G. The Core particle formation in chimeric DNA polymerase constructs
V. CONCLUSION
Hepatitis B virus (HBV) is a human pathogen and a causative agent of serious global health problem. HBV DNA polymerase, a multifunctional protein, has four domains, TP, spacer, RT, and RNaseH. Since HBV DNA polymerase is very unstable and rapidly degradable, it has been very difficult to study the biochemical aspect of it.
To identify the critical domains or motifs of HBV DNA polymerase for HBV replication, the approaches using chimeric viruses have employed in this study. To accomplish this goal, polyclonal antiserum against each domains of DNA polymerase were generated from rabbit by immunizing the expressed fusion proteins from E. coli.
The distribution of HBV polymerase was analyzed first to evaluate the specificity of polyclonal antiserum against DNA polymerase. TP specific antiserum found to be useful and used in this study since HBV DNA polymerases were detected in the polymerase construct or HBV wt transfected cells using this antiserum. HBV polymerase was exclusively localized in the cytoplasm and was not co-localized with ER, Golgi, and peroxisome. After the alignment of amino acid sequences of HBV and DHBV DNA polymerases, the TP, RT, and RNase H domains of hepadnavirus polymerase were subdivided into N- and C-terminus for the constructions of chimeric DNA polymerases. Then series of chimeric DNA polymerases were constructed and replication rescue of P deficient mutant by chimeric DNA polymerases was tested. Unfortunately, all of the chimeras could not complement the HBV DNA polymerase since encapsidation of pgRNA and DNA synthesis were not
47
observed. To examine why these chimeras cannot rescue the HBV replication, RNA expressions of chimeras were examined. All of the chimeras, except DRN that has DHBV RT N-terminal sequence, expressed chimeric RNA, and 2.4 and 2.1 Kbp of surface mRNAs. Since RNA expressions were confirmed, chimeric protein expressions were tested by IFA. The expressions of chimeric DNA polymerase proteins were not detected by IFA. However when P deficient mutant and chimeras were co-transfected, there were significant differences in core particle formations.
Since P deficient mutant can form core particles without polymerase, it can be postulated that RNAs from chimeras might influence the core particle formations.
48
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56 - 국문요약 –
B형
형형 간염형 간염간염 바이러스간염 바이러스바이러스바이러스 DNA 중합효소의중합효소의중합효소의 발현중합효소의 발현발현발현 및및및 기능분석및 기능분석기능분석 기능분석(사람과
사람과사람과사람과 오리의오리의오리의오리의 chimeric B형형형 간염형 간염간염 바이러스간염 바이러스바이러스바이러스DNA 중합효소의
중합효소의중합효소의중합효소의 발현발현발현) 발현아주대학교 대학원 의학과 박 길 순
(지도교수: 김 경 민)
목적목적
목적목적: B형 간염 바이러스는 세계적으로 주요한 pathogen중 하나로 그들의 복제 기전과 구성물의 정확한 생물학적 역할을 이해하는 것은 중요하다. Chimeric DNA 중합효소를 이용하여 B형 간염 바이러스 DNA 중합효소의 어느 부위가 복 제에 중요한지를 알아보고자 하였다.
재료 재료 재료
재료 및및및 방법및 방법방법방법: 사람 B형 간염 바이러스와 오리 B형 간염 바이러스 DNA 중합 효소의 아미노산 서열을 alignment하여 상호적으로 교체할 부위를 결정하여 in frame으로 사람 B형 간염 바이러스 DNA 중합효소에 상응하는 오리 B형 간염 바이러스 부위를 cloning 하였다. 부위별 기능 분석을 위해 사람 B형 간염 바이 러스 DNA 중합효소의 각 domain을 박테리아에서 발현시켜 토끼에 주사하여 polyclonal 항체를 얻었다. 우선 얻은 항체로 wt 사람 B형 간염 바이러스 DNA
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중합효소의 세포네 발현과 위치를 immunofluoresence assay를 이용하여 알아보 았다. Chimeric DNA 중합효소가 B형 간염 바이러스의 복제와 encapsidation을 재개 할 수 있는지 endogenous polymerase assay와 encapsidation assay를 수 행 했으며 chimeric DNA 중합효소 RNA와 단백질의 발현은 Northern blot analysis와 immunofluoresence assay를 통해 알아 보았다. 또한 chimeric DNA 중합효소의 발현이 B형 간염 바이러스 core particle 형성에 미치는 영향을 알아 보기 위해 core particle western blot analysis를 시행하였다.
결과 결과결과
결과: B형 간염 바이러스 DNA 중합효소의 TP (terminal protein) 부위에 특이적 인 항체를 얻었으며 이 항체를 이용하여 B형 간염 바이러스 DNA 중합효소의 세포네 위치가 cytoplasm 전반에 걸쳐서 존재 하였으며 endoplasmic reticulum, Golgi 그리고 peroxisome에 분포하지 않음을 알 수 있었고 복제를 수행하고 있 는 세포에서도 B형 간염 바이러스 DNA 중합효소와 B형 간염 바이러스의 spliced 단백질을 확인 할 수 있었다. 그러나 chimeric DNA 중합효소 construct 는 단백질로서 발현하지 않아 B형 간염 바이러스 복제와 encapsidation이 일어 나지 않았다. Chimeric DNA 중합효소 RNA는 core particle 형성에 영향을 주는 것으로 확인 되었다.
