進階搜尋


下載電子全文  
系統識別號 U0026-3107201310362100
論文名稱(中文) 利用親和性串聯純化為基礎的蛋白質體技術探索腸病毒結構蛋白可能有作用的細胞蛋白網絡
論文名稱(英文) Investigating the cellular interactome of EV71 structural protein by tandem affinity purification based proteomic approach
校院名稱 成功大學
系所名稱(中) 分子醫學研究所
系所名稱(英) Institute of Molecular Medicine
學年度 101
學期 2
出版年 102
研究生(中文) 連芸吟
研究生(英文) Yun-Yin Lien
學號 T16001029
學位類別 碩士
語文別 中文
論文頁數 63頁
口試委員 指導教授-王憲威
口試委員-王貞仁
口試委員-陳舜華
中文關鍵字 免疫共沉澱法  共軛焦免疫螢光細胞染色  腸病毒71型  串聯親和性蛋白純化技術  病毒結構蛋白 
英文關鍵字 Co-immunoprecipitation  Confocal Imaging  Enterovirus 71  Globular Heads of C1q Receptor  Melanoma differentiation-associated gene  ProteinsTandem Affinity Purification  Viral Structure 
學科別分類
中文摘要 腸病毒71型具有單股正股RNA基因,為不具外套膜的小RNA病毒,是導致手足口症的主要病原體。已知其主要感染對象為10歲以下兒童,並可能併發中樞神經系統疾病。腸病毒71型其病毒顆粒由60個外鞘蛋白(capsid)組成二十面體構造,每一個外鞘蛋白係由四個病毒結構蛋白(structure proteins),VP1、VP2、VP3、VP4組成。結構蛋白VP1主要參與於病毒感染宿主細胞的受體結合,且與某些宿主細胞蛋白的交互作用在病毒的複製及致病機轉扮演著重要角色。結構蛋白VP2及VP3也曾被報導可能影響病毒感染力,另外,也有文獻指出和腸病毒71型同屬的小兒麻痺病毒其結構蛋白VP4於病毒感染細胞時,扮演協助病毒進入細胞的角色。因此,我們認為腸病毒71型的結構蛋白在病毒的致病機轉上應該都有著舉足輕重的影響,但目前為止除了VP1,其餘結構蛋白的相關研究仍不清楚。本篇我們首先利用串聯親和性蛋白純化技術及蛋白質體學進行尋找和腸病毒71型結構蛋白可能有交互關係的細胞蛋白,並期望進一步藉著研究宿主細胞蛋白與病毒結構蛋白的交互作用關係,加深瞭解病毒的致病機轉。在本篇研究結果中,我們藉由一維電泳圖譜與蛋白質體學分析,找出一些和腸病毒71型結構蛋白VP4有交互作用關係的宿主細胞蛋白,其中大多為具有解旋酶或剪接活性的RNA結合蛋白。之後進一步利用免疫共沉澱法及共軛焦免疫螢光細胞染色,我們確認病毒結構蛋白VP4和其中一個宿主細胞蛋白GC1qR有明確的交互作用。並發現雖然在腸病毒71型的急性感染狀態下,GC1qR蛋白的表現量似乎不受影響,但利用shRNA將GC1qR剔除後,卻發現細胞內腸病毒71型的病毒結構蛋白產量有下降的趨勢。先前曾有文獻指出GC1qR會抑制依靠MDA5的抗病毒訊息傳遞反應作用,且近期也有報導指出腸病毒71型病毒感染,會增強MDA5的降解。因此,綜合以上結果,我們認為腸病毒71型的病毒結構蛋白VP4和宿主細胞蛋白GC1qR有交互作用關係,並且可能參與調控細胞內抗病毒訊息傳遞的機制,但相關的作用機制仍有待進一步釐清。此研究提供一個與腸病毒結構蛋白VP4可能有交互作用的細胞蛋白組與探討其交互作用的前鋒性基礎驗證模式,未來期望能藉由瞭解此病毒蛋白與宿主細胞蛋白的交互作用關係,作為發展抗病毒試劑或作為設計基因治療的一個參考方針。
英文摘要 Enterovirus 71 (EV71) is a small, nonenveloped virus with positive single strand RNA genome. It is a major causative agent of hand, foot, and mouth disease (HFMD), and known to cause severe neurological disease mainly on young children. EV71 is typified by an icosahedral outer capsid structure consisting of 60 capsomers that is composed of four structural proteins VP1-VP4. The VP1 has a well-documented function in receptor binding, and is likely to have multiple roles with other host factors in viral multiplication and pathogenesis. However, the functions of other capsid proteins remain largely unknown. A study has shown that the structure proteins VP2 and VP3 are also related to viral infection. Another study has shown that the participation of VP4 is required during cell entry of the poliovirus, a close relative of Enterovirus. We therefore speculate that the functions of all the EV71 capsid proteins are important for viral multiplication and pathogenesis. In this study, we adapted a modified murine stem cell virus (MSCV) packaging vector to express EV71 structure proteins, either individually as a functional unit or together as a polyprotein, as baits for the pull-down of cellular proteins. This vector allows efficient expression and recovery of bait proteins for tandem affinity purification (TAP) of interacting protein complexes in mammalian cells. By using a modified purification strategy and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), we report here an VP4-interactom . Most of them are RNA binding proteins with helicase or splicing activities, suggesting RNA is a critical mediator for VP4-associated complex. In addition, we verified here the interaction of VP4 and one of its candidate interacting protein GC1qR by co-immunoprecipitation and confocal imaging studies. We also found that during acute EV71 infection, the level of GC1qR expression was not significantly altered. When GC1qR was knockdown by shRNA, it would decrease intracellular EV71 production. Since GC1qR had been shown to inhibit the MDA5-dependent antiviral response and that MDA5 plays a crucial role in EV 71 RNA-mediated IRF3 activation, our result in GC1qR knockdown may reveal a possible mechanism of the interaction of VP4 and GC1qR in antagonizing MDA5-mediated antiviral response for EV71 associated pathogenesis. Collectively, our study here provides a cellular ineractome of VP4 and a pilot investigation model for the verification of their cellular interaction. Further investigation on the functional relevance of the VP4-interacting cellular proteins, such as the VP4 and C1qR, may facilitate the development of anti-viral agents or gene therapy-based therapeutic interventions.
