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研究生:李宜佳
研究生(外文):Yi-Ija Li
論文名稱:竹嵌紋病毒複製酵素之功能特性分析
論文名稱(外文):Functional analysis of the capping enzyme and helicase like domain of the ORF1 of bamboo mosaic potexvirus
指導教授:孟孟孝
指導教授(外文):Menghsiao Meng
學位類別:博士
校院名稱:國立中興大學
系所名稱:農業生物科技學研究所
學門:農業科學學門
學類:農業技術學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:英文
論文頁數:103
中文關鍵詞:病毒複製酵素
外文關鍵詞:RNA virusreplicasecapping enzymehelicase
相關次數:
  • 被引用被引用:11
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竹嵌紋病毒(bamboo mosaic virus, BaMV)主要感染綠竹,歸屬於potexvirus group。其第一個轉譯架構(open reading frame 1, ORF1)可以轉譯出分子量為155-kDa的蛋白質,根據胺基酸序列的比對,推測此蛋白質的功能應與基因體複製及在5¢端形成cap結構有關。而capping酵素推測是位於ORF1的N端。為了確認ORF1的N端是否與capping有關,本實驗室經將相對的cDNA插入表現載體pYES2並送進酵母菌(Saccharomyces cerevisiae, INVSc1)內,利用半乳糖誘導的方式,進行蛋白質的表現。並且發現被誘導表現的N端區域蛋白質大部份都會和細胞膜結合在一起。利用蔗糖梯度密度離心,可以部分純化到表現的蛋白。且此一表現的蛋白具有AdoMet-dependent guanylyltransferase的活性且具有methyltransferase的活性,在AdoMet及GTP的存在下,可行成 mGMP-enzyme共價結合的中間產物。因此以酵母菌表現的N端蛋白的確具有capping enzyme的活性。 接著,在本實驗中繼續針對病毒複製酵素中間端的部分,RNA helicase-like domain,繼續研究,發現以大腸桿菌表現的RNA helicase like domain蛋白不但具有NTP水解的活性,還具有RNA 5’-triphosphatase的活性。且經由蛋白質定點突變NTPase的活化位,不旦會影響NTPase的活性,也會影響RNA 5’-triphosphatase的活性。另外根據此二活性對金屬的喜好性一致且在活性上會互相競爭,因而推測此二活性的活化位有重疊。更進一步的,我們將以RNA 5’-triphosphatase處理過的RNA來當作capping的受質,加到capping enzyme的反應液中,發現RNA會被cap,且形成典型的cap結構,mGpppG。我們分別表現有活性的病毒的capping enzyme及RNA 5’-triphosphatase,並在in vitro中成功的展現了病毒完整的複製機制。
Bamboo mosaic virus (BaMV), a member of the potexvirus group, infects primarily members of the Bambusoideae. Open reading frame 1 (ORF1) of BaMV encodes a 155-kDa polypeptide that has long been postulated to be a replicase involved in the replication and formation of the cap structure at the 5¢ end of the viral genome. Analysis of the amino acid sequences reveals several conserved motifs suggesting that three functional domains, methyltransferase, helicase, and RNA dependent RNA polymerase on the order of N to C termini, are organized in the 155-kDa polypeptide. In our previous study, we have demonstrated the RdRp activity of BaMV. In this study, we went on exploring the activities associated with the putative methyltransferase and helicase domains. We used the putative methyltransferase domain expressed in yeast to investigate the capping mechanism of BaMV. In the regard of methyltransferase domain, both methyltransferase and guanylyltransferase activities were identified. The methylyltransferase activity transfers the methyl group from AdoMet to GTP, subsequently, the guanylyltransferase activity catalyzes the mGTP to mGMP and forms the intermediate mGMP-enzyme complex. Therefore, the putative methyltransferase domain is indeed a RNA capping enzyme. The capping activity of BaMV is AdoMet dependent and follows a similar role as the cap formation of Semiliki Forest virus that differs from eukaryotic mRNA cap formation. Regarding the putative helicase domain, we demonstrated that it could hydrolyze nucleoside triphosphate to nucleoside diphosphate in the presence of Mg2+. In addition to nucleoside triphosphatase, the helicase-like domain also possesses the RNA-5¢ triphosphatase activity. The g-phosphate of nascent RNA is removed by helicase-like domain, which facilitates the capping enzyme forming the cap structure mGpppG. This is the first report of the helicase domain of a