B형 간염 바이러스 유전자형 C의 역전사효소 내 rtL269I 변이주의 제 1형 인터페론 신호전달을 통한 HBeAg 음성 감염 및 간질환 악화기전에 관한 연구

학위논문(박사)--서울대학교 대학원 :의과대학 의학과,2020. 2. 국윤호.Background and Aims: Hepatitis B virus infection is a serious global health problem and causes life-threatening liver disease. In particular, genotype C shows high prevalence and sever liver disease compared with other genotypes. However, the underlying mechanisms regarding virological traits still remain unclear. This study investigated the clinical factors and capacity to modulate Type I interferon (IFN-I) between two HBV polymerase polymorphisms rt269L and rt269I in genotype C. Methods: The clinical factors between rt269L and rt269I in 220 Korean chronic patients with genotype C2 infections were compared. The prevalence of preC mutations between rt269L and rt269I was compared using this cohort study and the GenBank database. For in vitro study, plasmid DNA encoding HBV genome were transiently transfected into hepatocytes, and HBV virions were infected using HepG2-hNTCP-C4 and HepaRG systems. In addition, hydrodynamic injection of HBV genome into mice tail were conducted for in vivo experiments. Results: The clinical cohort study indicated that rt269I was related to HBV e antigen (HBeAg) negative serostatus, lower levels of HBV DNA and HBsAg, and disease progression compared to rt269L. Furthermore, the epidemiological study showed that HBeAg negative infections of rt269I were attributed to a higher frequency of preC mutations at 1896 (G to A). In vitro and in vivo study also suggested that rt269I caused mitochondrial stress mediated STING dependent IFN-I production, resulting in decreasing HBV replication via the induction of heme-oxygenase-1. In addition, I also found that rt269I enhanced iNOS mediated NO production in an IFN-I dependent manner. Conclusion: In this study, I found that there are two polymorphisms at polymerase RT region, rt269L and rt269I, in patients infected with genotype C, which is only found in genotype C. These data demonstrated that rt269I can contribute to HBeAg negative infections and liver disease progression in chronic patients with genotype C infections via mitochondrial stress mediated IFN-I production.B형 간염 바이러스는 세계적으로 심각한 감염성 질환을 유발하며, 특히 B형 간염 바이러스에 의한 만성 감염은 생명을 위협하는 간질환을 일으킨다. 그 중 유전자형 C는 다른 유전자형에 비해 높은 병원성, 항바이러스제제 치료의 어려움, 인터페론 알파 치료의 저항성 및 간질환 악화의 급속한 진행 등의 특성이 관찰되었다. 이러한 특성은 유전자형 C에서만 발견되는 여러 유전자 변이주 및 유전자 다형성에 기인할 가능성을 제시하는 연구가 보고되었으나, 특성과 관련된 근본적인 기전은 여전히 분명하게 밝혀진 바 없다. 본 연구에서는 유전자형 C에서 HBV 중합효소 내 역전사 효소rt269L부위의 두가지 다형성 (rt269L과 rt269I) 사이의 제 1형 인터페론 (IFN-I)을 조절하는 특성과 감염 환자의 임상적 요인을 확인하였다. 이를 위하여 국내 유전자형 C rt269L과 rt269I에 감염된 환자 220명 사이의 임상적 요인을 비교하였으며, 감염환자 및 GenBank 데이터 베이스를 이용하여 두 다형성 사이의 preC 돌연변이 빈도를 분석하였다. 또한 임상적 특성의 원인을 규명하기 위하여 C57BL/6 마우스 및 간암세포 모델에서 감염시킨 후 바이러스 복제 양상, 제 1형 인터페론 신호 기전 및 미토콘드리아 스트레스, 활성질소 등을 비교 관찰하였다. 본 연구의 임상 데이터를 통하여rt269I 변이주가 rt269L에 비하여 혈청 내 외피항원 (HBeAg) 음성감염, 낮은 수준의 HBV DNA와 표면항원 (HBsAg), 그리고 질병의 악화를 야기하는 것을 확인하였다. 이러한rt269I 감염에 의한 외피항원 음성감염은 preC 내 1896번째 뉴클레오티드가 구아닌에서 아데노신으로 돌연변이가 생겨 일어나는 것임을 알 수 있었다. 또한 시험관 내 및 생체 내 연구에서는 rt269I가 미토콘드리아 스트레스 매개 제 1형 인터페론 생성을 유도하여 힘옥시게나제 (heme oxygenase)-1의 발현을 증가시키고, 그 결과 HBV 복제를 감소시킬 수 있다는 것을 확인하였다. 또한, rt269I 변이주가 제 1형 인터페론 의존적인 방식으로 산화질소활성효소 (inducible nitric oxide synthase) 매개 활성질소 생산을 향상시킬 수 있다는 것을 발견하였다. 이 연구를 통해 HBV 중합효소 내 역전사효소 부위에는 유전자형 C에서만 발견되는 rt269L과 rt269I 두가지 다형성이 있음을 확인하였다. 뿐만 아니라, 이 연구의 결과는 rt269I 변이주가 미토콘드리아 스트레스 매개를 통해 제 1형 인터페론을 생산하고, 이 기전이 만성감염 환자에서 외피항원 음성감염과 간 질환 악화에 영향을 줄 수 있음을 알 수 있었다.CONTENTS INTRODUCTION .. 