2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/37739Human hepatitis delta virus (HDV) has a single-stranded circular RNA genome that replicates by RNA-directed RNA synthesis. The virus encodes only a single protein product, the hepatitis delta antigen (HDAg). The HDAg lacks sequence homology to known RNA polymerases, suggesting that the virus employs a cellular polymerase for HDV replication. It has been proposed that replication of HDV is mediated by RNA polymerase II, a cellular DNA-directed RNA polymerase. Due to the lack of natural infection for in vivo model and a reliable in vitro transcription assay system, the hunt for the human cellular RNA-directed RNA polymerase (RdRP) is hampered. The central dogma of cell biology would be changed if the cellular RdRP could be identified from human cells. We have developed an in vitro RNA/protein (RNP) transfection system that initiates HDV replication without DNA contamination. This RNP transfection system is a cDNA-free system that avoids the possible cross-interaction with DNA directed polymerases. Therefore, we propose to use this RNP transfection system to establish a subtractive hybridization screening for the identification of cellular RdRP machineries from human cells. From this screening, it is likely to identify many transcription factors associated with the RdRP activity. Thus, this three-year research project will be focused on the characterization of the HDV-specific RdRP machineries in order to unmask the mystery of cellular RdRP observed in HDV viral life cycle. The RdRP activity is strongly associated with the pathogenesis of chronic HDV infection.Along the way to our study, there are three major goals that are expected to be accomplished:1. Identification of the cellular RdRP from human cell that responsible for HDV replication.2. Establish the HDV-specific in vitro transcription assay 3. Construction of a novel RNA-based human gene therapy vector for replicating RNA.The clinical application of the HDV-based gene therapy vector is not certain unless the cellular RdRP machineries could be identified. We like to conduct an investigation for the possibility of a chimeric HDV genomic RNA replicates in human cells. The advantage of this RNA-based vector is a replicating gene of RNA with less potential of cellular tranformation ability. Using purified HDAg that associated with in vitro transcribed genomic HDV RNA provides a system to examine the mechanism of cellular encoded HDV RNA synthesis. The commitment of this project would therefore reveal the significance of viral-host interactions through evolution.人類的D型肝炎病毒(HDV)具有一個環狀單股的核酸(RNA)做為它的基因體。因此，使用其RNA為鑄模來做為合成新基因體之用。到目前為止，HDV的基因體中，只有發現一個基因。這個基因的產物就是D型肝炎病毒的抗原(HDAg)。比較HDAg和已經知道的核酸聚合脢的序列，無法找到相似的地方和功能，所以，D型肝炎病毒之所以能夠在動物細胞內複製，一定使用到細胞攜帶的核酸聚合脢(RdRP)。曾經有文獻報告指出，核酸聚合脢的pol II可能是主要的核酸聚合脢用來複製HDV，可是pol II 只能使用DNA為鑄模，如何扭轉它的專一性，還未有任何合理的解釋。由於缺乏適合的模式研究被感染的細胞，以及可靠的試管內定性，使得尋找這個HDV專一的核酸聚合脢無法有效的進展。D型肝炎病毒是唯一的會感染人類而且只在染色體外複製的RNA病毒，而且D型肝炎病毒進入細胞之後的複製和B型肝炎病毒無關。我們知道細胞內未知的核酸複製組合可以複製D型肝炎病毒，但是其中所含有之成分以及它的運作機制並未清楚地被證明出來。我們已經建立了一個使用HDV的RNA/HDAg (RNP)transfection的感染細胞模式，在這個模式中完全避免了可能成為鑄模之HDV的DNA，因此適合使用於鑑定只用RNA為鑄模之核酸聚合脢的組成。為了尋找這個神秘地RdRP，決定使用細胞基因之精簡雜交之差異表達(Subtrac- tive hybridization)來對照出被RNP誘導的基因和被DNA誘導的基因之不同。從這個方法中應該會找到與HDV的複製相關的轉錄因子，進而推衍出真正的RdRP的基因。D型肝炎病毒的核酸聚合脢和D型肝炎的致病性具有重要關係，瞭解它的本質對於治療D型肝炎是有實質的幫助。如果能夠從動物細胞內鑑定出從未被承認過的RdRP將會對於生命科學帶來重大的沖擊。未來，除了確定這個神秘地RdRP的組合成員之外，對於為何只有HDV的RNA序列會被使用於RdRP的活動中也應該加以研究。所以，在本計劃中，我們希望能夠達到下列的主要目標：1. 從人類細胞中鑑識出與RdRP相關的組合成員。2. 建立起一個可靠地、具有HDV的RdRP專一性的試管內合成RNA之方法，以供分析。3. 建造一個可複製的，已經加入一個重組基因之D型肝炎病毒核酸載體。如果我們能夠把HDV之RdRP組合的成員多加以瞭解，並且分析了HDV的RNA在複製時必需的序列，就有可能製造出一個可複製的D型肝炎病毒核酸載體。使用這種RNA核酸載體的最大優點就是造成細胞變形和影響染色體功能的機會大為減少。我們使用試管內合成之D型肝炎病毒RNA和純化的重組型D型肝炎病毒抗原來組成核酸和蛋白質複合體，把這個核酸和蛋白質複合體轉殖到細胞內，可以偵測到D型肝炎病毒開始複製。使用這個方法來檢查隱藏在人類細胞內之只用RNA為鑄模之核酸聚合脢，以及D型肝炎病毒和宿主之間的互相牽制或利用的機制，或許可以揭開長久以來存在人類和病毒之間的一個演化上的答案。人類的D型肝炎病毒啟動子序列病毒複製HDVpromoterreplicationGFPProject