臺灣博碩士論文加值系統

Hepadnavirus, including hepatitis B virus (HBV), possesses a partial double-strand DNA genome, also known as relaxed circular DNA (RC-DNA), which is converted to covalently closed circular DNA (cccDNA), a critical template for hepadnavirus replication, after entry to the hepatocytes. However, little is known about the detailed mechanisms regulating the conversion from RC-DNA to cccDNA. Detection of cccDNA usually requires Southern blotting, which is quite labor-intensive. Therefore, a convenient in vitro cccDNA reporter system should facilitate the research of cccDNA. However, in the present there is still no in vivo and in vitro system that can support efficient HBV infection and cccDNA formation. In contrast, duck hepatitis B virus (DHBV) can efficiently form cccDNA in human cell lines as long as its pregenomic DNA is made. In this study, we utilized DHBV system to construct the cccDNA reporter for ready detection of cccDNA formation. We first showed that trans-complementation of core and polymerase could rescue the replication cycle of core- and polymerase-deficient DHBV mutants. We then generated an array of deletion mutations that spanning the region between the start codons of core and polymerase genes. Interestingly, we found that the region between the poly A signal and the start codon of polymerase gene could be deleted without significant reduction of cccDNA formation. Thus, we inserted two reporter genes, the zeocin-resistant gene and a fluorescent protein into this region. Using Southern blotting, we demonstrated that they both maintain the ability to produce cccDNA and RC-DNA, indicating the feasibility of our reporter system. Further experiments are required to prove its utility in serving as a reporter of hepadnavirus cccDNA.

中文摘要 ........................................ 5
ABSTRACT .................................... 6
TABLE of CONTENTS .................. 8
LIST of FIGURES ........................ 10
1. INTRODUCTION ................... 11
1.1 The history of hepatitis B virus .................... 11
1.2 The classification of hepatitis B virus ............................... 11
1.3 Epidemiology and the natural history of HBV infection .................... 12
1.4 The current challenges in treatment of chronic HBV infection ...... 14
1.5 Virion structure and genome organization ..................... 15
1.6 The replication cycle of human HBV .................................. 16
1.6.1 HBV replication cycle ....................... 16
1.6.2 From pgRNA to RC-DNA (Reverse transcription) .............. 17
1.6.3 From RC-DNA to cccDNA ................ 18
1.7 DHBV, a good model for studying cccDNA formation of hepadnaviruses .......... 19
1.8 DHBV cis-acting sequences that contribute to the replication cycle .................... 19
1.8.1 DHBV cis-acting sequences involved in encapsidation ........................... 19
1.8.2 DHBV cis-acting sequence that contribute to negative-strand DNA synthesis. .. 20
1.8.3 DHBV cis-acting sequence that contribute to RC-DNA positive-strand synthesis and cccDNA formation............................... 21
1.9 The effect of the size of the inserted DNA on DHBV replication ..... 21
2. SPECIFIC AIM ........................ 23
3. MATERIAL AND METHODS ...................... 24
3.1 Plasmids ........................................ 24
3.2 Cell lines and cell culture system ................. 25
3.3 DNA transfection ......................... 26
3.4 Modified Hirts’ extraction method .............. 27
3.5 Southern blot analysis ..................................... 28
3.6 DIG-labeled DNA probe synthesis ................ 29
4. RESULTS .................................. 31


4.1 The strategies to construct a DHBV cccDNA reporter ........................ 31
4.2 Production of RC-DNA and cccDNA from the DHBV expression plasmids .......... 32
4.3 Complementation of the core- and polymerase-deficient DHBV by the core- and polymerase-expressing vectors .................. 33
4.4 Construct of a series of core-deletion DHBV mutants ........................ 35
4.5 Construct of the cccDNA reporters D1SVC155 and D1SZeocin ........ 36
4.6 Construct of the DHBV1S dimer for analysis the activity of the DHBV promoter in Huh7 cells ....................................... 38
5. DISCUSSION ........................... 40
6.REFERENCE ............................ 44
7. FIGURE ................................... 49


LIST of FIGURES
Figure 1. The life cycle of HBV. .............. 49
Figure 2. Establishment of a cccDNA reporter system……………………50
Figure 3. Formation of RC-DNA and cccDNA in 293T and Huh7 cells transfected with DHBV and DHBV1S plasmids. ............................. 51
Figure 4. Complementation of core-deficient and polymerase-deficient DHBV mutants. ............................. 52
Figure 5. Complementation of series of core-deletion DHBV mutants. .................. 54
Figure 6. Bimolecular fluorescence complementation of Venus fluorescence genes. .................... 56
Figure 7. Complementation of D1SVC155. .................. 57
Figure 8. Complementation of D1SZeocin. .................. 58
Figure 9. Formation of RC-DNA and cccDNA in Huh7 cells transfected with DHBV1S dimer plasmid. ......................... 59

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