論文目次 中文摘要…………………………………………………………………………………...1
英文摘要…………………………………………………………………………………...3
誌謝…………………………………………………………………………………...........5
目錄………………………………………………………………………………………...6
表目錄……………………………………………………………………………………...8
圖目錄……………………………………………………………………………………...9
附錄目錄…………………………………………………………………………………..10
符號與縮寫………………………………………………………………………………..11
緒論
一、 腸病毒71型之病毒學概論……………………………………………………....12
二、 腸病毒71型之流行病學及臨床症狀……………………………………………13
三、 腸病毒71型之致病機轉及病毒生活史…………………………………………13
四、 Globular Heads of C1q Receptor (GC1qR)……………………………………….15
研究目的…………………………………………………………………………………..16
材料與方法
A. 實驗材料
一、 細胞株……………………………………………………………………………17
二、 病毒株……………………………………………………………………………17
三、 質體構築所需之引子序列………………………………………………………17
四、 shRNA質體……………………………………………………………………...17
五、 抗體………………………………………………………………………………18
六、 試劑………………………………………………………………………………19
B. 實驗方法
一、 質體構築(plasmid construct)…………………………………………………….20
二、 細胞培養及轉染法 (cell culture and transfection)……………………………...20
三、 串接親和純化技術(tandem affinity purification)……………………………….21
四、 腸病毒71型病毒產出與感染(generation and infection of Enterovirus 71 virus)……………………………………………………………………………...22
五、 病毒斑分析(plaque assay)……………………………………………………….22
六、 免疫共沉澱法(co-immunipreciption)……………………………………………23
七、 免疫螢光染色法(immunofluorescence staining)………………………………..23
八、 西方墨點法(western blotting)…………………………………………………...24
九、 Trypan blue染色法………………………………………………………………24
十、 慢病毒產出及蛋白剔除實驗(Lentivirus production and protein knockdown)…25
結果
一、 腸病毒71型結構蛋白質在293T、RD及SK細胞上的表達………………….26
二、 利用串接親和純化技術及蛋白質體學分析與腸病毒71型結構蛋白交互作用之宿主蛋白…………………………………………………………………………..26
三、 確認腸病毒71型結構蛋白VP4與宿主蛋白GC1qR的交互作用…………….27
四、 在腸病毒急性感染細胞時,GC1qR的表達量沒有受到顯著影響…………….29
五、 剔除細胞蛋白GC1qR後,腸病毒71型病毒結構蛋白在細胞內的產量下降...29
討論………………………………………………………………………………………..30
表…………………………………………………………………………………………..34
圖…………………………………………………………………………………………..35
參考文獻…………………………………………………………………………………..45
附錄………………………………………………………………………………………..51
參考文獻 Abdelmohsen, K. and M. Gorospe (2012). "RNA-binding protein nucleolin in disease." RNA Biol 9(6): 799-808.
AbuBakar, S., I. C. Sam, J. Yusof, M. K. Lim, S. Misbah, N. MatRahim and P. S. Hooi (2009). "Enterovirus 71 outbreak, Brunei." Emerg Infect Dis 15(1): 79-82.
Alexander, J. P., Jr., L. Baden, M. A. Pallansch and L. J. Anderson (1994). "Enterovirus 71 infections and neurologic disease--United States, 1977-1991." J Infect Dis 169(4): 905-908.
Arita, M., Y. Takebe, T. Wakita and H. Shimizu (2010). "A bifunctional anti-enterovirus compound that inhibits replication and the early stage of enterovirus 71 infection." J Gen Virol 91(Pt 11): 2734-2744.
Beatch, M. D., J. C. Everitt, L. J. Law and T. C. Hobman (2005). "Interactions between rubella virus capsid and host protein p32 are important for virus replication." J Virol 79(16): 10807-10820.
