Study of mutations on hepatitis B virus promoters and construction of a replication-competent hepatitis B virus clone.

Chan Ka Ping Sophie.Thesis (M.Phil.)--Chinese University of Hong Kong, 2006.Includes bibliographical references (leaves 140-144).Abstracts in English and Chinese.Thesis/Assessment Committee --- p.iAcknowledgements --- p.iiAbstract --- p.viii摘要 --- p.xAbbreviations --- p.xiList of Figures --- p.xiiList of Tables --- p.xivChapter 1 --- IntroductionChapter 1.1 --- Pathogenesis of HBV Infection --- p.1Chapter 1.2 --- Classification and Structure --- p.2Chapter 1.3 --- HBV Genome --- p.4Chapter 1.4 --- Replication Cycle --- p.7Chapter 1.5 --- HBV Genotypes and Nomenclature --- p.9Chapter 1.5.1 --- Asian prevalent genotypes --- p.9Chapter 1.5.2 --- Numbering system --- p.9Chapter 1.6 --- Identification of Markers in HBV Genome for HCC Development --- p.11Chapter 1.7 --- Project Objective --- p.13Chapter 1.8 --- Promoters of HBV --- p.14Chapter 1.8.1 --- Pre-S1 promoter --- p.14Chapter 1.8.2 --- X promoter and enhancer I --- p.14Chapter 1.8.3 --- Core promoter and enhancer II --- p.15Chapter 1.8.4 --- Pair of mutations at BCP --- p.17Chapter 2 --- Materials and MethodsChapter 2.1 --- Construction of pGL3-promoter Plasmids --- p.18Chapter 2.1.1 --- Templates selection --- p.18Chapter 2.1.2 --- Amplification of promoters --- p.19Chapter 2.1.3 --- Cloning into pGL3-basic vector --- p.21Chapter 2.1.4 --- Screening and plasmid preparation --- p.21Chapter 2.2 --- Construction of Mutant Promoter Clones --- p.23Chapter 2.2.1 --- Site-directed mutagenesis --- p.23Chapter 2.2.2 --- pPreS 1 /2712C mutant clone --- p.24Chapter 2.3 --- Cloning of Full-length HBV Genomes --- p.26Chapter 2.3.1 --- Replication-competent HBV clone --- p.26Chapter 2.3.2 --- Amplification of full-length HBV genome --- p.28Chapter 2.3.3 --- Cloning into pUC19 vector --- p.28Chapter 2.3.4 --- Screening for insert and sequence confirmation --- p.29Chapter 2.3.5 --- Excision of full-length HBV from plasmid --- p.29Chapter 2.4 --- Re-construction into a 1.3-fold HBV Clone --- p.32Chapter 2.4.1 --- Cloning of HBV fragment nucleotide 979-2617 (nt 979-2617) --- p.32Chapter 2.4.2 --- Screening for insert and sequence confirmation --- p.33Chapter 2.4.3 --- Cloning of HBV fragment (nt 905-2000) --- p.33Chapter 2.4.4 --- Construction of a 1.3-fold HBV genotype Cs clone --- p.34Chapter 2.5 --- Cell Culture --- p.37Chapter 2.5.1 --- Cell culture maintenance --- p.37Chapter 2.5.2 --- Transient transfection of promoter clones --- p.37Chapter 2.5.3 --- Transient transfection of HBV genomes --- p.38Chapter 2.6 --- Dual-Luciferase® Reporter Assay System --- p.40Chapter 2.6.1 --- Principle of the assay --- p.40Chapter 2.6.2 --- Cell harvest --- p.43Chapter 2.6.3 --- Luciferase assay --- p.43Chapter 2.7 --- Data Analysis --- p.44Chapter 2.8 --- Extraction of HBV DNA from Intracellular Cores --- p.45Chapter 2.8.1 --- Harvest of intracellular cores --- p.45Chapter 2.8.2 --- Phenol/chloroform extraction --- p.45Chapter 2.9 --- Southern Blotting --- p.47Chapter 2.9.1 --- Transfer of DNA to membrane --- p.47Chapter 2.9.2 --- Preparation of probes --- p.47Chapter 2.9.3 --- Hybridization with radiolabeled probes --- p.48Chapter 2.10 --- Detection of HBeAg and HBsAg --- p.50Chapter 2.10.1 --- HBsAg assays --- p.50Chapter 2.10.2 --- HBeAg assays --- p.51Chapter 2.11 --- SEAP Reporter Gene Assay --- p.52Chapter 3 --- ResultsChapter 3.1 --- Templates Selected --- p.53Chapter 3.2 --- Results of Luciferase Assays --- p.58Chapter 3.2.1. --- BCP mutation of genotype A as control --- p.58Chapter 3.2.2. --- Effect of C1165T mutation on Xpro/enhI activity of HBV genotype B --- p.60Chapter 3.2.3. --- Effect ofT2712C mutation on pre-S1 promoter activity of HBV Genotype B --- p.60Chapter 3.2.4. --- Effect of G1613A mutation on core pro/enhII activity of HBV Genotype Cs --- p.64Chapter 3.2.5. --- G1613A and BCP mutation --- p.67Chapter 3.3 --- Full-length HBV Genome Clones --- p.70Chapter 3.3.1. --- Construction of replication-competent full-length HBV genome clones --- p.70Chapter 3.3.2. --- Drawbacks of the system --- p.78Chapter 3.4 --- Construction of a Replication-competent 1.3-fold HBV Clone --- p.82Chapter 3.4.1. --- Construction of the HBV (nt 979-2617) clone --- p.82Chapter 3.4.2. --- Construction of the HBV (nt 905-2000) clone --- p.86Chapter 3.4.3. --- Construction of 1.3-fold genotype Cs HB V clone --- p.89Chapter 3.4.4. --- Test for replication competency --- p.92Chapter 4 --- DiscussionChapter 4.1 --- BCP Mutation as Control of the Luciferase Assay --- p.94Chapter 4.2 --- Promoter Activities Not Altered by T2712C and C1165T --- p.96Chapter 4.3 --- Mutation G1613A of Core pro/enhll --- p.98Chapter 4.3.1 --- Mutation resides in negative regulatory element of core promoter --- p.98Chapter 4.3.2 --- NRE and NRE-binding protein --- p.98Chapter 4.3.3 --- Relationship with BCP mutation --- p.101Chapter 4.4 --- HBV Constructs --- p.103Chapter 4.4.1 --- Rationale in re-construction of 1.3-fold HB V clone --- p.103Chapter 4.4.2 --- Replication competency --- p.104Chapter 4.5 --- Conclusion --- p.106Chapter 4.6 --- Future Work --- p.107Appendix --- p.108References --- p.14