1 MATERIALS AND METHODS 8 1. Patients 8 2. HBV DNA extraction and PCR amplification for polymerase RT region 8 3. HBV genotyping 8 4. Plasmid and site-directed mutagenesis 9 5. Cell culture and transfection 9 6. In vivo and hydrodynamic injection 10 7. Enzyme-linked immunosorbent assay 10 8. Covalently closed circular DNA extraction and real-time polymerase chain reaction 11 9. Total RNA extraction and real-time polymerase chain reaction 13 10. IFN-I luciferase reporter assay 13 11. IFN-I signal block assay 13 12. Preparation of HBV from transiently transfected cells and Infection assay 14 13. Flow cytometry and Confocal analysis 14 14. 8-OHdG ELISA assay 14 15. Measurement of NO2- and NO3- levels 15 16. Statistical Analyses 15 RESULTS 16 1. rt269I was related to enhanced disease progression in a Korean cohort with genotype C infections. 16 2. The higher frequency of preC mutation (G1896A) in patients with rt269I infections was responsible for the higher frequency of HBeAg negative infections. 21 3. rt269I led to lower levels of HBV replication in in vitro and in vivo experiments. 23 4. Hepatocytes activated IFN-I signaling against rt269I infection. 28 5. The reduced replication capacity and enhanced IFN-I expression of rt269I were also proved in two HBV infection models. 37 6. The replication of rt269I was inhibited via STING- IFN-I axis. 41 7. rt269I enhanced mitochondrial stress mediated IFN-I production and heme oxygenase-1. 44 8. rt269I enhanced iNOS dependent NO production. 52 DISCUSSION 58 REFERENCES 65 국문초록 74 LIST OF TABLES Table 1. Definition of HBV mutation and polymorphism at reverse transcriptase based on database and cohort data 7 Table 2. PCR primers used for this study. 12 Table 3. Comparison of the clinical features between patients infected with the two types of genotype C, rt269L and rt269I. 20 Table 4. The frequencies of BCP and preC mutations between rt269L and rt269I in a Korean cohort and from reference strains. 22 LIST OF FIGURES Figure 1. Phylogenetic analyses of 1,032-bp polymerase RT sequences showed that the representative 20 patients used in cohort study belonged to genotype C. 18 Figure 2. The viral replication of rt269I was restricted in vivo infection model. 24 Figure 3. rt269I reduced viral replication in vitro analyses. 25 Figure 4. The viral replication of rt269I was reduced viral replication in vitro transient transfection system. 26 Figure 5. rt269I enhanced IFN-I production in in vivo assay. 30 Figure 6. Hepatocytes increased transcription level of IFN-I against rt269I infection. 32 Figure 7. HepG2 cells activated IFN-I signaling pathway and produced IFN-I against rt269I. 34 Figure 8. IFN-I production was enhanced in hepatocytes infected with rt269I. 36 Figure 9. The reduced replication capacity of rt269I was shown in HBV infection models. . 39 Figure 10. The replication inhibition found in rt269L was mediated via STING-IFN-I axis 42 Figure 11. HepG2 cells enhanced HO-1 in transcription and translation level against rt269I infection. 46 Figure 12. rt269I induced mitochondrial reactive oxygen species production. 48 Figure 13. rt269I caused mitochondrial stress in infected hepatocytes. 50 Figure 14. rt269I enhanced iNOS dependent NO production. 54 Figure 15. rt269I enhanced iNOS dependent NO production in IFN-I dependent manner. 56 Figure 16. Schematic presentation indicating distinct mitochondrial stress mediated IFN-I production and its distinct contribution to disease progression in chronic patients with genotype C infections between rt269L and rt269I. 63Docto

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