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Initial Stages of Hepatitis B Virus Replication
| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | Kim, Kyongmin | - |
| dc.contributor.author | 김희영 | - |
| dc.date.accessioned | 2019-10-21T06:47:06Z | - |
| dc.date.available | 2019-10-21T06:47:06Z | - |
| dc.date.issued | 2005-08 | - |
| dc.identifier.other | 645 | - |
| dc.identifier.uri | https://dspace.ajou.ac.kr/handle/2018.oak/16648 | - |
| dc.description | 학위논문(박사)--아주대학교 대학원 :의학과,2005. 8 | - |
| dc.description.abstract | B형 간염 바이러스 DNA 중합효소의 역전사 기능 B형 간염 바이러스 (HBV)의 DNA 중합효소는 RNA나 DNA를 주형으로 하여 DNA를 합성하는 기능을 갖고 있으며, 바이러스의 음성가닥 DNA를 만드는 과정에서 단백질 프라이머로서 작용하기도 한다. 이전의 연구에서 HBV DNA 중합효소의 시작 결손 변이주가 말단 단백질과 첫번째 디옥시올리고뉴클레오티드 사이의 공유결합 없이도 짧은 DNA 조각을 합성한다는 것을 밝힌 바 있다. 이 사실은 HBV DNA 중합효소가 프라이머 없이 DNA를 합성하는 RNA 중합효소의 특성을 갖고 있을 가능성이 있다는 것을 제시한다. 본 연구에서는 NTP 결합능력을 관찰함으로써 HBV DNA 중합효소에서 RNA 중합효소의 특성을 조사하였다. HBV DNA 중합효소의 dNTP 결합부위에 부피가 큰 아미노산을 부피가 작은 글라이신이나 발린으로 변이시킨 후, NTP 결합능력을 획득하는 지를 확인하였다. 그 결과 HBV DNA 중합효소의 dNTP 결합부위에 위치하는 부피가 큰 436번 페닐알라닌을 글라이신으로 변이시킨 중합효소 변이주가 HBV DNA를 합성할 수는 없었지만 NTP 결합능력이 있음을 확인하였다. 이 결과는 HBV DNA 중합효소의 dNTP 결합부위에 있는 436번 페닐알라닌이 dNTP 선택에 있어서 공간적 관문으로 작용한다는 사실을 의미한다. 본 연구의 결과는 단일 아미노산의 치환에 의하여 DNA 중합효소와 RNA 중합효소 특성의 경계가 불분명해질 수 있음을 나타낸다. | - |
| dc.description.tableofcontents | TABLE OF CONTENTS ABSTRACT = ⅰ TABLE OF CONTENTS = ⅶ LIST OF FIGURES = xi PART 1 = 1 Ⅰ. INTRODUCTION = 1 Ⅱ. MATERIALS AND METHODS = 4 A. DNA construction = 4 B. Cell culture and transfection = 7 C. Isolation of core particles = 7 D. RNase protection analysis(RPA) = 7 E. Northern and Southern Blotting = 8 F. Western blotting = 8 G. Endogenous polymerase assay = 9 H. Immunofluorescence assay(IFA) = 9 Ⅲ. RESULTS = 11 A. Construction of HBV mutants that represent various stages of HBV replication = 11 B. HBV RNA transcription and core particle formation = 12 C. HBV pgRNA encapsidation = 15 D. HBV DNA replication = 15 E. Intracellular trafficking of HBV core particle in various stages of DNA replication = 20 Ⅳ. DISCUSSION = 25 Ⅴ. CONCLUSION = 29 REFERENCES = 31 국문요약 = 38 PART 2 = 40 Ⅰ. INTRODUCTION = 40 Ⅱ. MATERIALS AND METHODS = 42 A. HBV plasmid DNA construction = 42 B. Cell culture and transfection = 44 C. Isolation of core particles = 45 D. RNase protection analysis(RPA) = 45 E. Northern and Southern Blotting = 46 F. Core particle Western blotting = 46 Ⅲ. RESULTS = 47 A. Core protein and chimeric core protein construction = 47 B. HBV RNA transcription = 48 C. Chimeric core particle assembly = 53 D. HBV pregenomic RNA encapsidation by chimeric core proteins = 54 E. HBV DNA replication in chimeric core particles = 57 Ⅳ. DISCUSSION = 60 Ⅴ. CONCLUSION = 63 REFERENCES = 64 국문요약 = 68 PART 3 = 70 Ⅰ. INTRODUCTION = 70 Ⅱ. MATERIALS AND METHODS = 73 A. DNA construction = 73 B. Cell culture and transfection = 74 C. Isolation of core particles = 74 D. RNase protection analysis(RPA) = 75 E. Northern and Southern Blotting = 75 F. Core particle Western blotting = 76 G. Endogenous polymerase assay(EPA) = 76 Ⅲ. RESULTS = 77 A. Construction of HBV DNA polymerase mutant = 77 B. HBV RNA transcription and core particle formation = 81 C. HBV pgRNA encapsidation = 83 D. HBV DNA replication by polymerase mutants = 85 E. NTP incorporation by dNTP binding cleft mutant polymerase = 87 Ⅳ. DISCUSSION = 90 Ⅴ. CONCLUSION = 93 REFERENCES = 94 국문요약 = 98|LIST OF FIGURES PART 1 Fig. 1. Schematic diagram of HBV replication stages and HBV wild type (wt) and mutants constructs = 13 Fig. 2. HBV RNA transcription and core particle formation = 14 Fig. 3. HBV pgRNA encapsidation = 17 Fig. 4. Southern blot analysis = 18 Fig. 5. Endogenous Polymerase Assay = 19 Fig. 6. Intracellular traffickingof HBV core particles = 24 PART 2 Fig. 1. Schematic diagram of core protein chimera constructs = 51 