency. Data presented in chapter 6 along with other studies suggested the presence of a quasispecies composition in the 5'UTR of the HCV genome (Soler et al., 2002). Previously, it has been shown that different 5'UTR sequences existing in an individual showed significant differences in their ability to promote translation both in vitro and in vivo (Laporte et al., 2000). Studying IRES activities from matched clones with the relevant majority sequence obtained from each patient was important because it reflects the behaviour of the major population within the quasispecies in the 5'UTR. I therefore ensured that only clones which matched exactly the sequence obtained by direct sequencing of the PCR product were examined.In designing the constructs for this study, we included the sequences within the HCV RNA that were included in the earlier study by Collier et al. (1998) as representing the minimum IRES element. Our constructs contain the entire 5' UTR and 15 nts of the core coding sequence. There is controversy whether the HCV protein coding sequence downstream of the IRES is essential for IRES function. Reynolds et a l (1995) suggested that 14 to 32 nts of the HCV capsid-coding sequence are absolutely required for efficient IRES activity in in vitro and in vivo systems. In contrast, other studies have shown efficient IRES activity in the absence of core coding sequence (Rijnbrand et al., 1995; Wang et al., 1993). One explanation for the discrepancy between these studies was suggested by Honda et a l (1996a). The fusion of reporter gene sequences with the 5'UTR may form an RNA structure that is unfavorable for translation initiation. The inclusion of the 5' core sequence which constitutes part of domain IV may prevent base pair formation between the HCV sequence and the beginning of the firefly luciferase coding sequence (reviewed by Rijnbrand and Lemon, 2000). In the present study, the core coding sequences needed to form the proximal stem-loop of domain IV were included as in the study carried out by Collier (1998).83Chapter 3The data presented in this study showed that deletion of 17 nts from the 5’ end of the 5’ UTR resulted in a two fold reduction in IRES activity. This is consistent with the findings reported by others that stem loop I contains RNA elements required for optimal HCV RNA translation (Friebe et al., 2001; Fukushi et al., 1994; Luo et al., 2003). It should be noted that deletion of stem loop I in our original construct (nt 1-17) was partial whereas these studies examined the construct lacking the entire domain I including nucleotides 1- 20. Our result appeared to contradict the observations made by other groups which suggest an inhibitory role for stem loop I on translation by the IRES (Honda et al., 1996b). It has been suggested that the inhibitory effect of stem loop I (nts 1-22) may be cell type specific (Kamoshita et al., 1997). They reported that IRES activity of constructs lacking stem loop I was equally efficient in HeLa cells, but less efficient in African monkey kidney cells.Of 8 GT 3 isolates tested for IRES activity in BHKsinT7 cells, patient RJ with a substitution C121U in the pyrimidine tract (nts 120-130) with an activity of 40% had the least activity. It is interesting that mutational analysis of the single stranded poly C region between domains II and III, consisting of nucleotides 120-125, suggested that this region played an essential role in maintaining the proper spacing of specific elements within the IRES and substitutions in this region could lead to the disruption of translation initiation (Varaklioti et al., 1998). No other isolate in our cohort had changes in this region.When the different IRES activities were compared in each separate experiment using BHKsinT7 cells, the mean IRES activity of GT 1 isolates was significantly higher than GT 3 isolates in 3 out of 5 experiments. It should be noted that the mathematics of the Mann- Whitney test make it “harder” to reach significance than with the student t test. However, in HuH7 cells which had been pre-infected with a recombinant vaccinia virus expressing the T7 RNA polymerase, the mean IRES activity of GT 1 isolates was not significantly greater than GT3 isolates. Studying IRES activity in cells of hepatic origin may be more appropriate than other cell lines, since those cells are the main site of HCV replication. However, it should be noted that HuH7 cells are derived from tumour cells, not normal hepatocytes. Buratti et a l (1997) reported that the GT 3a IRES was only 50% as active as lb in COS-1 (African Green Monkey kidney cells). However, the GT 3a constructs of84Chapter 3Buratti et al. contained the first four residues of GT 1 core sequence rather than GT 3. Collier et al. (1998) reported similar activity for GT la and 3a in BHK and HuH7 cells. However, their constructs contained only nts 18 to 356 of the HCV genome thereby including 15 nt of the core coding region. The construct used in this study contained the full length 5’UTR and 15 nucleotides of the core coding region (nts 1-356). All these reports examined only one isolate as representative o f each genotype. Therefore, it is not clear whether the IRES activity observed was actually isolate specific. Also, no attempt was made to ensure that the sequences tested for IRES activity were matched with the majority sequence found in the patient. It is conceivable that the observed IRES activity was that of a minor variant in the quasispecies or contained errors generated by Taq polymerase or other enzymes.A basic difficulty in performing statistical analysis of data from this study was that experiments were not performed in a randomised order. In each experiment, the order of testing of isolates was the same with GT1 isolates preceding GT3 isolates. This may have led to unrecognised bias. In the second experiment using BHKsinT7 cells, the renilla luciferase activity obtained from the reference sequence H77c, which was used for normalisation of data, did not meet the criteria set down for achieving an adequate renilla activity. Therefore, comparison of IRES activities of isolates was not possible between experiments. It was decided to compare the mean IRES activity of GT1 and GT3 isolates within each experiment in BHKsinT7 cells. It was shown that the difference between mean IRES activities of GT1 and GT3 isolates was statistically significant in 3 out of 5 experiments. However, the biological significance is more uncertain. The renilla luciferase activities in the third experiment were approximately 10 fold lower than the other experiments in BHKsinT7 cells, possibly due to a low level of transfection or poor quality of the transfected cells, although the cells appeared as healthy as in the other experiments. It should be noted that all values remained clearly within the linear range of the luciferase assays except for the excluded values for OS, which, if included would have reduced the likelihood of the difference reaching statistical significance. Our feeling overall is that the low level of statistical significance of the differences in mean firefly to renilla luciferase ratios does not represent a real biological difference between GT1 and85Chapter 3GT3 IRES elements in BHKsinT7 cells but this could be resolved only by conducting further similar experiments.The relative firefly/renilla ratios obtained from a single transcript differed between experiments when using the same cDNA on different days. This variation was more noticeable in BHKsinT7 cells than HuH7 cells. This causes some concern and, in the future, it would be preferable to standardise cell culture conditions more closely than was the case in this work. The exact reason for the observed variation in ratios obtained from a single transcript is not clear. During this project, different batches of BHKsinT7 cells were used and no synchronization of cells was carried out prior to transfection. It is conceivable that T7 RNA polymerase activity may differ under certain circumstances such as the confluency, quality of cells or their passage number and history, which could result in generation of different levels of transcripts. If cellular trans-acting factors are available in limiting amounts, the presence of more IRES-containing transcripts would exhaust the /nms-acting factors needed for cap-dependent translation and as result the relative level of cap-independent to cap-dependent translation would increase. Another possible explanation is the position in the cell cycle of the majority of cells in the culture, which has been shown to play a role in the activity of certain IRES elements. Compared with cap-dependent translation, the activity of the IRES was greatest in actively growing cells and relatively reduced in resting synchronised HuH7 cells using bisictronic luciferase constructs similar to those used in this project (Honda et al., 2000). This suggests that HCV translation is regulated by cellular proteins that vary in abundance during the cell cycle. Differences in transfection efficiency may also influence the firefly/renilla ratios by an unknown mechanism although it appears to affect the translation of both reporters in the same DNA. In order to overcome the differences between ratios of each isolate in separate experiments, DNA from H77c was used in order to normalise the ratios. Finally, it is possible that the delay in adding one or other substrates in the luciferase assay could influence the results. This was not applicable during my experiments as the addition of substrates were carried out according to programme set up in the luminometer according to manufacturer’s instructions.86Chapter 3Our study was not designed to address whether the response of individual patients to interferon therapy was related directly to the structure of the individual's viral 5'UTR. However, overall, GT3 infected patients have been shown repeatedly to respond to interferon-based therapies markedly better than that those with HCV GT1. If, on average, the 5'UTRs of GT3 isolates were less efficient at directing translation of the polyprotein, this could contribute to the greater effectiveness of interferon therapy. Our study results do not support this hypothesis. The lack of relation between 5’UTR sequence and response to interferon has been reported by other groups (Saiz et al., 1999; Soler et al., 2002).Dumas et a l (2003) reported the presence of a strong cryptic promoter within the 5’UTR DNA sequence. They concluded that results obtained from using cells transfected with bicistronic DNA constructs containing the HCV 5’UTR should be analysed at both translational and transcriptional levels. I carried out a preliminary experiment to compare IRES activity in BHKsinT7 cells expressing T7 RNA polymerase and BHK-21 cells lacking T7 RNA polymerase. In the absence of T7 RNA polymerase, no RNA transcripts from the construct should be produced. Therefore, if there was a promoter activity in the 5'UTR cDNA in our system, we would expect to have similar firefly luciferase activities in both cell lines. The data from this experiment was not able to exclude the presence of a possible cryptic promoter in the cDNA of the 5’UTR, as the constructs tested were circular and the presence of any promoter in the 5’UTR sequence could result in transcription of the entire plasmid including the renilla and firefly luciferase genes. However, it was concluded that the presence of such weak firefly luciferase activity was insufficient to invalidate the results of translation in BHKsinT7 cells by the authentic RNA IRES in this study. However, if time had permitted, I would also have performed Northern blot analysis and/or RT-PCR analysis to determine how much RNA transcript was produced in each experiment.87Chapter 4Chapter 4 Comparison of IRES activity of 5’UTR plus core regions from GT 1 and 3 infected patients4.1. IntroductionThe HCV core protein is believed to form the viral nucleocapsid. The core protein has been reported to have a wide range of biological activities such as interaction with host cell proteins including members of the tumor necrosis receptor family, apolipoprotein All and heterogeneous nuclear ribonuclear protein K (McLauchlan, 2000). It has been suggested that core protein modulates sensitivity to apoptosis (Ray et al., 1996) and induces liver steatosis (Fujie et al., 1999). It has been shown that the HCV core protein contains a unique domain which plays a vital role in the localization of core to lipid droplets (Hope and McLauchlan, 2000).Interaction of the core protein with the genomic RNA of HCV and its effects on cap- independent translation mediated by the IRES have been studied by several groups (Kim et al., 2003; Reynolds et al., 1995; Rijnbrand et al., 2001; Shimoike et al., 1999; Wang et al., 2000; Zhang et al., 2002). The 3' boundary of the IRES is uncertain. Early studies demonstrated that the inclusion of nt 1 to 32 of the core protein coding sequences was essential for efficient IRES activity (Lu and Wimmer, 1996; Reynolds et al., 1995).The data reported by Santolini et a l (1994) showed that core protein is an RNA binding protein and RNA binding domains have been localized to the N-terminal 75 amino acids. Shimioke et al. (1999) constructed a series of monocistronic plasmids containing HCV88Chapter 45’UTR and 5’UTR plus core fused to the firefly reporter gene and found that expression of core suppressed the firefly activity in a dose dependent manner in an in vivo system. It was also reported that amino acids 34-44 of core interact with the IRES RNA and contribute to the inhibition of translation (Zhang et al., 2002). This suppression was eliminated by frameshift mutations introduced into this region which abolished expression of the core protein, suggesting that it is the core protein rather than the core-coding nucleotide sequence that downregulated the efficiency of the HCV IRES activity. More recently, Li et a l (2003) reported that a synthetic peptide representing amino acids 1-20 of the core protein inhibited IRES activity in HepG2 cells.In contrast, Wang et a l (2000) reported that suppression of IRES-directed translation resulted from an RNA-RNA interaction between the core coding nt sequence and the IRES RNA. They constructed a dicistronic reporter system containing the 5’UTR plus 66 nt from core coding sequence (nt 1-407) and the 5’UTR plus 518 nt of core (nt 1-860) coding sequence fused in-frame directly with firefly luciferase along with two other constructs containing similar lengths of the core, but with frameshift mutations in the core sequence that resulted in translation of a nonsense sequence. A lower efficiency of translation was obtained from constructs containing nt 1-860 (17%) than in