2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/37787HDV has been studied since 1977, and all the evidences suggest that the life cycle of HDV is tightly incorporated with mammalian host cells. Although it is a very small RNA virus, it is inheriting an unknown ability to mediate the host genes to synthesize the RNA genome of HDV in infected cells. This RNA-dependent RNA synthesis machinery has only been isolated from other RNA viruses and plants, but not from the cells with HDV infection. The mechanisms of HDV replication have been studied extensively with most of them using the cDNA plasmid of HDV, but the uncertainty of interference by the DNA-dependent polymerase II has raised certain concern. We have successfully applied a RNP and a mRNA cotransfection methods for studying the mechanism of HDV replication without the cDNA interference. Therefore, we propose to identify and characterize the cis- and trans-elements that are required for HDV replication in vivo with two cDNA-free transfection methods and in vitro with HDV-specific nuclear extract transcription assay. In this five-year project, we will focus at the following targets with a time-table sequence. 1. Identification of the minimal-length of the HDV RNA genome sequence that is essential for HDV replication in Cos7 cells with deletion analysis. 2. Characterization of the RNA promoter sequence by site-directed mutagenesis in vivo. 3. Establishment of the in vitro RNA synthesis system for HDV-specific transcription with nuclear extract. 4. Characterization of the HDV RNA promoter and cellular protein interactions by UV-crosslinking assay. 5. Characterization of the HDV associated cellular RNA polymerase activity.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。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。包括HDV之核酸啟動子及細胞之調節基因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。D型肝炎病毒複製機制HDVreplicationcDNA-freeProject