Fig. 2. HBV RNA expression from HuH 7 cells co-transfected chimeric core protein and C deficient mutant constructs = 52 Fig. 3. Core particle formation from chimeric core protein = 55 Fig. 4. HBV pgRNA encapsidation by chimeric core proteins = 56 Fig. 5. HBV DNA replication by chimeric core proteins = 58 Fig. 6. Summary of results = 59 PART 3 Fig. 1. Alignment of HBV polymerase with DHBV polymerase, HIV-1 and MoMLV reverse transcriptase = 79 Fig. 2. Schematic diagram of HBV wt and mutant constructs used for transient expression = 80 Fig. 3. HBV RNA transcription and core particle formation = 82 Fig. 4. HBV pgRNA encapsidation = 84 Fig. 5. HBV DNA synthesis by mutant polymerase = 86 Fig. 6. Endogenous Polymerase Assay = 89 | - |
| dc.language.iso | eng | - |
| dc.publisher | The Graduate School, Ajou University | - |
| dc.rights | 아주대학교 논문은 저작권에 의해 보호받습니다. | - |
| dc.title | Initial Stages of Hepatitis B Virus Replication | - |
| dc.title.alternative | 초기 단계의 B형 간염 바이러스의 증식 | - |
| dc.type | Thesis | - |
| dc.contributor.affiliation | 아주대학교 일반대학원 | - |
| dc.contributor.alternativeName | Kim, Hee-Young | - |
| dc.contributor.department | 일반대학원 의학과 | - |
| dc.date.awarded | 2005. 8 | - |
| dc.description.degree | Master | - |
| dc.identifier.localId | 564871 | - |
| dc.identifier.url | http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000000645 | - |
| dc.subject.keyword | hepatitis B virus core particle | - |
| dc.subject.keyword | replication stages | - |
| dc.subject.keyword | intracellular trafficking | - |
| dc.subject.keyword | chimeric core protein | - |
| dc.subject.keyword | hepatitis B virus replication | - |
| dc.subject.keyword | HBV polymerase | - |
| dc.subject.keyword | dNTP binding cleft | - |
| dc.subject.keyword | NTP incorporation | - |
| dc.subject.keyword | B형 간염 바이러스의 캡시드 입자 | - |
| dc.subject.keyword | 복제단계 | - |
| dc.subject.keyword | 세포 내 분포 | - |
| dc.subject.keyword | 키메라 캡시드 단백질 | - |
| dc.subject.keyword | B형 간염 바이러스의 복제 | - |
| dc.subject.keyword | HBV DNA 중합효소 | - |
| dc.subject.keyword | dNTP 결합부위 | - |
| dc.subject.keyword | NTP 결합 | - |
| dc.description.alternativeAbstract | PART 3 Reverse Transcriptase Activity of Hepatitis B Virus Polymerase Hepadnavirus DNA polymerase functions DNA synthesis from RNA or DNA template and acts as a primer for minus-strand DNA synthesis. From previous studies, endogenous polymerase activity in priming-deficient mutant core particles was found. This result suggest that the priming-deficient mutant polymerase has the ability to synthesize oligomers (presumably nascent minus-strand DNA) in the absence of covalent linkage between TP and the first deoxynucleotide, which means the primer independent initiation by HBV DNA polymerase. This raises a very important question that HBV DNA polymerase may have RNA polymerase feature. In this study, the RNA polymerase activity of HBV DNA polymerase was explored by testing the NTP incorporation capacities. The bulky amino acid, phenylalanine 436, at supposedly dNTP binding cleft of HBV DNA polymerase was changed to smaller amino acids, glycine or valine. NTPs incorporation capacity of HBV DNA polymerase was tested by endogenous polymerase assay with 32P-labeled ATP and cold dNTPs with isolated core particles. HBV DNA polymerase incorporates NTPs by F436 substitution to glycine which is smaller than valine. This result indicates that bulky amino acid in dNTP binding cleft acts as a steric gate in dNTP selections. Even though authentic DNA synthesis was not detected, it was found that HBV DNA polymerase incorporate NTPs by substituting F436 to G. Taken together, these results indicate that single amino acid substitution can blur property of DNA polymerase in the direction of RNA polymerase | - |
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