transcripts containing only 66 nt (nt 1-407) of core in HepG2 and HuH7 cells (60%). However, the translational analysis of the frameshift mutants showed similar results suggesting that this effect was due to the inclusion of the RNA sequence and not to the expression of the core protein. Ito et a l (1999) identified a region at the 3’ end of the core sequence which interacted with the polyprimidine tract binding protein (PTB) resulting in the inhibition of translation activity by the IRES. This inhibition was relieved by the presence of the X region at the end of the 3’ untranslated region. The presence of an RNA- RNA interaction between the IRES and the core coding sequence was further supported by study published by Kim et a l (2003). They demonstrated that nucleotides 428-442 of the core coding sequence annealed to nucleotides 24-38 of the 5’ UTR resulting in downregulation of cap- independent translation by the HCV IRES.89C1 C2 1 1i 7 7 v 7 7 7 ~ 7 7I S / / / S J / 2. p 2 3E 2 2 p’«)Domain2 -£aQ.C20 40 60 90 100 120 140 160 180Amino acid positionFigure 4.1. Hydrophobicity profile of the core polypeptide and the three proposed domains of core.p23 is the product o f cleavage at C2 (position 191/192) and p21 is the product of cleavage at Cl (between residues 174 and 191). (Taken from McLauchlan, 2000).Chapter 4Core protein (p23) is processed by a host signal peptidase at aa 191 to produce an immature form of the protein which is further processed by a host signal peptide peptidase (SPP) between aa 174-191 to produce what is thought to be the mature form of the protein (p21)(Figure 4.1). This latter processing event is important for conferring localization of core protein to lipid droplets (Hope and McLauchlan, 2000). Core is highly conserved between the 6 genotypes and the hydrophobicity profile of core identifies 3 domains within the protein. Domain I (aa 1-122) contains a high proportion of basic residues. Domain 2 (aa 123-174) is more hydrophobic than domain 1, while domain 3 (aa 175-191) is highly hydrophobic and acts as signal sequence for directing the viral El glycoprotein to the ER for further maturation prior to cleavage at aa 191 by a host signal peptidase (Santolini et a l 1994). One possible explanation for downregulation of translation observed with inclusion of full-length core is that when the ribosome reaches a signal sequence the RNA and translational machinery are translocated to the ER for further processing which slows down translation.The data presented in this chapter are preliminary. The original plan was to construct 5’UTR plus core clones matched with the majority sequence obtained from GT 1 and 3 infected patients similar to those described for the 5’UTR alone. This was in order to compare translation efficiency by the IRES in HCV isolated from GT 1 and 3 infected patients using constructs containing 5’UTR plus core to determine whether there is a consistent difference between the 2 genotypes.4.2. Amplification of 5’UTR plus core regionsI designed oligonucleotides to amplify the entire 5’UTR of HCV and core gene (nt 1-914). The sequences of these primers and the nucleotide positions to which they bind can be seen in Table 4.1. A. BamHI restriction sites were incorporated into the 5’ ends of the primers for second round amplification to facilitate future cloning. Samples from seven GT 1 infected patients with unique majority sequences were chosen for cloning. PCR products made by P. Preikschat as described in section 2.2.8.2 were used as template to amplify the 5'UTR plus core using sense primer MMCG1 and antisense primer MMCG2.90CO CO05 05o wAft ^T3s ^fl CQ f l s - ^ ’ i— l C/5ft ^^ oor-Xa2 f t flO »HX 2aU o ft °Vi ^OcS f^l'fl “ AC/5flA C/5OX) § f l .w3 'SS §8 Ji a ^OX) „fl fl -A c/5f t flAPQaa2 &A ^fi °• fl cG a ft OQI0 5 *c/5 'fl Sf l C/5O flfl o=I >—1 f t° fl6 B\—yr CjChapter 4From GT 3 infected samples, 7 unique sequences were identified. Amplification of GT 3 samples was carried out using primers PP5’UTR and PPCORE-R. High fidelity DNA polymerase (HF2, Clontech) was used for amplification to reduce errors resulting from misincorporation of bases by Taq DNA polymerase. PCR products from two identical reactions were pooled and gel purified as described in section 2.2.9.24.3. Sequencing of core coding sequences from patientsIn order to find clones matched with majority sequences from patients, the first step was to obtain the majority sequence from those patients. Since only incomplete or no core PCR product majority sequences were available, sequencing of the core coding region from studied patients was carried out using the sense and antisense primers shown in Table 4.I.B. All sequences were edited using “Sequence Navigator” (Applied Biosystems) and then exported into GCG (Wisconsin Package, version 10.2-Unix). Several sequences from the 5’UTR and core regions were assembled, using “GelMerge” and “GelAssemble” programs to obtain the majority sequence for each patient. Alignments of the full core majority sequences from 7 GT1 and 7 GT 3 isolates are shown in Figures 4.2. and 4.3. respectively. Figure 4.4 shows the amino acid sequences of core protein observed in the patient samples analysed. In GT1 samples, only 5 out of 73 nucleotide differences were nonsynonymous. Of 72 nucleotide differences observed in GT3 samples, 4 were nonsynonymous.4.4. Construction of plasmids containing 5'UTR plus core (pRLNC)Amplified PCR products containing the 5'UTR plus entire core region (nt 1-914) obtained from serum samples were cloned into the pRL vector (Collier et al., 1998) using the unique BamHIrestriction site as shown in Figure 4.5.A. Individual clones were cultured overnight and plasmid DNAs were extracted using an in-house miniprep method as described in chapter 2.2.1.2. Plasmids were digested with Ncol and Hindlll restriction91fl^3cdc .■oi-Jo.4>sl-HAV Oh£OflV OX)o*'Ofla*oflH o-=-Moa/ufla>fla"pflflOX!u3OX)Pu<<5Cco’wc/o•3oc,flcdo•fl • < 0 0 < 0 0 0: : : : : : : : 3 0 0...............4-> • • 0. . . jj . . . . U. . . o . . . . 0. . . . (0 . . . 0 0............................................................................. 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Comparison of mean IRES activity of GT1 and GT3 pRLNC constructsin BHKsinT7 cells.Firefly/renilla luciferase ratios obtained from each constructs were normalised against that obtained from pRLNC H77c in each experiment. The data from two experiments were combined and analysed using both student t and Mann-Whitney tests.The data are presented as +/- standard deviation.>, •rIS<*»oo400350300250200150100500GT 1 GT3p>0.05Chapter 44.7. The effect of core on IRES activityThe results of luciferase assays revealed that the inclusion of the additional core coding sequence substantially reduced firefly luciferase light output. It is possible that fusion of the core protein to the firefly protein reduced the enzymatic activity of this protein resulting in lower production of light. In this case, the inhibitory effect of core would be overestimated. To assess better the extent to which the inclusion of the core-coding sequence affected translation, the next experiments were carried out. BHK-21 cells were infected with vTF7-3, a vaccinia virus expressing T7 RNA polymerase. This system was used in preference to the BHKsinT7 system because the proteins of interest are expressed at a higher level. The cells were then transfected with plasmid DNAs containing the 5'UTR alone (pRLN) and 5TJTR plus entire core (pRLNC) of H77c and after 16 hours, the cells were lysed and cell extracts obtained. The pGL3 vector which contains the firefly gene driven by the SV40 promoter was also transfected as a control for the correct band size of the firefly protein. Luciferase assays were carried out using extracts of cells which had been transfected with pRLN and pRLNC constructs. The ratio of firefly to renilla observed from pRLNC was 5% of that obtained from the pRLN construct (Figure 4.9.D). However, the renilla luciferase activity, which indicated the transfection level, was 4 fold lower in pRLNC than that of pRLN constructs. In order to verify these results, western blot analysis was performed to assess the amount of downstream firefly protein expression using the same cell extracts tested by the luciferase assay. Figure 4.9.A shows the SDS- PAGE analysis of the translation products which clearly demonstrated the presence of each expected protein. pGL3, pRLN and pRLNC all showed expression of the firefly protein. In cells transfected with pGL3 and pRLN the protein size (61 kD) was as expected, but pRLNC gave expression of a protein with larger size in comparison to pRLN. Figure 4.9.B shows the expression of core protein from the pRLNC construct which is similar in size to the control full length core provided by Dr. J. McLauchlan. The presence of full length core indicated that cleavage of core protein by signal peptidase at the core/firefly junction has occurred. A lower abundance of the firefly protein was produced from pRLNC compared to pRLN. Densitometric analysis of bands showed that the firefly protein obtained from pRLNC was only 60% of that from pRLN (Figure 4.9.A).94minoooLL.O CDin cn00 CNJ_JQ.Q q2 2o mousat_o3c«■QJ+*C/5QJ?oCo©X)o-ooT3S3>.co*»■JCs_ocuaxa>cu-33s 3 ^ ©•— ^ CO >D 33•a ^* oCOCO3!-’>atoo3>SOCD3o33a0303O 3^,» O c ^ a g |^ o Iu oco CO CD •— aX— CDW) ^ o& Z'of Xas ar -1-a 3 c o3 £U "3 Z 6hJ ^2 333 £oo G^ 8 o £_ « o44 °> 33 ^ O -acj -a"3 aCU2 ■£ g. £O CD2 »- 3 iS ■— 373 O-' ."3 -*-> P co 3 ^ O O X — O^ MCD 3 3 33 2 2sX 3 Cy - l cZ 3 ^_J coCD > XJ . O 2z ^d o£2 atCD 2 3- X 3^ CD ^O c 3 -G 3 3o £ 5z 8 ? •—1 2 CDN- X3CDCD< J3 P3 ,3> o• 33•a c3 33 X! O - H G 0JD •- D«i ^ O 3 3 03_C co"O '—3 CD<3 wU 5 Z ^J 3 2 « G. o ^ u-Z »■2 a * oCD 3 G 73 ch 2M-H H-H.3 O 03 37 O £C3d O 3co D> 3 O 3 ID 2* § 2 (D — .330 ^ 1 ^ ^ o 2 =En »i -o = i 3- O 73 .3 7J a CD o CD7 33 3 P O 7(T\ ^p ! ^8 2 § D) Firefly and renilla luciferase light outputs observed from pRLN and pRLNC constructs and their ratio were shown.Chapter 4Anti P-Actin antibody was used as an internal control to normalize the amount of total cell lysate loaded in each lane (Figure 4.9.C). Taking into account the 4 fold less transfection level of pRLNC in comparison to pRLN construct, it would be expected that the abundance of firefly protein in pRLNC construct would be only 25% of that from pRLN. From these data, it would appear that no downregulation of firefly luciferase expression occurred in the presence of core but rather, increased amount of downstream reporter protein has been produced. It should be noted that western blot analysis was carried out twice whereas, transfection and preparation of cell extract for western blot was carried only once.4.7.1 Glycosylation of firefly luciferase proteinAs described earlier, a very low level of firefly activity was detected by luciferase analysis of pRLNC constructs in comparison with equivalent pRLN constructs and, as a result, the firefly to renilla ratio was only 3-7% of that observed from the pRLN construct suggesting that inclusion of core into the 5’UTR reduced IRES activity sharply. In contrast, more of the firefly protein was detected by western blot analysis of pRLNC in comparison with pRLN once the transfection level was adjusted for. One important difference between the translation of the two constructs is that translation of pRLNC would be expected to occur in the ER due to presence of the signal sequence at the 3’ end of the core protein, whereas, pRLN would be translated in the cytoplasm. Therefore, pRLNC may be subjected to post translation modifications such as glycosylation and formation of disulfide bonds. Therefore, the amino acid sequence of the firefly protein was further examined. It was noticed that 3 predicted N-glycosylation sites were present at positions 21, 90 and 161 (Figure 4.10). This suggests that firefly protein would be further modified by N- glycosylation in the ER resulting in the production of a heavier protein. This would explain the presence of bigger firefly band obtained from pRLNC constructs in western blot (Figure 4.9.A). Unfortunately, I was not being able to perform the experiment of removing the carbohydrate portion from the protein (deglycosylation) using glycosidases. It was also shown that 4 cysteine residues were present in the firefly amino acid sequence95MKRYALVPGTIAFTDAHIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIWCSENSLQFFMPVLGALFIGVAVAPANDIYo o o o oo \D CN o coCO i—1 CN] mQ H H P
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CO>■> OO 5*33 roo1)P>Oa o fl u 3OX) _r m ^fe 00COUXu2UJUXu2wroCns'O-cC o uozUXN-oCCUz•NIT)cuT3cuCQos>uXoJ2o•_a.