NESI蛋白質參與核輸出路徑之生化特性分析

Biochemical Characterization of NESI Protein Involved in the Nuclear Export Pathway

李雅萍(Ya-Ping Li)

指導教授 ： 張明富

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2005年)

https://doi.org/10.6342/NTU.2005.01131

摘要

D型肝炎病毒 (hepatitis delta virus, HDV) 為B型肝炎病毒的衛星病毒，需要B型肝炎病毒表面抗原組成其外套蛋白質，是一環狀RNA基因體病毒。在病毒顆粒內，delta抗原有兩種：分別是大型delta抗原(HDAg-L; 214個胺基酸)及小型delta抗原(HDAg-S;195個胺基酸)。比較胺基酸序列發現，大型delta抗原在C端比小型delta抗原多出19個胺基酸，大型delta抗原具有與B型肝炎表面抗原組成D型肝炎病毒顆粒的能力，小型delta抗原則為病毒RNA基因體複製所需。
D型肝炎病毒在感染細胞中的核內進行複製，在病毒感染後期，病毒基因體與抗原形成一複合體，進而被輸送出核外，以包裹為完整的病毒顆粒。根據本實驗室研究發現，大型delta抗原C端具有一短序列為proline-rich的核輸出訊號(nuclear export signal; NES)，將之命名為NES(HDAg-L)。此核輸出訊號透過CRM1-independent pathway調控大型D型肝炎抗原之核輸出。此外，一細胞因子NESI 【NES(HDAg-L)-interacting protein】與NES(HDAg-L)之細胞核輸出有關。
分析NESI序列，其第4到第13個胺基酸為可能與actin 結合之區域；而第193到第209個胺基酸具有bipartite nuclear localization signal。本研究利用GST pull-down assay，發現NESI與actin有專一性結合。刪除分析顯示，NESI之N端185個胺基酸即可和大型D型肝炎抗原做專一性結合。另外，本研究製造NESI之專一性抗體，以間接免疫螢光染色法證實內生性NESI位於哺乳動物的細胞核中。這些結果暗示著位於細胞核中的NESI蛋白質與actin之結合能力可能與CRM1-independent 之核輸出路徑有關。

關鍵字

NESI ； 核輸出路徑

並列摘要

Hepatitis delta virus (HDV) is a satellite virus of hepatitis B virus and requires hepatitis B virus surface antigens for the production of virus particles. HDV contains a circular RNA genome. Inside HDV particles, there are two distinct delta antigens, the small form (HDAg-S; 195 a.a.) and the large form (HDAg-L; 214 a.a.). Both HDAgs are identical in the N-terminal region, but the large form has extra 19 amino acid residues at the C-terminus. HDAg-S is essential for the viral RNA replication, whereas HDAg-L is required for virus assembly.
HDV replicates in the nucleus. In the late stage of the viral life cycle, the RNA genome is associated with delta antigens as ribonucleoprotein (RNP) complexes that are exported from the nucleus to the cytoplasm for assembly. Our laboratory has previously identified a proline-rich nuclear export signal (NES) located at the C-terminus of HDAg-L, designated NES(HDAg-L). The NES(HDAg-L) mediates the nuclear export of HDAg-L via a chromosome region maintenance 1 (CRM1)-independent pathway. In addition, a cellular factor NES(HDAg-L)-interacting protein (NESI) was identified to be involved in the nuclear export of HDAg-L and the assembly of HDV.
Sequence analysis of NESI revealed a putative actin-binding site from amino acid residues 4 to 13 and a bipartite nuclear localization signal from amino acid residues 193 to 209. In this study, the specific interaction between NESI and actin was demonstrated by GST pull-down assay. Deletion analysis indicated that the N-terminal 185 amino acid residues of NESI were sufficient to interact with HDAg-L. In addition, antibodies specific to the N-terminal 185 amino acid residues of NESI were generated. Indirect immunofluorescence staining with the NESI-specific antibodies demonstrated that endogenous NESI is localized in the nucleus of mammalian culture cells. These results suggest that the actin-binding activity of the nucleus-localized NESI protein may be involved in the CRM1-independent nuclear export pathway.

並列關鍵字

NESI ； nucelar export pathway

參考文獻

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