plant virus involved in cap formation. Further, we found that the helicase Walker A motif (GxGKS) mutation abolishes not only nucleoside triphosphatase but also RNA 5¢-triphosphatase activity. This result suggests that the catalytic sites of nucleoside triphosphatase and RNA 5¢-triphosphatase are overlapping. At the last, we looked into the duplex unwinding activity of the helicase-like domain of BaMV. To date only few cases of superfamily 1 viral helicase, to whom the BaMV helicase belongs, have been demonstrated its duplex unwinding activity such as nsP2 of Semiliki Forest virus (SFV). We used duplex-RNA with 3¢ or 5¢ single strand tails as substrates. Marginal activity was found when using a 5 ¢-to-3¢ polarity RNA as substrate. However, no significant differences between the wild type and Walker A motif or motif V mutants. From these results, we assume that the putative helicase domain might need cellular factors to help processing the duplex unwinding activity, or follow the role of duplex unwinding in a different way from others RNA virus in virus replication.
TABLE OF CONTENTS
ABSTRACT……………………………………………………………………….1
INTRODUCTION………………………………………………………………...3
Chapter 1 Characterization of the AdoMet-Dependent Guanylyltransferase Activity That Is Associated with the N Terminus of Bamboo Mosaic
Virus Replicase…………………………………………………………..…6
1.1 Abstract………………………………………………………………..7
1.2 Introduction……………………………………………………………8
1.3. Materials and methods…….....………………………………..11
1.4 Results
1.4.1 Viral protein expression in yeast cells……….....………15
1.4.2 Detection of guanylyltransferase activity……....……...15
1.4.3 Characteristics of BaMV guanylyltransferase activity..16
1.4.4 TLC analysis of the guanylate moiety....……………………17
1.4.5 Detection of methyltransferase activity…………………..18
1.4.6 Discussion ……………………………………………………..….19
Chapter 2 The Helicase-Like Domain of Plant Potexvirus Replicase
Participates in Formation of RNA 5¢ Cap Structure by Exhibiting
RNA 5¢-Triphosphatase Activity………………………………………..31
2.1 Abstract………………………………………………………………..32
2.2 Introduction……………………………………………………………33
2.3 Materials and methods………………………………………………36
2.4 Results
2.4.1 Protein expression and purification…………………………40
2.4.2 NTPase activity…………………………………………………..41
2.4.3 RNA 5¢-triphosphatase activity………………………………..42
2.4.4 Factors affecting NTPase and RNA 5¢-triphosphatase
activities....................................................43
2.4.5 Formation of cap structure at the 5¢ end of RNA…….…..44
2.5 Discussion …………………………………………………………..46
Chapter 3 Identification of Duplex-Unwinding Activity of Helicase-Like
Domain of Bamboo MosaicPotexvirus……………………………………………60
3.1 Abstract……………………………………………………………...61
3.2 Introduction………………………………………………………….62
3.3 Materials and methods………………………………………………64
3.4 Results
3.4.1 Protein expression and purification………………………...72
3.4.2 RNA duplex-unwinding activity of E. coli expressed helicase like domain….................………………………..….73
3.4.3 RNA binding properties of the helicase-like domain…....74
3.4.4 RNA duplex-unwinding activity of E. coli. expressed and yeast expressed helicase like domain………………………….…...75
3.5 Discussion ……………………………………………………….……77
References..……………………………………………………….……...92
Publications.…………………………………………………………….103
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