■ossc1*1CUa .cus.o%->scsCUc/3cuu.aoJ-X-scECUJSuC/5r j»riou.3ox.2 §> • f-«cC + -o C/3oCU£ u'O X £CCCJ ^ X ° O 6 2 2P w jbc uCC r 3-* 0) ^52 ^53 £c/3 a c/3 C * § PU ^> £ U 5Mri i—ioCJ «JS -2 w <£2 Z 2 Xx > a oo oO X3 CC O X cd x eX 3 3P ° BC/5 3X5 £ O XO' Q.U " cuCD—3 c / i U - 3O C/5 _ CCC/5 Oo_ oC lC/5D3‘ob opI— ( )(D3 X> t t_ l ^ r i§3C/3p oDL -O 3XOX) cDts O3_ o3-5_ _ "cC> Id 'C< c o DX «3 00 w CC . > wI g *«— • — n 5-t—«;> T3 3 >Q)'a.oooT“U)o(/)u.COuCQ£csQ*u—uXc033irioM3OX)XJ SCD^ B-73 °ODc3~occ3 C3CD•o g § ^ -,5 u03 ■■ X co'O03T3T3Co3 > X *2, ^ CL HE OI ^E 553 *—iB noo OTable 5.2. Ishak and steatosis scores in liver biopsies from patients infected with GT1 and GT3.NF=No biopsies availablePatient GenotypeGrading StagingSteatosisInflammation FibrosisMA 1 3 0 0TW 1 2 1 2BA 1 5 0 0OS 1 5 0 0BK 1 2 0 0MH 1 3 0 0PE 1 NF NF NFSA 1 4 0 0CD 1 5 3 1LA 1 3 0 1BH 1 3 0 0FV 3 3 1 2MO 3 2 0 0GR 3 5 0 1RJ 3 4 0 3ME 3 5 0 2MF 3 5 1 3U 3 4 0 2BC 3 NF NF NFJN 3 5 0 1MW 3 NF NF NFSJ 3 3 0 1BJ 3 4 0 1RI 3 4 0 1JA 3 NF NF NFCP 3 NF NF NFChapter 5In addition, biopsy sections were evaluated for the presence of steatosis. Steatosis was graded as absent or minimal (less than 1% of hepatocytes) (score 0), mild (<30% hepatocytes involved) (score 1), moderate (between 30 and 60% of hepatocytes involved) (score 2) or severe (>60% of hepatocytes involved) (score 3). Steatosis of the liver was observed in all GT3 infected patients except one, with scores ranging from mild (5 cases) to moderate (3 cases) to severe (2 cases). In GT 1 infected patients, only 3 out of 10 patients had steatosis (2 mild, 1 moderate).5.5. Analysis of the correlation between IRES activity, viral load and histological featuresTable 5.3 summarises the data used for statistical analysis. Data analyses were performed using SPSS version 11.0 (SPSS, Inc., Chicago IL). The correlations between translation efficiencies, Ishak scores and HCV RNA titres were analysed by the nonparametric Spearman rank-order correlation coefficient. A p value less than 0.05 was considered statistically significant.The relationship between the translation efficiency of the 5'UTR obtained from patients in HuH7 cells as described in chapter 3 and HCV RNA concentrations in serum of patients was investigated. In order to assess correlations, all patients having an identical 5'UTR sequence were considered to have the same relative IRES activity as the representative 5'UTR isolate in HuH7 cells. The mean IRES activity (normalised) obtained from 3 separate experiments from each isolate was used for analysis. The IRES activity did not correlate with serum HCV RNA levels in the patients studied (Correlation Coefficient (CC) = -0.34, p> 0.05).The relationship between the IRES activity of isolates obtained from the translational study and the liver histology from patients containing the identical 5'UTR to those isolates were analysed. Similarly, no association was found between the grade of liver inflammation and the corresponding translation efficiency (CC= 0.17, p> 0.05). There was109Chapter 5also no significant correlation between circulating HCV RNA titres and the degree of inflammation (CC= -0.3, p> 0.05).When we compared the steatosis scores with the HCV genotype, we found a significant correlation (p=0.008) with the presence of GT 3.Table 5.3. Patient data used for statistical analysisPatientGTInflam m ationgradeFibrosisstageSteatosisgradingV iral load (IU/m l)RTE in H uH 7 cell %MA 1 3 0 0 4 .7x l05 100TW 1 2 1 2 1.51xl07 110.6BA 1 6 0 0 1.3xl04 100.7OS 1 6 0 0 3.1xl05 110.6BK 1 2 0 0 3.1xl06 110.6MH 1 2 0 0 4 .8x l06 100PE 1 NF NF NF 8.3 xlO5 97SA 1 4 0 0 7.6 xlO5 107CD 1 5 3 1 4 x 105 110LA 1 2 0 1 4.3 xlO6 97.4BH 1 2 0 0 7.8 xlO4 115.3FV 3 3 1 2 3.9 xlO6 90.4MO 3 1 0 0 2.6 xlO6 120GR 3 6 0 1 5.5 x10s 120RJ 3 4 0 3 2.9 xlO7 62.8ME 3 6 0 2 2.7 xlO7 Not doneMF 3 5 1 3 1.5 xlO6 120LJ 3 4 0 2 1.6 xlO6 90.4BC 3 NF NF NF 9.6 xlO6 107.8JN 3 6 0 1 4.8 xlO4 114.9MW 3 NF NF NF 3.3x 107 120SJ 3 4 0 1 NF 92BJ 3 4 0 1 8.8 xlO6 120RI 3 4 0 1 6.3 xlO5 82.3JA 3 NF NF NF 4.7 xlO5 123.6CP 3 NF NF NF 3.4 xlO5 107.8RTE= relative translation efficiency NF= not availableFor details of scoring system, see text110Chapter 55.6. DiscussionThe aim of this part of the study was to address whether there was a correlation between the degree of histological damage, the translation efficiency of the IRES and the HCV RNA titres in patients infected with GT1 and GT3. Using the TaqMan real time PCR assay, the HCV RNA levels in serum samples from 25 patients in our cohort were measured. All liver biopsies in this study were assessed by a single histopathologist to prevent inter-observer error.In this study, using synthetic RNA, the linearity of the assay was conserved over a wide range of HCV copy numbers ranging from 104 to 1010. Therefore, it was ensured that no sample with a higher viral load than our internal HCV positive control was missed. None of our samples was measured at less than 104 IU/ml. In comparison to other quantitation methods, real-time PCR measures a much wider range of viral loads. The dynamic range of any assay is determined as how much target concentration can vary and still be quantified with equal sensitivity and specificity. Using TaqMan system, a wide range of detection has been reported ranging from 10 1 to 1010 (Enomoto et al., 2001).The result of this study concurs with other studies in finding no significant differences in virus load among patients infected with GT1 and GT3 (Lau et al., 1996; Smith et al., 1996). The presence of a higher viral load in serum of patients infected with GT 1 in comparison to GT2 and GT3 has been reported by several studies (Mahaney et al., 1994; Orito et al., 1994) and it has been argued that this difference might be related to the increased probability of achieving response to treatment observed in GT 2 and 3. These analyses require that the assays used to determine virus load are equally sensitive for each genotype. There is evidence that this was not the case for the original branched-DNA (bDNA-1) assay with a reported 2 fold reduction in the efficiency of detection of GT3 compared with those of GT1 (Hawkins et al., 1997). The current version of the Amplicor assay has been shown to amplify all known genotypes with equal efficiency. The111Chapter 5quantitative PCR assays used in this study ought to measure accurately viral load in all samples with the same efficiency because the 5’UTR sequences which were used in design of primers and probe are completely conserved in all isolates of all genotypes in GenBank. In order to confirm this, 4 serum samples including 2 patients with highest and 2 patients with least viral load quantified by TaqMan from each group of GT1 and GT3 patients were quantified by COBAS Amplicor HCV system by an independent worker blindly. Similar results to those obtained from TaqMan were reported using COBAS Amplicor test with a significant correlation between the results obtained from the two assays.Sequence variability in the 5'UTR may cause variations in the efficiency of viral translation. It is conceivable that variation in translation efficiency may also correlate with clinical features such as response to interferon, serum viral load or extent of the liver damage. It had been previously been shown that the sequence variability of the IRES did not appear to correlate with any difference in serum HCV RNA concentration (Yamamoto et al., 1997). They examined pre-treatment sera from 25 patients with GTlb infection and found no correlation between the number of nucleotide changes in the IRES with viral load or interferon response. We believe this is the first study in which correlations between translation efficiency, serum viral RNA and liver histology has been studied. The data presented here did not support such correlations. However, an association may have been masked in our study, as, by chance, most patients had very mild disease on liver biopsy. Against this, the range of viral loads seen was similar to that seen in patients in general with hepatitis C including those with more severe liver disease (Personal communication, Dr. E.A.B, McCruden).Studies assessing the relationship between serum viral titres and the severity of histological abnormalities have reported conflicting results. Some found no correlation between HCV viral loads and the extent of histological damage (Lee et al., 2001; McCormick et al., 1996; Zeuzem et al., 1996). On the other hand, Fanning et al. (1999) in a study on 77 Irish women who acquired their HCV infection through the administration of contaminated anti-D immunoglobulin, observed a significant correlation between112Chapter 5serum HCV viral loads and the degree of hepatic inflammation in liver biopsy samples using the Ishak scoring system. In the present study, no correlation was observed between serum HCV RNA titres and the degree of hepatic inflammation supporting the idea that the severity of liver disease was independent of serum levels of hepatitis C virus. It is also possible that the number of cases studied in this project was not sufficient to show a correlation. The precise mechanism by which hepatitis C virus damages the liver is not known. It has been reported that a direct cytopathic effect of the virus is responsible for the primary form of liver injury. However, it is widely accepted that the degree of liver damage is more likely to be the result of an interaction between the virus and immune response of the host (Rehermann, 2000). The results of the present study along with others argue against a direct cytopathic effect of HCV.Patients infected with GT 3 showed a higher prevalence of steatosis than those infected with GT1 in our study. It has already been shown that the prevalence of steatosis among the HCV genotypes is significantly different; patients infected with GT 3 showing the highest prevalence followed by type 2 infection and lastly type 1 infection (Adinolfi et a l , 2001a; Rubbia-Brandt et al., 2004). The mechanisms responsible for development of steatosis in HCV infection are not understood. In transgenic mice, it has been shown that the HCV core protein induces hepatic steatosis (Moriya et al., 1998). Core protein expression within the mitochondria alters the double membrane structure and causes an impairment of lipid oxidation, which produces steatosis (Moriya et al., 1998). It has been reported that steatosis may influence liver fibrosis progression in GT 3 infected patients. In patients with HCV genotypes other than 3, progression of liver disease may depend on other factors, such as prolonged alcohol abuse or being overweight (Rubbia-Brandt et al., 2004).Our data demonstrate that the observed differences in circulating viral loads and biopsy scores could not be explained by the translational activity by the IRES measured in our system in Huh7 cells.113Chapter 6Chapter 6 Quasispecies composition of 5’UTR in serum and liver6.1. IntroductionA characteristic of the HCV genome, like those of other RNA viruses, is sequence heterogeneity. The HCV population in each individual consists of mixture of a genetically different but closely related variants called a quasispecies (Martell et al., 1992). New variants are generated during virus replication as a result of errors made by the viral RNA- dependent RNA polymerase, which lacks proofreading activity. A quasispecies distribution within a population can be studied using different methods. The sequencing of cloned RT PCR products was the methodology first used to analyse HCV quasispecies (Martell et al., 1992). However, both cloning and sequencing are time-consuming and may not accurately reflect the true nature of the quasispecies makeup. Therefore, some other techniques based on differential gel electrophoresis mobility were developed such as single-strand conformation polymorphism analysis (McKechnie and McCruden, 2001), and heteroduplex tracking analysis (Gretch et al., 1996).In the use of cloning and sequencing for determination of quasispecies composition, nucleotide sequence information is obtained through PCR amplification of virus-specific cDNA produced by reverse transcription of viral RNA. The enzymes used through these two steps, a reverse transcriptase (RT) and a thermostable DNA polymerase, exhibit relatively high error rates. Therefore, such error rates have to be taken into account together when considering the actual heterogeneity within a viral population (Smith et al., 1997b).114Chapter 6The 5' UTR, even though the most highly conserved part of the virus genome, shows a quasispecies distribution (Laporte et al., 2000; Lu et al., 2000; Malet et al., 2003; Soler et al., 2002; van Leeuwen et al., 2004). Soler et al (2002) sequenced 360 clones from 6 patients infected with different HCV genotypes and showed that most substitutions were in unpaired regions of 5’UTR or clustered such that base pairing was maintained. Substitutions in paired regions of the 5’UTR would be expected to alter the stem loop structure of the IRES if there were not compensating substitutions such that base pairing was maintained. Alteration of the stem-loop structure would be expected to result in effects on the translation efficiency of the IRES. No nucleotide sequence changes were observed after interferon-a therapy suggesting that the quasispecies distribution of IRES sequences does not play a role in HCV resistance to interferon. Laporte et a l (2000) examined the sequence of 5' UTRs from the quasispecies characterized in the serum of a chronic HCV GT la infected patient and its corresponding translational activity. They showed that sequence heterogeneity between IRES elements led to important changes in their translation efficiency both in vitro and in different cell lines.Differences in the composition of the quasispecies between that circulating in the blood and that in the liver within an infected patient have been reported in several studies. However, most of these studies were conducted on the E2 HVR1 region, in which the number of variants within quasispecies is expected to be especially high (Cabot et al., 2001; Cabot et al., 2000; Navas et al., 1998). Only one report (Cabot et al., 1997) was found in which the 5’UTR quasispecies was compared in paired serum and liver samples from 1 patient. They cloned and sequenced the 5’UTR from paired serum (9 clones) and three different parts of liver explant (33 clones) of a patient undergoing liver transplantation and showed the presence of the same master sequence in both tissues. The proportion of master sequence in the three liver samples was variable, while the frequency of any other variants never surpassed 5% of total clones sequenced. In serum, the Pn ratio (the ratio of number of polymorphic sites to number of nucleotides sequenced) was lower than those in the liver samples. As can be seen, the comparison between 5’UTR in serum and liver has not been investigated properly.115RNA extracted from serum or liverReverse transcription PP2-REV (GT1) (nt 1324-1304) MSQ10(GT3) (1300-1280)HCV viral RNA5’cDNABamHI PP-AC5 (nt 18-33)PCR 1 product/PP-AC5 (nt 18-33)5’3’PCR 2 product5’UTRPCR 1PCR 2CoreBamHI3’PP3 (G Tl)(nt 988-972)PP1 (GT3) (nt 988-972)PP-AC8 (GT1/3) (nt 752-738)3’5’Figure 6.1. Schematic representation of 5’UTR/core RT-PCR.This shows the amplification o f the 5’ UTR and core region from serum and liver ffon HCV GT 1 and 3 infected patients by reverse transcription and amplification o f cDNa by seminested PCR. Bases corresponding to a BamHI digestion site were added to fie 3’ ends of the relevant primers to permit easy cloning and are represented by dotted lines. PCR 2 (seminested) was carried out only on serum and liver samples from patient LA and the liver sample from patient BH.Chapter 6The 5’UTR is the most conserved region of the HCV genome and contains an internal ribosomal entry site which directs translation of viral proteins (Honda et al., 1996b). Therefore, any change in the nucleotide sequence of 5’UTR may influence the amount of protein that is expressed. Primary work carried out by P.Preikschat in our laboratory showed that majority sequences obtained from serum and matched liver samples were identical in 26 HCV infected patients studied. It was hypothesized that the liver might contain minor variants with lower translational activity which allows sufficient protein expression for viral replication but insufficient levels to induce immune recognition of infected cells resulting in persistent infection. However, the majority sequencing data did not support or refute this hypothesis. The work described in this and the next chapter were carried out in order to examine the above hypothesis using two techniques. In this chapter, using a cloning and sequencing strategy, I have compared the circulating and intrahepatic quasispecies composition