2010-08-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/702962摘要：在真核細胞內發生的各種事件及病毒的生活史中，以細胞因子作為媒介進行細胞質與核間之物質輸送是相當重要的。對於具有富含白氨酸核輸出訊息之蛋白質而言，CRM1 是其核輸送過程中的典型受器。然而，目前陸續有不同型式的核輸出訊息及路徑被發現。D 型肝炎病毒為負股之RNA 病毒，但不同於一般的RNA 病毒，D 型肝炎病毒並不表現RNA 依賴性RNA 聚合酶。在感染早期，D 型肝炎病毒在核中進行轉錄，以宿主第二型DNA 依賴性RNA 聚合酶合成小型D 型肝炎抗原之mRNA。在感染晚期，宿主細胞質中以病毒反股基因體經編輯修飾後得到之mRNA 模板合成病毒組合所需的大型D 型肝炎抗原。位於大型D 型肝炎抗原C 端之色氨酸和isoprenylation 的修飾對於其與B 型表面抗原結合及病毒組合是重要的。小型及大型D 型肝炎抗原皆具有核位訊息，當B 型表面抗原不存在時主要位於核中。然而，當B 型表面抗原存在時，大型D 型肝炎抗原會同時分布於細胞核和細胞質中。我們先前發現大型D 型肝炎抗原之C 端有一個富含脯氨酸之核輸出訊息，大型D 型肝炎抗原藉此核輸出訊息經由CRM1 非依賴性路徑進行核輸出。最近我們進一步找到可與大型D 型肝炎抗原之核輸出訊息結合的細胞蛋白質NESI。NESI 是D 型肝炎病毒RNA 藉由大型D 型肝炎抗原為媒介進行核輸出時所必須。我們相信NESI 也參與宿主蛋白質核輸出的過程。然而，大型D 型肝炎抗原及宿主蛋白質如何經由NESI 及細胞內之其它系統當媒介，以進行核輸出，則不甚明瞭。本研究計畫之特定目標說明如下:1. 進行NESI 的肌動蛋白質結合區及其他次功能區參與大型D 型肝炎抗原核輸出之功能分析，2. 分析CAS、lamin A/C、RanGTP 及核孔蛋白質在大型D 型肝炎抗原核輸出上可能的功能，3. 決定HDV 不同基因型之大型D 型肝炎抗原在進行核輸出時所需之胺基酸序列，並分析其在作用機制上可能的差異性，4. 鑑定經由NESI 路徑進行核輸出的宿主蛋白質。<br> Abstract: Cellular factor-mediated nucleocytoplasmic transport is critical for diverse cellularevents in eukaryotes and the life cycle of viruses. Chromosome region maintenance 1 (CRM1)is a classical receptor for the transport of proteins with nuclear export signal bearingleucine-rich sequence. However, alternative nuclear export signals and nucleocytoplasmictransport pathways are beginning to emerge. Hepatitis delta virus (HDV) is a negative-strandRNA virus. However, different from most RNA viruses, HDV does not encode its ownRNA-dependent RNA polymerase. In the early infection, HDV transcription takes place in thenucleus of host cells and the viral mRNA that encodes the small delta antigen (HDAg-S) issynthesized by the host DNA-dependent RNA polymerase II. In the late infection, large deltaantigen (HDAg-L) that is required for the viral assembly is synthesized from a modifiedtemplate resulting from RNA editing of the viral antigenomic strand. Tryptophan residues andisoprenylation modification at the unique C terminus of HDAg-L are important for theinteraction with HBsAg and the assembly of HDV in the cytoplasm of host cells. BothHDAg-S and HDAg-L possess nuclear localization signals and are mainly localized to thenucleus in the absence of HBsAg. Nevertheless, in the presence of small HBsAg, HDAg-Llocalizes to both the nucleus and the cytoplasm. We have identified a nuclear export signal(NES) with proline-rich sequence at the C terminus of HDAg-L. HDAg-L transports from thenucleus to the cytoplasm through CRM1-independent pathway. A cellular protein, NESI, wasidentified that specifically interacts with the NES of HDAg-L and is essential for theHDAg-L-mediated nuclear export of HDV RNA. We believe that NESI is also involved in thenuclear transport of host proteins. However, how the nuclear export of HDAg-L and hostproteins is mediated by the NESI protein and additional cellular machinery is not clear.Specific aims of this study are described as follows.1. Functional analysis of the actin-binding and other subdomains of NESI that are involvedin the nuclear export of HDAg-L,2. Analysis of the possible functions of CAS, lamin A/C, Ran-GTP, and nucleoporins in thenuclear export of HDAg-L,3. Determination of sequence requirements of HDAg-L for nuclear export in various HDVgenotypes and analysis of potential differences in the control mechanisms,4. Identification of host proteins that undergo NESI-dependent nuclear export.