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研究生:周嗣堯
研究生(外文):Sih-Yao Chow
論文名稱:痘病毒寄主範圍蛋白質CP77與宿主細胞Rrp42蛋白質之間交互作用的研究
論文名稱(外文):Structural and Biochemical Studies of the Interaction between the Cowpox Viral Host Range Protein CP77 and the Human Host Protein Rrp42
指導教授:袁小琀
指導教授(外文):Hanna S. Yuan
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生命科學暨基因體科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:英文
論文頁數:54
中文關鍵詞:痘病毒痘病毒寄主範圍蛋白質
外文關鍵詞:PoxvirusHost Range Protein
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痘病毒寄主範圍蛋白質(host range protein),可以幫助痘病毒在原來無法支持病毒的細胞(non-permissive cells)中生長複製,並完成其病毒生命週期。CP77為牛痘病毒(cowpox virus)的寄主範圍蛋白質,在其幫助之下,痘苗病毒(vaccinia virus)可以在其原來無法生長的細胞株當中生長,並完成其病毒生命週期。前人利用酵母菌雙雜交篩選法的研究指出,CP77會與宿主細胞當中的蛋白質Rrp42交互作用。進一步的研究發現該交互作用會導致病毒早期(early)與中期(intermediate)RNA的分解。Rrp42為宿主細胞exosome聚合體的成員之一,而exosome聚合體主要負責細胞中RNA的代謝。本論文主要在利用生化與結構生物學的方法去了解CP77與Rrp42之間的交互作用,並探討CP77與宿主細胞蛋白質的交互作用,如何導致病毒RNA的分解。
我們在大腸桿菌中大量表現及純化CP77與Rrp42蛋白質。研究發現,這兩個蛋白質都不具備有核酸水解酵素的活性。另外我們發現CP77為一雙聚體蛋白質,而Rrp42為一單體蛋白質。藉由凝膠過濾法(gel filtration)與GST pull-down實驗可以發現,CP77與Rrp42並無直接的交互作用。此外CP77會與細胞當中exosome聚合體成員Rrp42與Rrp46交互作用。以上的實驗結果指出,CP77可能直接或間接的與exosome聚合體交互作用並導致病毒RNA的分解。
Many poxviruses can enter host cells and complete their life cycle with the aid of viral host range genes. CP77 is a cowpox virus host range protein, and the expression of CP77 gene in the Vaccinia virus allows the virus to grow in non-permissive cells. Previous studies showed that degradation of the viral early and intermediate mRNA is enhanced in the virus-infected cells in the presence of CP77, which interacts with a host protein Rrp42, as assayed by the yeast-two-hybrid and GST pull-down experiments. Rrp42 is a component protein of cellular exosomes, which play important roles in RNA processing, decay and quality control in eukaryotic and archaeal cells. Here we study the interactions between CP77 and Rrp42 by biochemical and structural approaches to elucidate how CP77 is involved in viral RNA degradation through its interactions with host proteins. The recombinant CP77 and Rrp42 were over expressed and purified in E. coli. We found that CP77 was a dimeric protein, whereas Rrp42 was a monomeric protein in vitro. Both proteins did not have any detectable nuclease activity. No direct interaction was identified between the recombinant CP77 and Rrp42 by gel filtration chromatography and GST pull-down assays. However, pull down assays indicated that CP77 interacted with the exosome component proteins, Rrp42 and Rrp46 in cell lysate. These results suggest that CP77 interacts with the exosome directly or indirectly via other adaptor(s), to promote viral RNA degradation.
論文摘要1
Abstract 2
1 Introduction 3
1.1 Poxviridae superfamily viruses 3
1.2 Host range proteins are identified in poxviruses 3
1.3 CP77 is a host range protein from cowpox virus 4
1.4 CP77 mediates several intracellular pathways 6
1.5 Rrp42 is a component protein in the exosome 6
1.6 Specific aims 8
2 Materials and Methods 9
2.1 Protein expression and purification 9
2.1.1 Plasmids 9
2.1.2 CP77 protein expression and purification 9
2.1.3 Rrp42 expression and purification 9
2.2 Dynamic light scattering (DLS) 11
2.3 Protein crystallization 11
2.4 RNase activity assays 11
2.5 RNA/DNA binding assays 12
2.6 Fractionation of cell extracts and western blot
analyses
2.7 GST-pulldown assays 13
3 Results 14
3.1 Recombinant CP77 is a dimeric protein 14
3.2 Rrp42 is a monomeric protein in vitro but it
associates with a complex in vivo 14
3.3 Neither Rrp42 nor CP77 Possesses RNase Activity 15
3.4 CP77 does not interact directly with Rrp42 16
3.5 CP77 interacts indirectly with the exosome
component protein Rrp42 and Rrp46 17
4 Discussion 19
5 References 22
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