of the HCV 5’UTR derived from serum and liver tissues of six chronically infected patients to answer whether there are any differences in quasispecies makeup o f the 5'UTR region between serum and liver.6.2. Study patients and samplesWe chose, at random, paired serum and liver samples from 3 patients infected with GT1 and 3 with GT3 from our cohort. None of the patients had received interferon treatment before the samples were obtained. Patients SA, LA and BH were infected with GT 1 and patients MO, RJ and ME with GT 3.6.3. Amplification of 5’UTR/core from patients and comparison of resulting “majority” sequenceAll the following steps were carried out by P. Preikschat in our laboratory. As shown in Figure 6.1, extracted RNA from non-thawed paired serum and liver samples was reversed116Chapter 6transcribed to generate cDNA using Superscript II RT (Invitrogen). Single rounds of PCR involving 38 cycles were carried out for patients SA, MO, RJ, ME and cDNA from serum from patient BH (Table 6.1. A). In order to increase the yield of PCR product, for serum and liver samples from patient LA and the liver sample from patient BH, seminested PCR involving 58 cycles was carried out using primers PPAC5 and PPAC8 (Table 6.1.B).Comparison of sequences obtained by direct sequencing of PCR products obtained from GT 1 infected patients with reference sequence H77c (EMBL accession number AF 011751) revealed that all had a difference with G at nt 107 instead of A (G107A). Patient LA had a mix of G and A at nt 233 indicated by (R) and patient BH had A204C (Figure 6.2A).Differences in sequence in GT 3 patients were G203A in patient MO, C121U in patient RJ and A340C in patient ME when compared with reference sequence NZL1 (EMBL accession number D 17763). (Figure 6.2B)6.4. Cloning of 5’UTR/core into pCR 2.1 vectorGel purified PCR products generated by P. Preikschat as described in the previous section containing the 5'UTR and core of HCV were ligated by me into the pCR2.1 TOPO vector (Invitrogen), as described in chapter 2.2.10.2. Fourteen to 27 independently isolated clones from PCR products were selected using X gal selection. Plasmid DNAs containing the 5'UTR sequences were purified using columns (Miniprep Kit, Qiagen). Clones were selected for the presence of the correct size insert by digestion with EcoRl, which is present on both sides of the cloning site. The orientation of the insert within the vector was determined by cleavage with Hind III and Clal restriction enzymes as can be seen in Figure 6.3. DNA from 202 clones was quantified by spectophotometry and nucleotide sequenced.117Table 6.1. Amplification of 5’UTR and core region from six patients.(A) The number o f PCR cycles for each sample is shown. Seminested PCR was carried out for serum and liver samples from patient LA and the liver sample from BH. B) Oligonucleotide primers for RT-PCR of genotype 1 and 3 5’UTR and core regions.ABPatient GT Sample ! PCR cycles , CommentS. A. 1 Serum ; 38--------1 LiverL.A. 1 Serum 58 \ Seminested1 Liver ! 58 ' SeminestedB.H. 1 Serum 381 Liver 58 SeminestedM.O 3 Semm 1 38 13 Liver 38RJ. 3 Semm 383 Liver i 38M E 3 Semm 383 Liver 38Primer Sequence (5’ to 3’) Genotype Position UsePP2-REV (as) GACCAGTTCATCATCATATCC 1 1304-1324 RTMSQ10 (as) GCCATT CGGTGT C CTGAGAG 3 1280-1300 RTPP-AC5 (s) TTGCTGGATCCGCGACACTCCACCAT 1+3 18-33 PCR 1/2PP-AC8 (as) CCGACGCTGCAGATGTACCCCATGAG 3 752-738 PCR 2PP1-REV (as) AGCAAGGATC CT CATACACAATACT 3 988-972 PCR 1PP3-REV (as) AGCAAGGATCCGCCTCGTACACAATACT 1 988-971 PCR 1PP4 (as) CCGGGAACTTGACGTCCT 1+3 400-417 SequencingNucleotide numbering according to H77c (AF 011751) for genotype 1 andNZL1(D17763) for genotype 3 samples.s= sense, as= antisenseRT= reverse transcriptionBamHI restriction sites are underlined.Patient GT Sample nt-Position107 204 I 233SA 1ySerumLiverG -^ A ! |G-V A .............“..7LA 11SerumLiverG > A j ! G-^R G A * | G-^RBH 11SerumLiverG -► A I A -> C ! ..G ^ A f A ^ C !B Patient GT Sample nt-Position121 203 340MO 3 Serum ! G-^A !3 Liver G -*A !RJ 3 Serum c - * u3 Liver c -► uME 3 Serum ! A -*C3 Liver A—► CFigure 6.2. Nucleotide differences between majority sequences of patients and reference sequences.Tables A and B show changes in 5’UTR region in genotype 1 and 3 patients studied respectively. H77c (AF 011751) andNZLl (D17763) were chosen as reference sequences for GT1 and GT3 respectively.Chapter 66.5. Sequencing of clonesAll clones containing insert were sequenced in both directions by the Microarray and DNA Analysis Unit of the Sir Henry Wellcome Functional Genomics Facility (IBLS), University of Glasgow, using an ABI 377 sequencer (Applied Biosystems) based on the dideoxy method developed by Sanger (Sanger et al., 1992). In this system, each dideoxynucleotide contained a specific fluorescent dye which could be excited by a laser. Dideoxynucleotides (ddNTP) contain a hydrogen group (H) on the 3’ carbon instead of a hydroxyl group (OH) which prevents the addition of further nucleotides after integration into DNA chain. The reactions were performed as linear amplification on a PCR machine by Dr. G. Riboldi-Tunnicliffe, using BigDye Terminators v 1.1 (Applied Biosystems), in a single tube containing all four ddNTPs which are each labelled with dyes which fluoresce at different wavelengths. The contents of tubes were subjected to clean up by ethanol precipitation and electrophoresis on a slab polyacrylamide gel in order to separate the different sized bands. The signals were then collected and analysed by the Sequencing Analysis 3.4.1 programme (Applied Biosystems). A coloured electropherogram was produced. In order to sequence only the 5’UTR region in the insert containing 5’UTR plus core, clones with correct orientation (5' to 3') were sequenced using M l3 Reverse primer (5’CAGGAAACAGCTATGAC3’) and PP4. For clones in the opposite orientation, M l3 Forward (5’GTAAAACGACGGCCAG 3’) and PP4 were used (Figure 6.3). A fragment (nt. 33-336) was examined from the 5'UTR of HCV including stem loop II and III and part of IV. Reference sequences H77c and NZL1 were used to align the sequences obtained for GT1 and 3 respectively.6.6. Comparison of liver and serum quasispecies composition in patientsThe populations of 5’UTR sequences in paired serum and liver samples from 6 patients (3 GT 1 and 3 GT 3) were analysed. Figures 6.4 to 6.9 summarise the results obtained. The same majority sequence was present in both the liver and serum in each patient and this118CD CD-acscoa.OHfNC*us.s"w■w3CDcn ao« .£ -5 o■*-’ r—3 C • — 3in O'ro cnr— $“■- CDc3 £S J) ’ E .£> *0 8 £ O S ^ 'S3^ -S■5 uKw. 5—uChapter 6sequence was the same as that observed by direct sequencing of the PCR product. The proportion of the predominant sequence varied from 26% to 73% of clones in serum samples and 25% to 60% in liver samples and in most patients, no other clone was detected more than once. The exception to this was patient LA in whom a mixed population of G and A was observed at nt position 233 (Figure 6.4). Forty six percent of clones from serum and 35% clones from liver had G at position 233. A change from U to C was found at positions 56 and 63 in two serum clones (d and f) but clone f had two other changes which were not present in clone d. A change of A to G was present in clones n and q from the liver but clone n had three other changes therefore the variants were not the same. One clone (clone k) had an insertion of C at position 119/120.In patient BH, (Figure 6.5), a change from U to C at position 37 was observed in clones d and e, however, clone d had an additional change of A to G at position 66. Clone f from the serum sample had a deletion of C at position 120.In patient MO, (Figure 6.8), with GT 3, from 37 clones analysed from serum and liver, clone e from serum and clone n from liver had an insertion of C at position 117/118. Another insertion of U was found in clone q from the liver sample at position 279/280. Clones r and n had deletion of G at position 118 and 227 in comparison with the majority sequence for this patient.6.7. Artefactual sources of variationOne difficulty with interpreting this kind of nucleotide sequence data is that both reverse transcriptase and Taq polymerase have relatively high error rates, and for the latter this is compounded by multiple cycles of PCR. Errors produced during RT PCR amplification of the virus genome will be present in clones and therefore be interpreted as virus heterogeneity.119CNCOCO < CD CD310 < CD291 CD <281 h- O269 \— O259h - O243 < CD CD238 o H-233 CD < < O CD CD < < < < < CD CD CD CD CD212 h - Oi noC N < CDC O03T- h- OsT—h- O185< CD182 < CD173 < CDC Oco < CD128 o119/120*OCDT—< CD107< CDo h- o3"r ~ < CDcoh ~ < CDCOC O H O OC Oin H O ON -co I- OS e r ii nt 1 5 clones 26%13%Li v e r: 14 clones 28% 14%nt Positioncl one a (4)clone b (2)clone c ( 1 )clone d ( 1)clone e ( 1)clone f ( 1 )clone g ( 1)clone h ( 1)clone j ( 1)clone k ( 1)clone m ( 1)cl one b (4 )cl one a (2 )clone n (1)cl one p (1)cl one q ( 1)cl one r ( 1)cl one s ( 1)clone t (1)cl one v (1)cl one w ( 1)CLot■ oc C /3 \ J 33 £ oo 3C L C O£ w 3O > -L= 5 «“ 2 . 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Oo O £ ' £o 3 CDDc/5o 4 3 _3£>>t2oa .■4—*_ 3•4—»CD43>3CDoxc x3CDC/543CD£ c00 Iclone b ( 1)Icl one c ( 1)Iclone d ( 1)Iclone e ( 1)Icl one f (1)Iclone e ( 1)Iclone h ( 1)Iclone i ( 1)Iclone k ( 1)Icl one ni ( 1)1 clone a (7)1 cl one n (1)1 cl one d (1)\clone a (1)1 cl one r (1)1 cl one s (I)1 clone t (1)1 cl one v Cl)HOgc*-Ccs—o£■Do>0/■aC/20>s2”_c-oa/>a/C/2J2oC/2ous0/.4)■oa/2-*•»o_ oasZassoa/M3OXc/2 'O O £ £c 3 C e 03 Co w , 3O 8 C£ 2 oO ’S MJ3 w cc n > —— 2 ®^ ^ 55 8 -B,0 oo D-3o3U 3 > \ c X > CD 3cr* 8>%cO3 cn 3 03 3 a> c r* £flj o-2 5 Jg e 2 -Sa> P c5 & 800 d (DW 3 a clones from this patient.Chapter 6The frequency of differences from the majority sequence observed in the HCV 5' UTR target region was calculated as:No. of differences in each group of sequencesNo. of clones x sequence length in bp x No of PCR cyclesand expressed as the number of unique polymorphism sites per nucleotide per cycle (Smith et al., 1997b)In total, 131 differences including 124 substitutions, 4 insertions and 3 deletions were observed amongst 202 clones sequenced. According to the manufacturer’s information, the error rate of Superscript II was 1/15000 bases sequenced. Again, according to the manufacturer, the error rate of AdvanTaq Plus was 0.66 errors/1000 bp/25 cycles or 2.6 xl0'5per nt sequenced per cycle of PCR. The expected number of substitutions in the PCR reaction was calculated as the error rate of Taq (2.6 xl0'5) x length of fragment (303) x number of PCR cycles x number o f clones sequenced. As a result, the expected number of changes would be higher for those samples (serum and liver from patient LA and liver from Patient BH) involving more PCR cycles. This was confirmed by the number of observed changes in those two patients. As shown in Table 6.2, the predicted number of errors generated by enzymes for sequences obtained after 1 round of PCR was 50 bases and that for those generated using 2 rounds of PCR including serum and liver samples from patient LA and liver sample from patient BH was 19.7 bases. Instead, 84 substitutions were observed in 125 clones involving 1 round of PCR and 40 substitutions amongst 43 clones generated by 2 rounds of PCR. In total, the expected number of changes were 69.6 nt, whereas, 124 nt changes were observed. Therefore, the number of observed substitutions obtained from cloning in this study was higher than errors predicted by RT and DNA polymerase used for amplification. In only one sample (SA, serum) the observed number of substitutions was lower than predicted.120Table 6.2. Frequency of nucleotide substitutions in the 5’UTR region derived from patient samples.The expected number o f substitutions in each sample was calculated as the sum of errors produced by reverse transcriptase and Taq polymerase. Errors resulting from Taq polymerase were calculated as the error rate of Taq (2 .6 x 10'5) x length of 5’UTR (303) x number o f PCR cycles x number o f clones.' Number of Expected Observed i Observedclones per PCR number of number of ! substitutionPatient : Tissue sample cycles substitutions I substitutions rate x(10 5)MO Serum 22 38 7 10 3.9’Liver 15 38 4.7 7 4RJ Serum 27 38 8.5 14 4.5Liver 14 38 4.3 10 6.2ME Serum 19 38 6 13 5.9Liver 17 38 5.3 10 5.1SA Serum 15 38 ; 4.7 4 2.3Liver 15 38 4.7 9 5.2LA Serum ; 15 58 7.1 -17 6.4Liver 14 58 6.3 13 5.2BH Serum.. 1538 4.7 7 4i Liver 14 58 | 6.3 ! 10 4Total 202 69.6 124Chapter 66.8. Characteristics of observed 5f UTR changesAltogether, compared to the 5’UTR sequences obtained from majority sequences from each individual, 124 substitutions, 4 insertions and 3 deletions were found in 202 cloned sequences. Only 8 (6%) substitutions were transversions (changes from purine to pyrimidine or vice versa): A to C (2), C to A (1), G to C (2), A to T (2), T to G (1) and the reminder were transitions. These were substitutions of purine to purine: T to C (39), C to T (14), or pyrimidine to pyrimidine: A to G (45), G to A (18). Three out of 4 insertions were observed at position 119/120. The fourth insertion was at position 270/271. Three insertions and 2 deletions were observed in one patient (MO). All insertion and deletions were in regions predicted to be unpaired except one (nt 227).6.9. Comparison of heterogeneity of sequences in serum and liverIn order to determine whether the liver samples contained more complex populations than serum samples, quasispecies complexity was calculated for all patients. Quasispecies complexity can be quantified using Pn (the ratio of the number of polymorphic sites to the number of nucleotides sequenced (Cabot et al., 2000).To correct for differences in numbers of cycles of amplification in the calculation of Pn, the number of observed substitutions in serum and liver samples from patient LA and liver sample from patient BH which involved 58 cycles of PCR, were recalculated as:No. of observed substitutions x 38 58Table 6.3. summarises the results obtained for the 6 patients. Clones obtained from liver samples from patients RJ and SA were more complex than clones obtained from paired serum samples. For patients MO and BH the complexity of clones obtained from serum samples was very similar to those from liver samples. In patients ME and LA, the121Table 6.3. Sequence complexity of the 5’UTR in the liver and serum quasispecies of patients.Appearances refers to the number o f times each sequence was observedin the clones analyzed. Pn: the ratio o f number of polymorphic sites to number ofnucleotides sequenced.Patient TissueNumber of clones analyzedNumber of changesNumber of identical sequencesIAppearances ; i (no of clones ) < PnM.O Serum 22 10 13 (59%) 1 (13), 9(1) ; 1/666Liver 15 7 9 (60%) : 1 (9), 6 (1) 1/649R.J. Serum 27 14 18 (66%) 1 (17), 8 (1) 1/584Liver 14 10 7 (50%) 1(7), 4(1 ) 1/303M.E Serum 19 13 10 (52%) 1(10), 9(1) 1/442Liver 17 10 10 (58%) 1(10), 7 (1) 1/515S . A. Serum 15 4 11 (73%) 1(11), 4 (1) * 1/1136Liver 15 9 6 (40%) 1(6), 9(1) . 