결론 결론결론
결론: 이 연구에서 제작된 TP 특이적 항체는 B형 간염 바이러스 DNA 중합효소 연구에 폭 넓게 이용될 수 있음을 보여 주었고 비록 chimeric DNA 중합효소가 B형 간염 바이러스 복제나 encapsidation을 재개 할 수는 없었지만 chimeric DNA 중합효소 RNA의 primary sequence가 B형 간염 바이러스 생활사중 core
58
particle의 형성에 영향을 미치는 결과는 이 바이러스 genome의 선천적 복합성 을 시사하고 이 부분에 대한 앞으로의 연구가 필요하다는 것을 알 수 있었다.
핵심되는 말: Polyclonal 항체, Chimeric DNA 중합효소, 복제, core particle formation
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PART II
Down-regulation of hepatitis B virus
replication by splicing event
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I. INTRODUCTION
Hepatitis B viruses (HBVs) are prototype member of hepadnaviridae family.
The viruses have relaxed circular, partially double stranded DNA genome with approximate 3.2 Kbp in size. The intracellular replication cycle of hepadnavirus involve in reverse transcription of an RNA pregenome (Summers and Mason, 1982).
Since they contain DNA genome in extracellular virus particles, they referred as pararetroviruses in comparison with retroviruses that contain RNA genome in virus particles. Among these viruses, mammalian hepadnaviruses, plant caulimoviruses and plant badnaviruses share the unique property of genome replication using reverse transcription (Rothnie et al., 1994).
The genome organizations of hepadnavirus and retrovirus are very similar.
Because RNA pregenome of hepadnavirus and RNA genome of retrovirus have multifunction such that their genomes also served as mRNAs for the capsid protein and RT, the polymerase, various specific regulatory mechanisms have evolved by producing polyprotein, by using subgenomic promoters, by adopting various ways of polycistronic translation, and by partial splicing to increase the number translation product, the number of mRNAs and/or the number of translation initiation sites per mRNA (Futterer and Hohn, 1996).
Another common property of these two viruses is that both have various spliced transcripts. For retroviruses, RNA splicing mechanisms are essential for the expression of the env-genes. Some retroviruses, such as HIV-1, produce several
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spliced RNAs to encode regulatory proteins which are either essential or modulating for the viral replication (Vaishnav and Wong-Staal, 1991). Even though all necessary viral proteins for hepadnavirus replication are translated from unspliced transcripts, spliced transcripts of HBV have been identified recently from HBV transfected cell lines, HBV-transgenic mice, and HBV-infected liver (Chen et al., 1989; Su et al., 1989; Choo et al., 1991; Kajino et al., 1991); (Wu et al., 1991) and from liver tissues of hepatocellular carcinoma patients (Lin et al., 2002). For HIV, the Rev regulate spliced and unspliced mRNA ratio post-transcriptionally (Emerman et al., 1989;
Hadzopoulou-Cladaras et al., 1989); (Malim et al., 1989). Because HBV RNAs have post-transcriptional regulatory element (PRE) that seems to be functionally similar to Rev-responsive element (RRE) of HIV, the synthesis of spliced and unspliced HBV transcripts seems to be reminiscent of the situation in HIV suggesting that the spliced transcript of HBV may play a role in the life cycle. It has been reported that the presence of spliced RNAs may be closely associated with the chronic course of HBV infection and viral multiplication in vivo (Rosmorduc et al., 1995). On the contrary, it has been reported that spliced RNAs have no effect on viral replication in transfected hepatoma cell lines (Su et al., 1989; Wu et al., 1991). However, recent study have reported that the 2.2Kbp HBV spliced RNA in tumor tissues could enhance the replication of full-length HBV genome (Lin et al., 2002). In fact, it has been reported that spliced mRNAs from genomic RNA of the DHBV are required for DHBV replication even though these spliced RNAs are not encapsidated and reverse transcribed due to the deletion of encapsidation signal through splicing (Obert et al.,
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1996). In other cases, spliced viral RNAs were packaged and reverse transcribed into the HBV particles (Terre et al., 1991; Davis et al., 2000) through the Arg-rich domain from 165 to 173 residues of HBV core protein (Le Pogam et al., 2005).
Also it has been reported that spliced HBV transcripts are expressed as new HBV proteins. From many spliced transcripts, two proteins were translated. One is an HBV splice-generated protein (HBSP) (Ducharme et al., 2000). The presences of HBSP and anti-HBSP antibodies are not related for viral replication and liver fibrosis in vivo (Ducharme et al., 2000). The meaning of the presence of HBSP in vitro are
not clear because HBSP may have affected the viral maturation or secretion (Ducharme et al., 2000). These reports suggested that HBSP proteins might be implicated on viral persistence. Another report demonstrated that a polymerase-surface fusion protein encoded by a spliced RNA are able to substitute the large surface (L) protein of Dane particles, the HBV virions, and the 22 nm subviral particles (Huang et al., 2000).
In this study, the importance of the spliced RNA or protein in the HBV transfected cells was investigated. Several spliced RNAs and polymerase-surface (PS) protein product from a spliced RNA were detected in HBV polymerase expressing cells. This PS protein was distributed in ER and nuclear pore complex (NPC) and also associated with microtubule and intermediate filament. The secretions of subviral particles as measured by surface protein antigen ELISA, the HBV DNA synthesis, and the formation of core particle were drastically inhibited by the expression of the PS construct. This phenomenon was attributed to the PS
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primary RNA sequence and the down-regulation to the expression of pgRNA. It suggested that the expression of the PS construct might modulate the HBV life cycles.
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