Skip navigation

DSpace JSPUI

DSpace preserves and enables easy and open access to all types of digital content including text, images, moving images, mpegs and data sets

Learn More
DSpace logo
English
中文
  • Browse
    • Communities
      & Collections
    • Publication Year
    • Author
    • Title
    • Subject
  • Search TDR
  • Rights Q&A
    • My Page
    • Receive email
      updates
    • Edit Profile
  1. NTU Theses and Dissertations Repository
  2. 生命科學院
  3. 生化科技學系
Please use this identifier to cite or link to this item: http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/7434
Title: A型流感病毒PB1-F2蛋白質於人類細胞株之穩定性與蛋白酶體降解效率之研究
Investigation of the Stability and Proteasome Degradation Efficiency of Influenza A Virus PB1-F2 Proteins from Different Viral Strains in Human Cell Lines
Authors: Yu-En Chiu
邱宇恩
Advisor: 張世宗
Keyword: 流感病毒,PB1-F2,蛋白?體,PA28,
Influenza,PB1-F2,Proteasome,PA28/PSME,
Publication Year : 2018
Degree: 碩士
Abstract: 已知A型流感病毒的PB1-F2蛋白質會調控病毒PB1聚合酶活性,亦會進入宿主細胞粒線體內外膜之間,促使膜電位下降,引發單核球細胞凋亡、調控細胞先天性免疫反應。此外PB1-F2也會抑制干擾素的生成,並增加二次細菌性肺炎感染的風險。然而,來自不同病毒株的PB1-F2在不同宿主細胞中的定位、表現量和功能都有不少差異,因此研究PB1-F2的穩定性、功能及調控機制,也許可以進一步了解不同流感病毒株致病力有所差異的原因。本研究將四株不同亞型的病毒株A/Puerto Rico/8/1934 (H1N1)、A/Udorn/307/1972 (H3N2)、A/Hong Kong/156/1997 (H5N1)、A/Taiwan/01/2013 (H7N9)的PB1-F2基因於HEK293細胞中表現時,發現其表現量有極大的差異,且皆會因加入蛋白酶體抑制劑MG132而有不同程度的上升,表示PB1-F2會受到蛋白酶體降解系統的調控。將四株不同亞型病毒株的PB1-F2進行部分胺基酸序列互換時,發現2、10、11、14和68-71會影響H1N1、H3N2和H7N9 PB1-F2在HEK293細胞中的穩定性,然而對於H5N1則沒有顯著影響。此外,細胞影像的結果顯示PB1-F2 68-71的胺基酸序列會影響PB1-F2在細胞中的分佈位置,其中ILVF有粒線體標的功能,會使PB1-F2位於粒線體。為了探究PB1-F2的穩定性是否受到泛素化的調控,將四株病毒株之PB1-F2上的所有離胺酸突變為精胺酸後,發現H1N1、H3N2、H7N9之PB1-F2皆因泛素化位點的突變而提升的在細胞中的穩定性,而H5N1 PB1-F2的穩定性則沒有因突變而有顯著提升,因此可知PB1-F2可經由泛素依賴型 (dependent) 或不依賴型 (independent) 路徑而被蛋白酶體降解。此外,分別將PA28α、PA28β或PA28γ與PB1-F2共轉染至HEK293細胞中,四株病毒株的PB1-F2的表現量皆有非常明顯的下降,顯示PA28可以促進PB1-F2的降解。
Influenza A virus (IAV) protein Polymerase basic 1-frame 2 (PB1-F2) regulates viral polymerase activity, induces apoptosis in host immune cells, interferes the host innate immune response and enhances the pathogenesis of secondary bacterial pneumonia. Moreover, PB1-F2 derived from different virus strains may perform different functions, expression levels and cellular localization, leading to their various strain-specific virulence in host cells. Therefore, studying the principles which determine the stability, functions and regulation mechanisms of PB1-F2 might help us know further about the pathogenesis of various influenza A virus strains. In this study, HEK293 cells expressed PB1-F2 from four IAV strains (A/Puerto Rico/8/1934 (H1N1), A/Udorn/307/1972 (H3N2), A/Hong Kong/156/1997 (H5N1), A/Taiwan/01/2013 (H7N9)) in extremely various expression levels. Moreover, the expression levels of PB1-F2 were increased upon MG132 treatment and showed different sensitivity to MG132, indicating that PB1-F2 may undergo proteasome-mediated degradation. Swapping of equivalent amino acid residues 2, 10, 11, 14, and 68-71 of PB1-F2 among these four IAV strains may alter their protein stability. Moreover, the cell images showed that amino acid residues 68-71 might modulate the localization of PB1-F2. In addition, PB1-F2 residues I68, L69, V70, and F71 are mitochondrial targeting sequence. In order to clarify whether PB1-F2 stability is ubiquitination-mediated, all of the lysine residues of PB1-F2 were mutated to arginine residues to inhibit the possible ubiquitination of PB1-F2. It is found that the expression levels of H1N1, H3N2 and H7N9 PB1-F2 were greatly increased, but H5N1 PB1-F2 did not increase significantly, indicating that the ubiquitin-dependent and ubiquitin-independent degradation pathway are all involved in regulation of PB1-F2 stability. Furthermore, the expression levels of PB1-F2 were markedly decreased while the proteasome activator PA28α/PSEM1, PA28β/PSME2, or PA28γ/PSME3 was co-transfected with PB1-F2, suggested that PA28 can promote degradation of PB1-F2.
URI: http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/7434
DOI: 10.6342/NTU201803869
Fulltext Rights: 同意授權(全球公開)
metadata.dc.date.embargo-lift: 2023-08-21
Appears in Collections:生化科技學系

Files in This Item:
File SizeFormat 
ntu-107-1.pdf3.92 MBAdobe PDFView/Open
Show full item record


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.

社群連結
聯絡資訊
10617臺北市大安區羅斯福路四段1號
No.1 Sec.4, Roosevelt Rd., Taipei, Taiwan, R.O.C. 106
Tel: (02)33662353
Email: ntuetds@ntu.edu.tw
意見箱
相關連結
館藏目錄
國內圖書館整合查詢 MetaCat
臺大學術典藏 NTU Scholars
臺大圖書館數位典藏館
本站聲明
© NTU Library All Rights Reserved