1/505L.A. Serum 15 11 4 (26%) 1 (4), 1(2), 9(1; 1/413Liver 14 8.5 4 (28%) 1(4), 1(2), 8 (i; 1/499B.H Serum 15 7 9 (60%) 1(9), 6(1) 1/649Liver 14 6.5 6 (42%) : 1 (6), 8(1) , 1/652Chapter 6quasispecies complexity of clones derived from serum was higher than those obtained from liver samples. Figure 6.10.A shows the number of changes, which occurred per base in sequences obtained from paired serum and liver from each patient. No significant difference was found between complexities of sequences studied pooling all the data for liver and serum derived clones. (p> 0.05 Student’s unpaired t test). (Figure 6.10.B)6.10. Comparison of variation between paired and unpaired regionsAll substitutions were positioned onto the predicted 5’UTR secondary structure. Seventy- five (58%) were found in paired regions whereas the total number of nucleotides in paired regions predicted by the 5’UTR secondary structure was 197 (65%). Fifty six (42%) changes were positioned in unpaired regions whereas 106 (35%) of the total nucleotides were predicted to be unpaired. (Figure 6.11 and 6.12)The number of changes occurring in nucleotides predicted to be unpaired was higher than in paired regions. Differences in Pn values were analysed by unpaired Student’s t test.The rate of change (Pn) in paired and unpaired region was calculated. As shown in (Figure6.10.C) the rate of change per nt in predicted unpaired regions was 0.0028 against 0.0018 for paired regions. The difference was not significant (p>0.05).6.11. DiscussionBy cloning and sequencing PCR products spanning the 5’UTR, we demonstrated the existence of a quasispecies with the majority of clones corresponding exactly to the sequence obtained by sequencing the PCR product directly. Additionally, in each patient sample, there appeared to be a number of minor variants suggesting that the quasispecies nature of HCV includes variants in the 5’ UTR.122U d UE6|/\JooAcu_____________CQud ,L O L O ^ - L O C O L O C N L O ^ — LO O ^ f O C O O C N O x — O Oo o o o o o o o o ouai-4/C/5Eoc-ocu#c*sJSoC /5cuos«u3ccuc/5u-O—o.EoUCU33uHDC^HoCu 00 3 2s ^E 2a. ° c3 3° T 3•c =e3 03 a. 3s3 333033CUo oCU ^ 3 3 03,2 CQc/503 3~ OC/5 r - 3 3C U •CGG UCACGUGGCCA GGG A QCC GC GC CG150 — GCIjOt.rU57 — AGA'CG kAUG ^ I C G U UCUU A AGAA G UGC C U A _ U CG^ CGGCCA GACACUCCACCAUAGAtfCACUCC GC ICG 30CGU"\ U jACG CG U U G AUA CG UA GCGU „ 250 UG /GC XAU 4/. k/UA r AG ^ u u GCCU UAGUG GCGGA AGCGC GC /A U Go ° * ^CG GU• A Jz , 295ACC CCC CCUCCC GGG GCCU A GGAGGGCCC UGGG U U CG , /AGCXf%Figure 6.11. The location of nt differences in sequences studied from 3 GT 1 infected patients on the secondary structure of the 5’UTR All differences are indicated by arrows. Arrows preceeded by for insertion and “O ” for deletion. Black and red arrows indicate differences from consensus observed in clones from serum and liver samples respectively, (modified from Honda et al 1996 by P. Preikschat)GT3 samplesCGc^AU ,,^r°cAC W U A3 UaGCGCg cu aCG -----> 9A^ -U i2 2 0- ^ A AC G ° - * cG GCCG^ C7 3 G U ^ T- ^ - A U A A A GG UAU "!* A U C G ^ f C G U . . UCUU<—->-UA^Uy ° c >A i U A GGGCACCGG GUGGCC A G CCC U GCG GC^ U A 9 ^ CG ~Z------UA^ T -^■Gu2 4 3Gc ^Ua^ TGC , /AU ILUA AG r U UGCCg UAGUG GCGGA AGCGC G —U lUC AUG IA CG |G - ^ C GC^^AU^" U G265aC C u GACACUCCACCAUgGAUCACUCC GC |CGSiCG U c U AGUA A .CG ^ ^ G c /CG - 5 ^ A u G.CCCCCCCUCCCGGG GCCU A GGAGGGCCC U2 9 3GC CUaaAcCUCAAAGA... AU.^ ■GC UA GC CG A AFigure 6.12. The location of nt changes in sequences studied from 3 GT 3 infected patients on predicted secondary structure of the 5’UTR.All differences indicated by arrow. Arrows preceded by for insertion and “Q ”for deletion. Black and red arrows indicate changes observed in serum and liver samples respectively, (modified from Honda et al 1996 by P. Preikschat).Chapter 6The study of HCV quasispecies composition is problematic. Sequencing of cloned PCR products is the most commonly used technique for quasispecies analysis. However, it is prone to artificial polymorphism introduced during RT-PCR. Both reverse transcriptase and Taq polymerase have high error rates. Therefore any errors resulting from RT PCR could be interpreted as virus heterogeneity. One approach to minimise the rate of errors introduced by DNA polymerase is to use proofreading DNA polymerases. It has been shown that the error rate for Taq polymerase is seven times higher than for proofreading DNA polymerases (Malet et al., 2003). In this study we used “Advan Taq Plus” polymerase which according to manufacturer’s instruction has an error rate of 2.6 x 10'5 per nt sequenced per cycle of PCR.It is possible that all variants present in quasispecies in each sample do not amplify with equal efficiency during RT PCR. One problem could be differential hybridisation of primers due to mismatches at the primer binding site. This could serve as a negative selection mechanism contributing to under-representation of variants containing those mismatching sites. Primers used during RT PCR in this study were designed from a conserved regions of the genome found by aligning as many sequences of the appropriate genotype that were available at the start of the project. At that time, there were many more complete and partial GTla than GT3a sequences so bias may be more problematic for GT3a. I would have liked to use a different set of primers external to those used for amplification of the 5’UTR and to sequence a number of clones containing the new PCR products in order to find out whether the same variants would be detected as in the original experiment. However, this would be costly.There is evidence which suggests at least part of the observed differences in our clones reflected real variants within the quasispecies rather than errors produced during the amplification process. Firstly, the substitution rate in the 5’UTR was found to vary between the 6 patients and between serum and liver samples within the same patient in 4 cases. Secondly, the observed substitution rates (4.5 x 10‘5 for sequences obtained from 1 round and 5.3 x 10'5 for sequences obtained from 2 rounds of PCR) were higher than expected errors produced by reverse transcriptase and Taq polymerase (2.6 x 10'5) used123Chapter 6for amplification. Thirdly, although the data presented in this chapter showed that most changes were unique to each clone, there were altered bases which were shared between more than one clone e.g. positions 56 and 63 in clones d and f in patient LA and also the mixed position at nt 233. In addition, there were some nucleotide positions which were substituted more than once among different clones sequenced (e.g. position 37, 56, 63, 74, 99 and 243 in clones obtained from GT 1 patients and positions 101, 132 and 147 in clones derived from GT 3 patients). Most artefactual substitutions would be expected to be found in only one of the multiple clones sequenced (Smith et al., 1997b). Fourthly, the rate of substitution observed in predicted unpaired regions was higher than those observed in predicted paired regions. Similar findings were reported by Soler et a l (2002). These suggest that substitutions in unpaired regions are tolerated more than those in paired regions. Substitutions located in paired regions change the stem loop structure of the IRES which may cause translational and replicative disadvantage. Any change in a paired region requires a covariant substitution in the complementary base to preserve the secondary structure of 5’UTR. Only one such substitution was observed, in clone c from serum sample of patient LA, which had two complementary changes of A to G at position 185 and T to C at position 212. The non-random distribution of observed changes suggests that errors introduced by DNA polymerase during the PCR reaction were unlikely to account for all the observed changes.The data from our study support the presence of quasispecies in HCV 5’UTR which has been reported by other groups (Laporte et al., 2003; Martell et al., 1992; Soler et al.,2002). The first report of the quasispecies structure of HCV 5’UTR was by Martell et al (1992). They cloned and sequenced 20 5’UTR clones from a serum sample from a patient infected with GT1 and found that 60% of the sequences were identical to the master sequence obtained from direct sequencing of PCR product and only 40% contained single, nonrepetitive base substitutions. In our study, between 26-73% of sequences obtained from serum were identical to their corresponding majority sequence. Three out of 4 insertions were observed at position 119/120 which was also observed in other 5’UTR quasispecies studies (Laporte et al., 2000; Lu et al., 2000; van Leeuwen et al., 2004).124Chapter 6Differences in the composition of the quasispecies between serum and liver have been reported mostly in the HVR1 region (Navas et al., 1998; Shimizu et al., 1997). Greater complexity in the liver could be due to the existence of different functional compartments in infected liver cells. It may also explained by the existence of sequences in the liver which are not able to become mature virions to be released into the circulating pool. Greater complexity in the serum could be explained by the contribution of minor variants replicating in extrahepatic sites (Laskus et al., 1998).Only two studies were found to analyse the differences between quasispecies composition in the serum and liver in the 5’UTR (Cabot et al., 1997; Jang et al., 1999). However, the study by Jang et al. was carried out using SSCP analysis which will be discussed in the next chapter. Cabot et al, using cloning, examined the 5’UTR from three different biopsy samples from a liver explant from one patient with that from matched serum and found that the major sequence was present in a similar proportion in the 3 biopsies suggesting that quasispecies complexity in serum is not due to anatomical compartmentalization of virus replication. In their study, complexity (Pn ratio) of clones derived from serum was lower than those from liver. In our study, no significant difference was observed when the mean Pn ratio obtained from serum samples was compared with those obtained from liver from 6 patients.The data from this study showed a higher rate of transitions (94%) than transversions (6%) in the 5’UTR fragment studied. The higher ratio of transitions in 5’UTR compared to the coding regions has been reported by other groups (Tanaka et al., 1993; Vizmanos et al., 1998). However, these studies examined the variability of 5’UTR in different isolates and not in quasispecies. The 5’UTR is not a protein coding region therefore it cannot be under immune pressure as is the case for the HVR1 region. However, the influence of other selection processes on the 5'UTR in which the fittest variant for the environment would be selected cannot be excluded.It has been reported that the GGG triplet between nucleotides 260 and 268 of the apical loop of stem loop Hid was very conserved (Soler et al., 2002). Assuming no selection125Chapter 6against mutants, the expected number of mutations in this segment among clones studied would be 3.8 nts. Interestingly, this triplet was completely conserved among the clones studied here. It has been shown that this triplet is essential for IRES activity in vitro and in vivo in HCV as well in flaviviruses, pestiviruses, and GB virus B (Jubin et al., 2000) .We have shown that a proportion of the observed substitutions in the 5’UTR derived by cloning and sequencing must represent variants present in the patient providing evidence for a quasispecies distribution in the 5’UTR. The data presented in this chapter did not support the original hypothesis that there is a subpopulation of virus in liver cells which has a lower IRES activity than the predominant HCV population circulating in the blood.126Chapter 7Chapter 7SSCP analysis of quasispecies composition of 5’UTR inserum and liver7.1. IntroductionSingle-strand conformation polymorphism (SSCP) is one of the techniques widely used to identify a mutant sequence or a polymorphism in a known gene. In this technique (SSCP), the migration of single stranded DNA in non denaturing PAGE is analysed. The mobility of DNA fragment is characterised by the tertiary structure which it adopts. The PCR products are heat denatured and three dimensional structures emerge during cooling which depend on the primary nucleotide sequence. The technique allows the detection of single base changes which affect the folding of the DNA. Therefore, mixtures of DNA molecules of the same size, such as would be found when RT PCR is performed on material containing related RNA genomes as with hepatitis C, may be separated into bands of different mobility. The sensitivity of SSCP tends to decrease as fragment length increases.The success of any particular SSCP experiment depends on the optimisation of conditions to maximise differential migration among fragments. This is of particular importance when analysing viral quasispecies as the number of expected bands cannot be determined beforehand. The original SSCP protocol used the incorporation of radioactive label and polyacrylamide gel electrophoresis on sequencing gels for detection (Orita et al., 1989) which was labour intensive and time-consuming. Simpler methods using polyacrylamide gel electrophoresis and non-radioactive staining such as silver staining have been127Chapter 7proposed. To improve the resolving power of SSCP, a variety of methods have been suggested, for example adding glycerol, reducing temperature, increasing the length of the gels or the duration of the gel runs (Sentinelli et al., 2000).According to the manufacturer, MDE (Mutation Detection Enhancement Gel, Cambrex) gel solution is a polyacrylamide-like matrix that has a high sensitivity for DNA conformational differences. Since its introduction, MDE has been used in other studies including the work carried out in our laboratory in order to study variation in the NS5A gene in response to interferon treatment (McKechnie and McCruden, 2001).This technique has been used in many studies to examine the quasispecies nature of HCV, but in most, the target region was the HVR1 (Enomoto et al., 1994; Moribe et al., 1995). The 5' UTR is the most conserved region of the HCV genome. Only a few studies have compared the quasispecies composition between matched liver and serum samples from patients infected with HCV. Laskus et al (1998), using SSCP analysis, reported the presence