D型肝炎病毒抗原N端區域與核酸分子交互作用之研究

NdAg為含D型肝炎病毒抗原第1至第88個胺基酸之蛋白，其可與各式核糖核酸結合，並具有核醣核酸監護子的活性，可協助各式核醣核酸摺疊成新的構型。NdAg序列第2至第27個胺基酸可與HDV RNA結合，第13至48個胺基酸含leucine-zipper like序列，另第67至88個胺基酸為核位訊息。本研究欲深入了解NdAg上各個區域對蛋白結構、RNA結合能力及RNA 監護子活性的重要性。我們構築各式NdAg蛋白的突變體利用雙旋光分光光譜 (CD) 分析蛋白結構，再利用filter binding assay偵測蛋白的RNA結合力，同時由蛋白促進hammerhead ribozyme它割反應的能力評估其 RNA監護子活性。NdAg具有a-螺旋結構，刪除NdAg第2至第13或第60至第88個胺基酸，對此結構無影響。但若於leucine-zipper like序列上刪除第14至第23或第35至第43個胺基酸，或於第27-28個胺基酸間插入2個alanine，均會使a-螺旋結構消失。故可知leucine-zipper like序列對NdAg之結構十分重要。另經由RNA結合力的測試結果可知，刪除第2至第13個胺基酸會使RNA結合力大幅下降，另leucine-zipper like序列之刪除或插入突變亦會降低RNA結合力，刪除第35至第43個胺基酸對結合力影響最巨。蛋白RNA結合力的強弱與其RNA監護子活性的高低是呈正相關的。RNA結合力低之突變體亦可促進hammerhead ribozyme它割反應，唯需較高濃度的蛋白。最後，經由沉澱實驗得知，NdAg與RNA結合後可形成巨大複合物。

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並列摘要

NdAg contains the first 88 amino acids of hepatitis delta antigen (HdAg). NdAg interacts with a variety of RNAs, in addition, it facilitates RNA structural rearrangement by acting as an RNA chaperone. NdAg contains HdAg’s cryptic RNA binding domains (aa 2-27), leucine-zipper like sequence (aa 13-48), and nuclear localization signals (aa 67-88). In this thesis, I constructed NdAg mutants for investigating the role of different structural/functional domains on the structure, the RNA binding activity, and the RNA chaperone activity of NdAg. The a-helical structure of NdAg was not affected when aa 2-13 or aa 60-88 were deleted. However, the deletions of aa 14-23 and aa 35-43, as well as the insertion of two alanine residues to aa 27-28 disrupted the a-helical structure. These findings confirm the importance of the leucine-zipper like sequence for the structure of NdAg. The deletion of the N terminal cryptic RNA binding domain, as well as insertion and deletion mutations in the leucine-zipper like sequence decreased the RNA binding activity of NdAg. NdAg and all of its mutants constructed in the study promoted the assembly of the hammerhead catalytic domain between a trans-acting hammerhead ribozyme and its cognate substrate, but the RNA binding activity and the stimulatory activity of NdAg and its mutants are correlated. The formation of the RNA-protein complexes seems to be a prerequisite for the stimulatory activity on hammerhead ribozyme catalysis of NdAg and its mutants, and the sedimentation experiment suggests that there are more than one form of NRA-NdAg complexes.

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參考文獻

Chao, M., Hsieh, S. Y., and Taylor, J. (1990) Role of two forms of hepatitis delta virus antigen: evidence for a mechanism of self-limiting genome replication. J. Virol. 64:5066-5069. Chou, H. C., Hsieh, T. Y., Sheu, G. T., and M. M. C. Lai. (1998) Hepatitis Delta Antagen Mediates the Nuclear Import of Hepatitis Delta Virus RNA. J. Virol. 72:3684-3690. Glenn, J. S. and White, J. M. (1991) Trans-Dominant Inhibition of Human Hepatitis Delta Virus Genome Replication. J. Virol. 65:2357-2361. Herschlag, D. (1995) RNA chaperones and the RNA folding problem. JBC 270:20871-20874. Huang, A. S. and Wu, H. N. (1998) Identification and characterization of the RNA chaperone activity of hepatitis delta antigen peptides. JBC 273:26455-26461.

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D型肝炎病毒抗原N端區域與核酸分子交互作用之研究

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