Characterization of delta antigen and RB protein involved in the replication of hepatitis delta virus

• Main Authors: Lin, Ming-Der, 林明德
• Other Authors: Chang Ming-Fu
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/34282090006720778294

碩士 === 國立臺灣大學 === 生化學研究所 === 86 === D 型肝炎病毒是一種衛星病毒，它必須在B型肝炎病毒同時存在下才能感染宿主 。D88肝炎病毒顆粒的外套是由B型肝炎病毒表面抗原所形成。其基因體屬於一條單 股、環狀、長度約為一千七百個鹼基的RNA結構。Delta抗原是目前被確認唯一由 D型肝炎病毒所轉譯出的蛋白質。D型肝炎病毒的訊息RNA能夠轉譯出小型(24 kDa) delta抗原，而當其負股RNA基因體經由RNA editing之後，其訊息RNA就能夠轉譯出 大型(27 kDa) delta抗原。目前已知，在培養細胞內小型delta抗原為病毒複製所 需而大型delta抗原則有抑制病毒複製的作用以及為病毒包裹時所需。D型肝炎病毒 基因體整個複製的過程，至今仍然不是很清楚。目前認為病毒複製時可能是利用宿 主細胞的RNA polymerase II-like activity，但是否有其他細胞因子參與其中並 不清楚。 先前，本實驗室利用活體外共同轉譯系統所得之RB蛋白質(retinoblastoma gene product)與小型delta抗原進行共同免疫沈澱，證實小型delta抗原可以和 RB蛋白質結合。並在共同轉染的細胞培養實驗中，證明RB蛋白質可以促進病毒的 複製。本論文的第一部份將針對RB蛋白質與delta抗原的交互作用，進行更進一步 的探討。首先構築可表現glutathione S-transferase (GST)與D型肝炎病毒小型 delta抗原的融合蛋白質，並以此融合蛋白質以GST pull-down assay 證明RB蛋白 質與小型delta抗原在此分析系統下的確有交互作用。然後，分別構築RB蛋白質與 小型delta抗原的各種刪除式變異蛋白質，再進行GST pull-down assay。實驗結 果顯示RB蛋白質可能利用其A、B pockets或者其C端的部份與小型delta抗原結合， 而小型delta抗原的C端與RB蛋白質的結合無關。 本論文的第二個部份，則以另一個角度來探討D型肝炎的複製機轉，主要是利 用免疫螢光染色法和螢光原位雜交法觀察在病毒進行複製的轉染細胞內，delta抗 原與病毒RNA基因體的分佈狀況，試圖對D型肝炎病毒的複製場所作進一步的探討 ，並期望能夠對病毒的複製機轉有更進一步的認識。目前的實驗結果顯示，在複製 進行開始之後delta抗原會由核仁重新分布至核質，其位置緊鄰non-snRNP splicing factor SC35，且極有可能是在PF (perichromatin fibrils)的位置。 為了證明這種delta抗原重新分佈的現象的確和病毒的複製有關，於是以 a-amanitin處理細胞，結果發現經由a-amanitin處理過的細胞其delta抗原的分佈 與未處理過的細胞有所不同。由於a-amanitin在in vitro的實驗中能夠抑制D型肝 炎病毒的複製，而且在培養細胞內小型delta抗原對於D型肝炎病毒的複製又是必要 的，故這個結果間接的證明了delta抗原的重新分佈與D型肝炎病毒的複製有關。 為了更進一步的證明，於是利用螢光原位雜交法觀察複製中的病毒RNA之分佈。實 驗結果顯示複製中的病毒其RNA在細胞核內的分佈與delta抗原相同，這個結果更加 強了PF可能是病毒複製場所的假說。 Hepatitis delta virus (HDV) is a human pathogen that can greatly increase the severity of liver damage caused by an infection of hepatitis B virus (HBV).HDV is a satellite virus of HBV because co-infection or superinfection with HBV is required for the productive infection and transmission of HDV. The gemone of HDV is a single-stranded circular RNA of about 1,700 nt that contains more than 70 % self-complementarity; as a result, the HDV genme forms a highly base paired rod-like structure. The HDV antigemonic RNA contains an open reading frame, which encodes the small from of delta antigen(HDAg). The small HDAg (24 kDa) is indispensable for HDV genomic replication in cultured cells. Whereas, the large form of HDAg (27 kDa) is derived by RNA editing, it represses the viral replication and is required for virion assembly. The detail mechanism of HDV replication is unclear. HDV may undergo replication through the double rolling circle mechanism in which cellular factors including an RNA polymerase II-like enzyme activity may be involved. In our previous studies, RB protein was demonstrated to support the replication of HDV and it interacted with the small HDAg in in vitro coimmunoprecipitation experiments. In the first part of this study, GST pull-down assay was used to analyze the interaction domains between RB protein and the small HDAg. Results demonstrated that the C-terminal region of the small HDAg is not required for its interaction with RB protein and the A, B pockets or C-terminal region of the RB protein may be responsible for its interactoin with the small HDAg. In the second part of this study, indirect immunofluorescence staining and fluorescence in situ hybridization following transfection were applied to analysis intranuclear distributions of HDAg and HDV genomic RNA during viral replication. Results from immunofluorescence staining indicated that HDAg translocated from nucleolus to nucleoplasm during viral replication. The location of HDAg in HDV replicating Huh7 cells was next to SC35, a non-snRNP splicing factor, speckles in nucleoplasm, possibly at perichromatin fibrils where the RNA polymerase II transcription takes place. When HDV replicating Huh7 cells were treated with (-amanitin, HDAg redistributed to sites of nucleoplasm other than the perichromatin fibrils. The in situ hybridization experiments demonstrated that HDV RNA colocalized with HDAg in the nucleoplasm of transfected Huh7 cells during viral replication. Thus, the localization of HDAg in the nucleoplasm of the HDV replicating Huh7 cells seems to be the site of the viral replication.