of different 5'UTR variants in peripheral blood mononuclear cells (PBMCs) from those found in serum in 5 HCV infected patients co-infected with HIV-1 suggesting the presence of extrahepatic HCV replication in PBMCs. However, no difference in quasispecies makeup was observed between liver and serum samples. The work carried out by Jang et a l (1999) examined the differences between the quasispecies composition of 5'UTR sequences in matched serum and liver samples obtained from 6 chronically HCV infected patients using SSCP. They reported that liver samples from 4 patients contained variants which were not found in serum samples from the same individuals. SSCP analysis of PCR product amplified from serum and liver revealed consistent major band patterns, indicating the presence of an identical master sequence in four of the six patients studied. However, in each of these patients, additional bands specific to liver derived RNA were identified. In the remaining 2 patients, however, differences in master sequence were observed and it was concluded that the observed inconsistency was due to lower viral loads in those patients.128Chapter 7The work presented in this chapter was undertaken to analyse the composition of the HCV quasispecies in the 5'UTR region using SSCP to answer the following questions:1) Is there a difference in the composition of the 5'UTR quasispecies detectable between serum and liver from individual patients?2) Are the results of quasispecies analysis by cloning and sequencing comparable with SSCP regarding variation in the 5'UTR of HCV? Specifically, are any of the variants detected by cloning present in sufficient quantity to be visible by SSCP?7.2. Optimisation of SSCPIn order to determine the reliability and reproducibility of this technique, primary experiments were carried out using clones generated by V. McKechnie in our laboratory. The clones were obtained by blunt end ligation of the PCR product from the HVR1 region from a patient infected with HCV GT lb into the vector pUCl 19 (McKechnie and McCruden, 2001).Plasmids 2c, 2d and 3d.2 containing the HVR1 inserts were amplified with primers EN3 and EN4 (Table 7.1 .A) using the PCR conditions shown in Table 7.1 .B resulting in the amplification of a 352 nt fragment. The numbers of nt differences between clones are shown in Figure 7.1.A. The PCR products were analysed by SSCP as described in section 2.2.11.3 to determine the number of variants with detectable differences in mobility. As expected, amplification using sense and antisense primers resulted in 2 separate bands consistent with each strand of DNA adopting a different conformation (Figure 7.1.B).129Figure 7.1. SSCP analysis of HVR clones.A) The number of nucleotide differences between clones 2d, 2c and 2d, 3d.2 and 2c, 3d.2 are 33, 33 and 4 respectively. B) Individual clones 2d, 2c and 3d.2 were analysed by SSCP. Detectable mobility differences were seen for the three clones. Gel stained with ethidium bromide and viewed under UV light. MVI, molecular weight marker (Boehringer Mannheim) was loaded alongside the individual clones to show the relative mobility.As DNA does not migrate according to size in SSCP analysis, the bands of MVI were assigned letters only, not sizes (C,D)A3d.2 2c 2d3d.2 4 332c 4 332d 33 33MVI 2d 2c 3d.2Band C LBand DChapter 7Table 7.I.A. Oligonucleotides primers for PCR of HVR1 from clones.Primer Sequence 5 ’ to 3 ’ Position*EN3 sense EN4 antisenseGC T T GGGATAT GAT GAT GAAC T GGT C GGTGTGGAGGGAGTCATTGCAGTT1296 to 1321 1623 to 1646* Nucleotide positions are numbered according to the sequence HCV-1 (Choo et al., 1991). This resulted in the amplification of a 352 nt fragment.Table 7.I.B. Amplification conditions used for HVR1 region PCR.Primer Number o f cycles PCR ConditionsEN3/EN4 25 94°C/ 1min 60°C/ 1min 72°C/1min7.2.1. Sensitivity of SSCP when two variants present.In order to determine the limit of detection of rare subpopulations, an experiment described by Enomoto et a l (1994) was performed with some modification. Two plasmids, pRLN MO and pRLN H77c, containing different 5'UTR sequences with different SSCP mobilities were mixed at various ratios from 500pg:500pg down to 990 pg: lOpg per pi. Double stranded DNA was amplified by PCR using PPAC5 and NCR4 primers and amplified products were gel purified and subjected to SSCP analysis. This experiment was carried out twice. In the first experiment, as shown in Figure 7.2, the upper band from H77c (minor variant) in both experiments disappeared as expected as the concentration decreased. However, both H77c and MO give faster migrating bands which seem to split and this made it difficult to see the disappearance of the lower band of H77c, particularly in the second experiment. The minor subpopulation (H77c) was visible at clone mixtures of 500pg:500 pg, 900pg:100pg, 950pg:50pg and 975pg: 25pg. Bands130VOO''N©>SC■1 ■tu~a03CQtT3Cra—v=0 s-m,(NN=>O''x=oxor -r -X> IU(i 11 i iiit 11iT3d>t—1Ohcj gC/D o CO ~0 OS -2 O« q £4> ^ Oi u5 'trC/3 . 3 O' r r -a.f2 SCL XC/5 ^S2 d>1 g £ 8 > «03 jG•o H2 ^s.2 * £03 >O£•L*s© fc £•S3 < o 8"Or-53ms - 8C/5 r1(D C/5#> rSf ^•— F"- 3- ^ ■ =s 1 i(U X3 d> M § |° 2 I " Or -r-Xd>j 5o«+-,o03£ §C ' C03 Gd>c.XCDc cd>03.° ? 'cs1 a£ | - 32 H03a. i> E 2?o 2O G_ d>ou-d>a.03Or -r - •<—>G -5•” O on _c*© 7-<■“ .-Cc3 .a -© £ ,c 'a3 3 d)<+- .5O c3 3D In .2 d>cn O0) 0 U ^on (N'g c^O, 3 •£ u-0) cn £ ”0.3! cn 5 3 X! 3 « ^ X> ’— C t ; CL GG E S3 OJ rCE w = 'g S E a -g 2x > u-u 3 )*-* <— 3 jon . 33 r- !- d .C C .C >-GG *g3 a . 3 -C cn o^ O CQG 3 _8 ^ ^ond>3C-a .3c ^3 "O’C ^> =ou- cn O d)r * >—1.3 a.3 d>d)cnO-3C/5p 2r-^ 53r n cn CJ (U^ t-H3 ) G-d)a.xd>T 33Ood)• 3 d)^ r *'§ £M d>1d) X5 ^ 'cn cn >3 C 3E ‘g a-3 x« 8 ^ d> O cn — d) 3D rC 3'5 .£G _Oa. £cn 3g o0 *5 o o03E+—»■3D •'53 Q^ TD v- G53 eo3 O8 e o 2r*3 d)>*=d -O . 333 3d> 3 ). ^ 'cnC/5C CdBand DChapter 7representing clone H77c were not visible at 990pg:10pg. Therefore, the technique was able to detect a minor variant representing only 2.5% of total population.7.2.2. Sensitivity of SSCP analysis using Silver stainingIn an attempt to increase the sensitivity, silver staining was performed using the Silver Xpress® silver staining Kit (Invitrogen) as described in chapter 2.2.11.4. It was claimed that this technique was 20 times more sensitive than ethidium bromide for the staining of double-stranded DNA in polyacrylamide gels (Boulikas and Hancock, 1981). In our case, comparison of SSCP gels stained with both techniques revealed that bands were clearer using silver staining but that, in any given gel, no additional bands were detected (Figure 7.2).7.3. Study patientsThe same samples that were studied by cloning and sequencing as described in chapter 6 were studied by SSCP. Patients SA, LA and BH, were infected with GT1 and patients MO, RJ and ME with GT3.Comparison of direct sequences obtained from GT 1 patients with reference sequence H77c (EMBL accession number AF 011751) revealed that all had a difference with G at nt 107 instead of A (G107A). Patient LA had a mix of G and A at nt 233 and patient BH had A204C. (Figure 6.2.A)Differences in sequence in GT 3 patients were at position G203A in patient MO, C121U in patient RJ and A340C in patient ME when compared with the reference sequence NZL1 (EMBL accession number D 17763). (Figure 6.2.B)131Chapter 7Extraction of RNA from the samples followed by PCR had been carried out by P. Preikschat in our laboratory as described in the chapter 6. Single rounds of PCR involving 38 cycles were carried out for patients SA, MO, RJ, ME and the serum sample from patient BH. For serum and liver samples from patient LA and the liver sample from patient BH, seminested PCR involving 58 cycles was carried out using primers PPAC5 and PPAC8.7.4. Amplification of 5fUTR region for SSCP analysis7.4.1. Control clones:Plasmids, containing the majority sequence of the 5’UTR determined by direct sequencing of PCR products obtained by amplifying the serum from individual patients, were used as templates to generate control PCR product for SSCP. pRLN clones derived from samples H77c, SA, BH, LA (clone 1 and 6) from GT 1 and ME, MO and RJ from GT 3 were selected as templates for amplification of the 5’UTR using PPAC5 and NCR4 primers (Table 7.2. A). The band pattern of each product amplified from the relevant clone therefore represents the major variant within each sample in the absence of other variants in the quasispecies.7.4.2. Patients samples:For SSCP analysis of the PCR product from patient samples, I performed PCR using PCR products made by P. Preikschat as templates using primers PPAC5 and NCR4 (Table7.2.A and 7.2.B). This resulted in the amplification of a 306 nt fragment which covered all of changes in patient majority sequences except the difference of A340C in nt 340 of patient ME. Therefore, amplified product for SSCP from this patient had an identical nt sequence with the NZL1 sequence over the region studied. PCR products were purified by gel electrophoresis and subjected to SSCP analysis132Chapter 7Table 7.2.A. Oligonucleotides primers for PCR of 5’UTR.Primer Sequence 5 ’ to 3* Position*PP-AC5 sense NCR4 antisense VtagRT (RT)TTGCTGGATCCGGCGACACTCCACCATCACTCTCGAGCACCCTATCAGTTTTTCTTTGAGGTTTAGGA18-33 293 -313 372-353* Nucleotide positions are numbered according to the sequence HCV-1 (Choo et al., 1991). BamHI site is underlined.Table 7.2.B. PCR conditions used for amplification of 5’UTR region from clones and patient samples.Primer Number o f Cycles PCRPP-AC5/NCR4 3094°C/ 1min 60°C/1 min 72 °C/ 1min7.5. SSCP profile of representative clonesClones obtained from patients within one GT differ from each other by only 1-2 nt. There are 20 nt differences between the two genotypes in the amplified region. Figure 7.4 indicates that clones LA and SA sense and antisense strands had the same mobility and in clone BH, the two sense and antisense strand separated only slightly. To investigate whether SSCP detected the presence of mixed nts of A or G at nt 233 in patient LA, clones 1(A in nt 233) and 6 (G in nt 233) were examined. No differences were found between mobility of the two clones (Figure 7.3). Clone H77c containing the reference sequence for133Figure 7.3. SSCP analysis of clones matched with majority sequences ofpatients.Individual clones matched with majority sequence were subjected to SSCP analysis to determine if clones could be distinguished. MVI, molecular weight marker (Boehringer Mannheim) bands have been assigned the letters C and D. Clones from the two genotypes have different migration patterns. No detectable mobility difference was found between clones LA/1 with adenosine at position 233 and LA/6 with guanosine at that position (boxed).rGTl GT3MVI H77 LA/1 LA/6 SA BH ME MO RJ 2C 3d2Band CChapter 7comparison of direct sequences obtained from GT 1 patients was included. It is notable that two bands were seen with H77c.Detectable mobility differences were seen between clones ME, MO and RJ derived from GT 3 samples and sense and antisense strands separated.7.6. Effect of nucleotide 107 on SSCP patternBy comparing the SSCP pattern of DNAs derived from GT 1 clones, it was noticed that both sense and antisense strands of clones from 3 patients had the same relative mobility. As shown in Figure 7.3, clones LA, SA and BH have a difference at nt 107 (G to A) from the sequence in H77c.The effect of nt change at position 107 on the separation of sense and antisense strands was investigated using three other GT 1 clones which were available. Clones OS, BA and PE with known nt A at position 107 were used. These clones differ from H77c by 1 to 3 nt but A was present in each at nt 107. PCR products amplified from these clones showed similar mobility to H77c (Figure 7.4). In other words, the two sense and antisense strands showed different mobilities. Unfortunately no GT 3 clone with guanosine at the equivalent position was available to see if both positive and negative strands would migrate at the same speed under denaturing condition.It has already been suggested that the 3’ end of minus strand of 5’UTR RNA does not fold into its mirror image (Schuster et al., 2002). In the secondary structure model of the 3’ end of HCV GT la minus strand RNA proposed by this group, nt 107 (A) is the first nt of stem-loop SL-CI, a highly stable stem loop, which is located in a linker region between SL-BI and SL-CI (Figure 7.5). It should be noted that, with SSCP, we examine DNA not RNA secondary structure, but we were nevertheless interested in looking at this phenomenon further.134Figure 7.4. The effect of nucleotide differences at position 107 on SSCP.To analyse the effect o f nucleotide differences at position 107, seven clones with known sequences were subjected to SSCP analysis. Clones LA/1, SA and BH have nucleotide A at position 107 instead of G in H77c, OS, BA and PE. Clone BH has an additional change of A to C at position 204 which is not present in other two clones. Clone 2c was used as internal marker. Marker VI represents molecular weight marker and bands are assigned letters only and not size. Silver staining was not performed.MVI LA/1 SA BH H77c OS BA PE 2CBand C —►- %•o aO^• " • A a O " " %^ «o - 0 » SL-EIDomain IA #, C - C G - CA - 0G - C0-0oOc -c * AC-* S «t Xro aM *oCOSL-AI As u,.n \ . . .oO 00 c «' 0,-1SL-DISL-B1-100Reactivity to chemicals® • 0 •nd + + + + + +Reactivity to RNase VI> > *+ + + + + +Reactivity to Pba— D - » -+ + + + + +SL-CIFigure 7.5. Secondary structure model of the 3’ end of HCV GT la (-) strand RNA.Domain I, spanning nt 230 to 1 of 5 UTR, is shown. The third stem-loop of domain I,SL-CI is highly stable. Nucleotide 107 is indicated by arrow . (Adapted from Schuster et al 2002).Chapter 7I attempted to analyse the effect of the observed sequence difference at position 107 on the 5’UTR secondary structure of sense and antisense strands on using the Zuker MFOLD program version 3.1 (Zuker, 2003). This analysis was carried out on DNA and corresponding RNA sequences in the same nucleotide region that was examined by SSCP. In order to test minus strands, sense sequences were reversed and complemented. As can be seen in Figure 7.6. the substitution of guanosine by adenosine at position 107 in the H77c sequence resulted in a completely different predicted secondary structure from the sense strand of DNA. In contrast to the predicted secondary structure from H77c in which both sense and antisense strands were completely different, there was no difference in predicted structure in the sequence containing adenosine at nt 107. RNA analysis showed different folding pattern in antisense compared to corresponding sense strand (Figure 7.7). However, the substitution of G to A at nt 107 did not result in different secondary structures in the relevant