D型肝炎病毒蛋白之轉譯後修飾對於調節病毒核酸複製之研究

Abstract

D 型肝炎病毒利用宿主細胞的核醣核酸聚合酶來執行其核醣核酸基因組之複製。然而截至目前為止，我們尚不清楚其確切的複製機轉。以前的研究報告顯示，小型D 型肝炎病毒蛋白的蛋白質轉譯後修飾於D 型肝炎病毒核酸複製中扮演著重要角色。其中，小型D 型肝炎病毒蛋白的S-177 磷酸化、K-72 乙醯化及R-13 甲基化已被證明會影響病毒基因股核醣核酸的生成而不影響其反基因股核醣核酸的生成。在本篇論文的前半部，我們探討了小型D 型肝炎病毒蛋白的轉譯後修飾對於 D 型肝炎病毒蛋白的訊息核醣核酸生成之影響。我們的研究證明小型D 型肝炎病毒蛋白的S-177 磷酸化、K-72 乙醯化及R-13 甲基化亦會影響病毒蛋白訊息核醣核酸的生成。於本篇論文的後半部，我們探討了小型D 型肝炎病毒蛋白的SUMO 修飾(sumoylation)及其對於D 型肝炎病毒核醣核酸複製之影響。我們證明小型D 型肝炎病毒蛋白可被SUMO1 蛋白所修飾。利用融合蛋白之方式，我們發現小型D型肝炎病毒蛋白與SUMO1 蛋白的融合會增加D 型肝炎病毒基因股核醣核酸及病 毒蛋白訊息核醣核酸的生成，但卻不影響病毒的反基因股核醣核酸的生成。綜合上述，我們的研究結果支持D 型肝炎病毒基因股核醣核酸及病毒蛋白訊息核醣核酸的生成與其反基因股核醣核酸的生成乃由不同的核醣核酸複製機轉所執行之論點。

Hepatitis delta virus (HDV) contains a viroid-like, 1.7-kb circular RNA genome, which replicates via a double rolling circle model. However, the exact mechanism involved in HDV genome RNA replication and subgenomic mRNA transcription is still unclear. Previously, it has been shown that post-translational modifications of S-HDAg, such as phosphorylation, acetylation and methylation, can modulate HDV RNA replication. In the first part of this study (see Section 1), we focused on the issue of how these modifications affect the HDV mRNA transcription. Using a unique qRT-PCR procedure and a series of HDV mutants, we demonstrated that Arg-13 methylation, Lys-72 acetylation, and Ser-177 phosphorylation of S-HDAg are required for the HDV mRNA transcription. The results of this part of study have been published in the Journal of Virology (J Virol 2008; 82:9409-9416). In the second part of this study (see Section 2), we demonstrated that S-HDAg is a small ubiquitin-like modifier-1 (SUMO1) target protein. Mapping data showed that multiple lysine residues are SUMO1 acceptors within S-HDAg. Using a genetic fusion strategy, we found that conjugation of SUMO1 to S-HDAg selectively enhances HDV genomic RNA and mRNA synthesis but not antigenomic RNA synthesis. The results of this part of study have also been submitted for publication (manuscript in revision). Collectively, these results add to the growing list of different metabolic requirements between the HDV genomic RNA/mRNA synthesis and the antigenomic RNA synthesis (see Section 3, Table 3-1, p76) and support the hypothesis that the cellular machinery involved in the synthesis of HDV antigenomic RNA is different from that of genomic RNA synthesis and mRNA transcription (see Section 3, Fig. 3-3, p79). Finally, in Section 3 of this dissertation, the recent advances in the understanding of HDV RNA replication are reviewed.