臺灣博碩士論文加值系統

C型肝炎病毒 (HCV) 是造成非A非B型肝炎的主要致病原。C型肝炎病毒是一單股正向的RNA病毒，基因體全長約為9600個核苷酸，可轉譯出一個約3000個胺基酸的多蛋白質前驅物，經由宿主和病毒本身的蛋白質水解酶切割，得到結構性和非結構性蛋白質。C型肝炎病毒的非結構性蛋白質NS4B為一疏水性蛋白質，分子量約為27 kDa，其功能尚未全然明瞭。NS4B具有四個transmembrane domains，會引起細胞內的胞器膜構造產生變化，形成緊密的膜狀網構造，推測C型肝炎病毒NS5B RNA聚合酶會與其他非結構性蛋白質形成複製複合體，座落於此膜狀網構造上。
之前的研究發現，NS4B中間有一個nucleotide binding motif，可以和GTP結合並且可將其水解， NS4B具有的GTPase活性對於C型肝炎病毒複製是重要的。若使A motif (1840-GX1X2X3X4GK-1846) 的保留性序列K1846突變成serine或arginine，會造成NS4B(K1846S) 和 NS4B(K1846R) 與GTP的結合減少，使GTPase活性下降，進而抑制C型肝炎病毒的複製；但是另一適應性突變NS4B(K1846T) 卻會使C型肝炎病毒的複製增加達三十倍。
在本研究中，利用轉染表現的細胞經由免疫螢光染色，證實NS4B和NS4B(K1846T)均分布在細胞質中，而且較集中在細胞核周圍。為了測定NS4B和NS4B(K1846T)的NTPase活性，藉由誘導大腸桿菌大量表現GST-NS4B和GST-NS4B(K1846T)，以glutathione Sepharose 4B和PreScission protease將NS4B和NS4B(K1846T)　加以純化，結果顯示NS4B具有GTPase和ATPase活性，而與NS4B比較，K1846T的ATPase活性減少到40%，但是對GTPase活性幾乎沒有影響。以上結果顯示NS4B的nucleotide binding motif中lysine-1846，對於NS4B的NTPase活性並不是絕對必要的。推測適應性突變種NS4B(K1846T)可能藉由其他機制，促進C型肝炎病毒的複製。

Hepatitis C virus (HCV) is a major cause of liver disease worldwide. HCV contains a positive-stranded RNA genome of 9.6 kilobases encodes a polyprotein of about 3000 amino acid residues. The polyprotein undergoes cellular and viral protease processing to generate structural and nonstructural proteins. Nonstructural protein 4B (NS4B) is a relatively hydrophobic 27 kDa protein of unknown function. NS4B has four transmembrane segments and has the ability to induce a tight structure formation as membranous web. It is proposed that HCV NS5B RNA polymerase forms replication complex with other nonstructural proteins at the membranous web.
NS4B possesses GTPase activity. It contains a nucleotide binding motif (NBM) that mediates binding and hydrolysis of GTP previously demonstrated to be important for HCV replication. Substitutions at the conserved K-1846 with serine or arginine in the A motif (1840-GX1X2X3X4GK-1846) of NBM impaired GTP binding and hydrolysis, and resulted in inhibition of HCV RNA replication. Nevertheless, a cell culture adaptive mutant K1846T in which K-1846 was mutated to threonine enhances HCV RNA replication to 30 fold.
In this study, NS4B and NS4B(K1846T) were detected in the cytoplasm of transfected cells and preferentially localized at the perinuclear region by immunofluorescence staining assay. To examine the NTPase activities of NS4B and NS4B(K1846T), GST-NS4B and GST-NS4B(K1846T) were expressed in E. coli, purified by glutathione Sepharose 4B, and cleaved to NS4B and NS4B(K1846T) by PreScission protease. HCV NS4B protein possesses both GTPase and ATPase activities. K1846T mutation decreased the ATPase activity of NS4B to 40%, but had little effect on GTPase activity. The results indicate that the lysine-1846 in the nucleotide binding motif is not absolutely required for the GTPase activity of NS4B. Different from previous studies, the results suggest that the adaptive mutant K1846T may promote HCV replication by mechanisms other than the GTPase activity of NS4B protein.

中文摘要 ………………………………………………… I
英文摘要 ………………………………………………… II
縮寫表 …………………………………………………… III
緒論 ……………………………………………………… 1
材料來源 …………………………………………………14
實驗方法 …………………………………………………20
實驗結果 …………………………………………………36
討論 ………………………………………………………41
圖表 ………………………………………………………45
參考文獻 …………………………………………………65

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