RNA as it did with DNA. These data help explain the presence of different mobility patterns observed in sense and antisense strands of DNA samples sharing guanosine at nt 107 including H77c, OS, BA and PE but not when adenosine was present at the same location.7.7. SSCP analysis of 5’UTR from GT 1 patientsTo determine whether SSCP would reveal other variants within the quasispecies, fragments derived from the HCV 5’UTR were amplified from paired serum and liver samples from three patients (patients LA, SA and BH). SSCP analysis of PCR products amplified from clone, serum and liver revealed indistinguishable major band patterns, although the patients were known to have differences in the nt sequence of their majority variant (Figure 7.8). However, in patients LA and BH, SSCP analysis revealed the presence of an additional faint band present only in liver derived DNA and absent from clone or serum derived DNA. In all cases, SSCP bands were detected reproducibly in two separate SSCP runs using the same PCR products.135Figure 7.6 “Mfold” analysis of sense and antisense strands of the 5’UTR DNA.The predicted secondary structure of the positive strand of H77c (A) and the relevant minus strand (B). Figures C and D show the secondary structure models of same sequence in which the nucleotide guanosine at position 107 has been substituted with adenosine.Figure 7.7 “Mfold” analysis of sense and antisense strands of the 5’UTR RNA.The predicted secondary structure of the positive strand o f H77c RNA (A) and the relevant minus strand (B). Figures C and D show the secondary structure models of the same sequence in which the nucleotide guanosine at position 107 has been substituted with adenosine.Figure 7.8. SSCP analysis of HCV 5’UTR sequences amplified from serum andliver from GT 1 infected patients.Lanes “C” represents sequences from clones matched with majority sequences from patients. Lanes “S” and “L” represent viral sequences from serum and liver. Two patients, LA and BH, had additional bands in liver samples shown by boxes.Marker VI (Boehringer Mannheim) represents a molecular weight marker.The band are assigned letters (C and D) only and not size as DNA does not migrate according to size on SSCP analysis. Clone 2C was used as marker.The bands revealed by A) ethidium bromide and B) silver staining.MVI LA SA BHC S L C S L C S LBand D —Chapter 7Slight separation of both sense and antisense strands of sample BH was observed for the clone as well as serum and liver.7.8. SSCP analysis of 5’UTR from GT 3 patientsAmplified PCR products from serum and liver from three patients (patients ME, MO and RJ) were analysed by SSCP. The fragment derived from the clone matched with majority sequence from the respective patient in each case. As shown in the Figure 7.9, the band pattern for each patient is quite distinctive due to differing single strand DNA (ssDNA) mobilities. However, an additional band present only in liver derived samples was not found in any of these patients. In contrast to GT 1 patients, SSCP profiles revealed that sense and antisense strands had differing mobilities in all patients.7.9. SSCP analysis of DNA extracted from silver stained polyacrylamide gelThe following steps were carried out by S. Erwa, a student in our laboratory, as part of her short term project under my supervision. To further clarify the issue of the additional band in SSCP from two liver samples (patients LA and BH), the minor band was excised from the gel shown in Figure 7.8. For comparison, major bands were excised from two corresponding serum samples (LA and BH). Extraction of DNA from the excised band was performed as described in section 2.2.11.5. Purified DNA was amplified using primers PP-AC5 and NCR4. Amplification of gel extracted DNA from the serum sample from LA was not successful. The other bands were successfully amplified by PCR and purified by gel extraction.PCR products recovered from the gel were subjected to electrophoresis along with the relevant matched clone and the original PCR product from the liver for comparison. Silver136Figure 7.9. SSCP analysis of HCV 5’UTR sequences amplified from GT3 infected patients.Amplified DNA from serum and liver from three patients, ME, MO and RJ were analysed by SSCP. Lanes marked “C” represent sequences from clones matched with majority sequences from patients. Lanes “S” and “L” represent viral sequences from serum and liver. In all three patients, amplified DNA from clones, serum and liver showed identical mobility. Clone 2C were used as marker.Marker VI represent molecular weight marker and bands are assigned letters only and not size.Chapter 7staining of the gel revealed that all samples shared a major band with identical mobility with the relevant control clone DNA (Figure 7.10). None of the minor bands recovered from liver samples showed similar mobility to the additional band present in the original PCR product from liver samples. Interestingly, PCR products recovered from the major band from serum and the minor band from liver in patient BH showed migration patterns identical to each other. DNA recovered from the liver of patient LA was similar to patient BH with an extra band. No DNA recovered from the gel band from serum sample was available for comparison in patient LA.7.10. SSCP analysis of PCR product derived from new cDNAIn a further attempt to reproduce the minor band, we attempted to amplify a new cDNA template from the RNA extracted from the liver using the original RT-PCR primers used by P. Preikschat. However, PCR amplification was not successful. Therefore, RNA extracted from two sera and two livers were reverse transcribed using Vtag-RT primer. Two rounds of PCR were carried out. Firstly, the 5’UTR region was amplified using sense primer PP-AC5 and antisense primer Vtag-RT. For the second round of PCR, NCR4 was used as an anti-sense primer along with PP-AC5. SSCP analysis (Figure 7.11) revealed a major band identical to control cloned cDNA from the same sample. No additional bands were seen in DNA derived from liver samples. The data obtained by re-amplifying from cDNA and RNA together lead to the conclusion that the observed minor bands in liver derived samples in the primary experiment did not represent a real minor subpopulation. They were possibly attributable to the additional PCR cycles carried out on the two liver samples.7.11. DiscussionTo evaluate the cloning results and assess the profile of the HCV quasispecies in paired liver and serum samples from the same patients, SSCP analysis was performed as this137Figure 7.10. SSCP analysis of DNA extracted from Silver stained gel.The minor bands from the two liver samples (LA and BH) and major band (S*) from one serum sample (LA) were successfully extracted and amplified. Purified PCR products were loaded to SSCP and subjected to silver staining. Lanes C, L and S represent sequences obtained from matched clone, liver and serum respectively. L* and S* represent sequences recovered from silver stained gel. MVI, molecular weight marker, bands were assigned the letters C and D. Clone 2C was used as internal marker.Band C ■MVIrL. A B.H.2CIt***# v*Band D-L* C S* L*Figure 7.11. SSCP analysis of PCR product derived from new cDNA.SSCP analysis was performed using PCR products generated from new cDNA from serum and liver samples from patients LA and BH.PCR product from new cDNA (S* and L*) were run along withpreviously made products (S and L). MVI, molecular weight marker,bands were assigned the letters C and D. Clone 2C was used as internal marker.Chapter 7approach overcomes some of the problems associated with cloning of PCR products and the study of individual clones. For instance, it is more rapid and less prone to generate false heterogeneity as the result of misincorporation of bases by enzymes.Using standard clones, it was shown that this technique could detect a subpopulation comprising only 2.5 % of the RNA within a simulated quasispecies of two variants. The data from the previous chapter showed the presence of single, distinct variants along with the majority of identical clones with the consensus sequence obtained from each patient. However, none of variants detected by cloning was detected by SSCP. This suggests that the real proportion of variants in the population may be overestimated using the cloning system due to the limited number of clones sequenced. In other words, none of single variants detected by sequencing could constitute 2.5% or more of the total population.It is possible that some changes in nt sequence do not alter the secondary structure in a way that leads to a different mobility pattern in SSCP. This was seen in clones 1 and 6 from serum sample from patient LA which had only 1 difference at nt position 243. In contrast some changes appear to alter the secondary structure of DNA and resulted in a significant difference in mobility of single strand DNA (nt 107 in H77c). “Mfold” analysis showed that sense and antisense strands of H77c 5’UTR formed different secondary structures at DNA and RNA levels. However, substitution of guanosine with adenosine at position 107 resulted in a similar folding pattern for both sense and antisense strands, only in the case of DNA not RNA. These data explained the observed mobility difference between H77c and clones LA, SA and BH. It has been shown that that the 3’ end of the minus strand of 5'UTR does not fold into its mirror image (Schuster et al., 2002). In the secondary structure model of the 3' end of HCV GT la minus strand RNA proposed by this group, nt 107 is located in a linker region between two stem loops (SL-BI and SL-CI) (Figure 7.6). Therefore, it is conceivable that any change in this nt could lead to a drastic effect on the secondary and tertiary structure of the 5’UTR with possible functional consequences. However, “Mfold” analysis on the equivalent RNA structures suggested that substitution of G107A did not change the secondary structure as it did with DNA.138Chapter 7At the first attempt, SSCP analysis of the serum and liver samples revealed the presence of a major band pattern, and a minor band in the liver samples from 2 of the 6 patients studied. In the remaining patients, the band pattern of serum and liver derived sequences were identical. Interestingly, the minor bands in liver samples were observed in those samples which involved three rounds of PCR. The association between the number of PCR cycles and presence of spurious bands in SSCP has been shown previously (McKechnie and McCruden, 2001). As mentioned earlier in this chapter, 3 rounds of PCR were carried out for 2 liver samples (Patients LA and BH) as well as serum sample from patient LA. In contrast to liver sample, no additional band was found in the matched serum from patient LA.The presence of minor bands in the two liver samples was investigated further. Repeated SSCP analysis of PCR product resulting from amplification of extracted minor bands in two liver samples along with corresponding serum major bands, showed identical mobility pattern for major and minor bands extracted. To further investigate the reproducibility of minor variants observed in liver samples obtained from the two patients, SSCP analysis was repeated using new cDNA from the same extracted RNA samples. cDNA was generated using a different RT primer but still covering the entire region studied. No minor bands were observed in liver samples when reverse transcription using the different primer was performed. Therefore, it seems that the minor bands detected do not represent a real in vivo minor population of virus, but rather it was a part of the majority sequence with different migration due to an in vitro experimental factor probably Taq polymerase error during the additional rounds of PCR. We therefore decided not to proceed to the expense of sequencing the DNA from the minor band.Only two studies were found which investigated the differences in quasispecies composition of 5TJTR between serum and liver using the SSCP technique (Jang et al., 1999; Laskus et al., 1998). Jang et al showed indistinguishable major bands compatible with the presence of identical master sequences in serum, liver and liver-derived negative strand from four out of six patients. In the remaining two patients, the results were not consistent. They also found an additional band specific to liver-derived positive strand139Chapter 7RNA, which were not present in either serum or liver-derived negative strand RNA in 4 of the 6 patients. In contrast, Laskus et a l (1998) compared the 5’UTR sequences obtained from serum and various organs including lymph nodes and liver in 5 HCV infected patients co-infected with HIV1 and found no differences in mobility pattern of bands from serum and liver samples in each patient. The results of our study revealed the presence of identical major bands in PCR products derived from paired serum and liver from each patient. However, no band specific to the liver samples was identified in line with the finding reported by Laskus et al.One of the main problems with the use of SSCP in the analysis of quasispecies is that very few workers have rigorously standardised the method. Some groups did not report on the sensitivity of their assay and the amount of DNA loaded for SSCP analysis (Fujii et al., 1996; Toyoda et al., 1997; Vuillermoz et al., 2004). Loading a standard amount of DNA is important. If less DNA is loaded, it is possible that some of the minor variants in quasispecies will be missed. On the other hand, loading greater amounts of DNA would increase the thickness of bands and might mask some variants. Using MDE gel, SSCP profiles in this work were reproducible when run for 18 hours at room temperature as originally reported by V. McKechnie (2001). In addition, the presence of matched clones with the majority sequence from each patient allowed us to compare the band profile of each patient with its proper control.In summary, comparison of the single strand conformation polymorphism patterns revealed that the number and the mobility of bands were the same between the serum and liver in the 6 patients studied. Therefore, there was no evidence to support the existence of different variants of the 5’UTR in liver from those in serum which occurred at a level of more than 2.5% of the total population. However, this conclusion must be tempered by the knowledge that some majority sequences had the same mobility as others known to differ by one nucleotide. This means that I cannot exclude the presence of minor populations differing in nucleotide sequence but having identical mobility pattern to major population.140Chapter 8Chapter 8 General DiscussionThe first aim of this study was to compare the translation efficiencies of the IRES of HCV isolates from GT1 and GT3 infected patients. While several previous studies have examined the translation efficiency of a single sequence as a representative of one genotype, this is the first report, to our knowledge, in which the translation efficiencies of two genotypes were compared using a number of sequences from each genotype. There is some variation in the 5’UTR even between different isolates of the same genotype. One problem with using a single sequence is that it is not clear whether the observed translational activity is specific for particular sequence used or reflects real genotype specific activity. I ensured that all 5’UTR constructs used for this study were completely matched with the majority sequence obtained from each patient. This means that the observed translation activities were a true reflection of the translational activity of the majority of virions existing in each patient.I demonstrated in BHKsinT7 cells, which are of non hepatic origin, that the mean translational activity of GT1 samples was significantly higher than that of GT3 isolates in 3 out of 5 experiments. However, in HuH7 cells, which are of hepatic origin, no significant difference was observed. It might be expected that liver derived cells would be more relevant for the study of the HCV life cycle including protein translation. HuH7 cells are permissive for replication of HCV using the subgenomic replicon system. The discrepancy between results obtained from two cell lines may be because of the differences in the way in which T7 polymerase was generated. Renilla luciferase light output levels were much higher in HuH7cells in which T7 polymerase was generated from a vaccinia construct which may have meant that translation was maximally141Chapter 8stimulated and became limited by factors such as the supply of amino acids for incorporation into protein.It was shown that nucleotide differences between isolates led to different translation activities in the two cell lines studied. Some of the substitutions led to lower translation efficiency such as the substitution of cytosine to uracil at positions 121 in patient RJ.A bicistronic construct containing renilla luciferase as the upstream reporter gene and firefly luciferase as the downstream reporter gene was used for this study. It was based on that previously constructed by Collier (1998). Importantly, both reporter enzymes are assayed in the same cell lysate preparation. The upstream translation product acts to control internally for differences in transfection efficiency. IRES activities were studied in vivo using two different cell lines. Many authors have examined the IRES activity in cell free systems (in vitro) such as in rabbit reticulocyte lysates. One complication of the cell free system is that the optimal potassium ion concentration differs for cap-dependent and IRES dependent translation (Borman et al., 1995). Translations are carried out under conditions which favour either one or the other activity, making comparisons difficult.I would contend that studying IRES activity in cell lines is more appropriate since it is likely that the conditions are closer to those in which the HCV 5’UTR initiates the translation of the polyprotein in the infected liver. It is important, however, to bear in mind that this system is only an experimental model for studying IRES activity which may be different from that which occurs in actual infection. It is also possible that other regions in the HCV genome influence translation initiation in addition to the sequences included in the present study. For example, the effect of core protein on IRES has been reported by several groups (Kim et al., 2003; Wang et al., 2000). There are controversial reports regarding the influence of the HCV 3’UTR on translation efficiency. One study reported the enhancement of IRES activity by the 3’UTR (Ito et al., 1998), whereas, others reported downregulation (Murakami et al., 2001) or absence of regulation of IRES activity by the 3’UTR (Fang and Moyer, 2000).142Chapter 8Chapter 4 provides preliminary data regarding translational activity of the 5’UTR in the presence o f the core encoding RNA and translated protein in GT1 and GT3 infected patients. Attempts to make the 5’UTR plus core constructs match with the majority sequence of patients were unsuccessful. Therefore, as an alternative approach, it was decided to test the translation efficiency of 3 unmatched clones from each patient blindly. In total, 6 patients were studied. It was reasoned that if there was a consistent and significant difference in translation between genotypes with such constructs, it would be evident. The mean translational activity of all GT1 constructs was compared with that obtained from three GT3 infected patients and showed no significant difference. It was shown that the inclusion of core sequence resulted in a marked reduction in firefly activity in BHKsinT7 cells which, in turn, caused a sharp decrease in firefly to renilla ratio, as indicative of IRES activity. The results from western blot analysis for expressed core and firefly proteins suggested that there was no reduction of the expressed firefly protein in comparison with constructs containing only 5’UTR. Instead it was likely that the presence of core increased the relative production of firefly protein. It seemed that the results obtained from luciferase assay and western blot were contradictory. Translation of the 5’UTR (without core) constructs would be expected to occur in cytoplasm. The presence of signal sequences, however, in the nascent core polypeptide will result in the translocation of the translational machinery into the ER. This would result in other modifications such as the glycosylation and the formation of disulfide bonds in the firefly protein. Amino acid sequence analysis of the firefly protein showed the presence of three predicted glycosylation sites. An increased apparent size of the firefly protein detected by western blot was consistent with glycosylation occurring at the predicted sites. Therefore, the observed reduction in firefly activity in the luciferase assay could be explained by reduction of firefly protein enzymatic activity rather than downregulation of the IRES activity. Unfortunately, there was no time to carry out electrophoresis following deglycosylation prior to SDS-PAGE to confirm this speculation. Taken together, our results did not support the concept that the presence of core coding sequence suppresses IRES activity. The data presented here are in contrast to previous reports suggesting that the presence of core downregulates the IRES activity (Shimoike et al., 1999; Wang et al., 2000).143Chapter 8Chapter 5 provides data in which postulated associations between translation efficiency and viral load or liver histology in our patient cohort were investigated. Real time quantitation of serum viral loads in patients showed no significant differences between the two genotypes. The data did not support the presence of a correlation between the IRES activity and serum viral load. There was also no significant correlation between circulating HCV RNA titers and the degree of inflammation or fibrosis in the liver. Examination of liver biopsies showed the presence of mild disease in most patients. As reported elsewhere (Adinolfi et al., 2001a; Rubbia-Brandt et al., 2004), our data demonstrated that patients infected with GT 3a had a higher prevalence of steatosis than those infected with GTla.The work presented in chapter 6, investigated the quasispecies makeup of 5’UTR in paired serum and liver samples. One hypothesis to explain the development of chronic infection was that the selection of variants of virus which replicated at low level occurred which would allow survival of infected cells in the presence of an immune response. Selection of mutations in a number of genes including the RNA polymerase could result in the generation of low-replication variants, but selection of variants with a less efficient IRES would result in a reduced level of protein translation which would have a direct effect on the amount of antigen produced. The resulting lower level of protein expression would reduce the possibility of the antigens being recognised by the immune system. Direct sequencing analysis of matched serum and liver samples from 26 chronically HCV infected patients carried out by P. Preikschat in our laboratory showed the presence of identical majority sequences in both tissues suggesting that the majority of hepatocytes were infected with the same variants that were present in the circulation. However, majority sequence analysis is not able to detect minor variants in the quasispecies composition of liver samples. In order to investigate this hypothesis further, the quasispecies composition of the 5'UTR in serum and relevant liver samples were examined in 6 patients using two different approaches. Firstly, a cloning and sequencing strategy was used. The majority of clones obtained from serum and matched liver samples had the identical sequence to the consensus sequence obtained from direct sequencing in each patient. It was demonstrated that a proportion of the observed substitutions in the144Chapter 85’UTR derived by cloning and sequencing must represent real variants present in the patient. The data from this study provided evidence for a quasispecies distribution in the 5’UTR on which selection can occur for the fittest variants for the environment in which infection is occurring. Immune selection directly by antibody would not be an issue as this is not a protein coding region but the RNA sequence could be subjected to other selection processes such as change in secondary and tertiary structure altering the interactions with cellular proteins. No significant difference was observed in the complexity of sequences in serum and liver. The presence of quasispecies in the 5’UTR of HCV has been supported by some other studies (Laporte et al., 2000; Soler et al., 2002). Sequencing of cloned PCR products is the most commonly used method for quasispecies analysis. However, it has some limitations. One problem is that only limited number of clones can be sequenced due to the high cost of sequencing. This technique is also prone to artefactual polymorphism introduced during RT-PCR. Therefore, it was decided to use a different approach as an alternative method to examine the quasispecies makeup of the 5’UTR region.The work presented in chapter 7 was undertaken to evaluate the quasispecies composition of the 5’UTR in serum and liver using SSCP analysis. Although SSCP has its own limitations, some of the problems of cloning and sequencing individual clones are overcome. The SSCP technique performed in this work could detect minor variants present as only 2.5% of the total population. I demonstrated that the band profiles obtained from matched serum and liver samples were identical in the 6 patients studied. This means that none of observed unique variants detected by cloning and sequencing were present in more than 2.5% of total population. Taken together, the results of sequencing and SSCP analysis of the 5’UTR, did not support the presence of different minor variants in the liver. I conclude therefore, that the hypothesis that variants in the liver with low translational activity exist which escape from immune surveillance is not consistent with the data I obtained.145Chapter 88.1. Future studiesIt would be necessary to repeat the experiments using the pRLN constructs in BHKsinT7 cells to remove doubts that transfection levels were adequate. To establish whether any differences observed in translation levels were really related to the cell line or to the different methods for supplying T7 RNA polymerase in BHKsinT7 and HuH7 cells, pRLN constructs could be transfected into BHK cells preinfected with vaccinia virus expressing T7 polymerase.In order to discover whether a cryptic promoter exists in the 5’UTR DNA, it would be necessary to perform Northern blot and/or RT-PCR analysis in all cell lines used. The presence of shorter transcripts would suggest the presence o f promoter in the 5’UTR DNA.My preliminary data suggested that the presence of full-length core encoding RNA and protein did not suppress the IRES activity as reported by other studies. Instead, it was suggested that the presence of core reduced firefly enzymatic activity by translocation of the protein into the ER which resulted in post translation modifications. If time had permitted, I would have repeated the whole experiment and carried out electrophoresis following deglycosylation prior to SDS-PAGE to confirm this speculation.The most established biological difference between GT1 and 3 is the better response of patients infected with HCV GT 3 to treatment with interferon-a (IFN-a), either alone or in combination with ribavirin. The causes and mechanisms of HCV resistance to IFN-a treatment are not understood, but several viral genomic regions or viral proteins may affect the antiviral process induced by IFN (Pawlotsky, 2003b). No work has been done in order to address the differences of the effect of IFN-a on IRES activity in GT 1 and GT 3. As discussed in chapter 3, the bicistronic constructs made for the translation study included 8 different 5'UTR isolates for each genotype. If time had permitted, a number of these constructs from each GT would have been transfected into different cell lines146Chapter 8followed by addition of IFN-a in serial dilutions. However, our constructs contain the T7 promoter which drives the transcription of mRNA. Using vaccinia virus for production of T7 RNA polymerase in HuH7 cells is problematic as vaccinia virus is very sensitive to IFN-a. In order to avoid this, I suggest that plasmid DNAs could be transcribed in vitro to RNA before transfection into cells. Another suggestion would be the replacement of the T7 promoter with one which can be processed in mammalian cells such as the cytomegalovirus (CMV) promoter. The problem with this system is that the transcribed mRNA might be subjected to post transcription modification such as splicing in the nucleus. From this study, a number of questions could be answered:Does IFN-a have an inhibitory effect on IRES activity?If it does, is there a difference between the inhibition of GT 1 and 3 constructs?I would also be interested to study the IRES activity of HCV GT2 using a number of isolates obtained from patients. Previous reports suggested that GT2b contained the most efficient IRES (Collier et al., 1998; Kamoshita et al., 1997). However, these groups used only a single 5’UTR sequence as representative of the genotype activity. 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