Bedard, K. M. and B. L. Semler (2004). "Regulation of picornavirus gene expression." Microbes and Infection 6(7): 702-713.
Bible, J. M., P. Pantelidis, P. K. Chan and C. Y. Tong (2007). "Genetic evolution of enterovirus 71: epidemiological and pathological implications." Rev Med Virol 17(6): 371-379.
De Jesus, N. H. (2007). "Epidemics to eradication: the modern history of poliomyelitis." Virol J 4: 70.
Ghebrehiwet, B., P. D. Lu, W. Zhang, S. A. Keilbaugh, L. E. Leigh, P. Eggleton, K. B. Reid and E. I. Peerschke (1997). "Evidence that the two C1q binding membrane proteins, gC1q-R and cC1q-R, associate to form a complex." J Immunol 159(3): 1429-1436.
Ghebrehiwet, B. and E. I. Peerschke (1998). "Structure and function of gC1q-R: a multiligand binding cellular protein." Immunobiology 199(2): 225-238.
Gilbert, G. L., K. E. Dickson, M. J. Waters, M. L. Kennett, S. A. Land and M. Sneddon (1988). "Outbreak of enterovirus 71 infection in Victoria, Australia, with a high incidence of neurologic involvement." Pediatr Infect Dis J 7(7): 484-488.
Gregan, J., C. G. Riedel, M. Petronczki, L. Cipak, C. Rumpf, I. Poser, F. Buchholz, K. Mechtler and K. Nasmyth (2007). "Tandem affinity purification of functional TAP-tagged proteins from human cells." Nat Protoc 2(5): 1145-1151.
Ho, M., E. R. Chen, K. H. Hsu, S. J. Twu, K. T. Chen, S. F. Tsai, J. R. Wang and S. R. Shih (1999). "An epidemic of enterovirus 71 infection in Taiwan. Taiwan Enterovirus Epidemic Working Group." N Engl J Med 341(13): 929-935.
Hober, D. and P. Sauter (2010). "Pathogenesis of type 1 diabetes mellitus: interplay between enterovirus and host." Nat Rev Endocrinol 6(5): 279-289.
Hosoya, M., Y. Kawasaki, M. Sato, K. Honzumi, A. Kato, T. Hiroshima, H. Ishiko and H. Suzuki (2006). "Genetic diversity of enterovirus 71 associated with hand, foot and mouth disease epidemics in Japan from 1983 to 2003." Pediatr Infect Dis J 25(8): 691-694.
Huang, S.-W., Y.-F. Wang, C.-K. Yu, I.-J. Su and J.-R. Wang (2012). "Mutations in VP2 and VP1 capsid proteins increase infectivity and mouse lethality of enterovirus 71 by virus binding and RNA accumulation enhancement." Virology 422(1): 132-143.
Kiener, T. K., Q. Jia, X. F. Lim, F. He, T. Meng, V. T. Chow and J. Kwang (2012). "Characterization and specificity of the linear epitope of the enterovirus 71 VP2 protein." Virol J 9: 55.
Krainer, A. R., A. Mayeda, D. Kozak and G. Binns (1991). "Functional expression of cloned human splicing factor SF2: homology to RNA-binding proteins, U1 70K, and Drosophila splicing regulators." Cell 66(2): 383-394.
Kung, S. H., S. F. Wang, C. W. Huang, C. C. Hsu, H. F. Liu and J. Y. Yang (2007). "Genetic and antigenic analyses of enterovirus 71 isolates in Taiwan during 1998-2005." Clin Microbiol Infect 13(8): 782-787.
Kuo, R. L., L. T. Kao, S. J. Lin, R. Y. Wang and S. R. Shih (2013). "MDA5 Plays a Crucial Role in Enterovirus 71 RNA-Mediated IRF3 Activation." PLoS One 8(5): e63431.
Lin, J. Y., T. C. Chen, K. F. Weng, S. C. Chang, L. L. Chen and S. R. Shih (2009). "Viral and host proteins involved in picornavirus life cycle." J Biomed Sci 16: 103.
Liu, C. C., M. S. Guo, F. H. Lin, K. N. Hsiao, K. H. Chang, A. H. Chou, Y. C. Wang, Y. C. Chen, C. S. Yang and P. C. Chong (2011). "Purification and characterization of enterovirus 71 viral particles produced from vero cells grown in a serum-free microcarrier bioreactor system." PLoS One 6(5): e20005.
Lleres, D., M. Denegri, M. Biggiogera, P. Ajuh and A. I. Lamond (2010). "Direct interaction between hnRNP-M and CDC5L/PLRG1 proteins affects alternative splice site choice." EMBO Rep 11(6): 445-451.
Lum, L. C. S., K. T. Wong, S. K. Lam, K. B. Chua and A. Y. T. Goh (1998). "Neurogenic pulmonary oedema and enterovirus 71 encephalomyelitis." The Lancet 352(9137): 1391.
Matthews, D. A. and W. C. Russell (1998). "Adenovirus core protein V interacts with p32--a protein which is associated with both the mitochondria and the nucleus." J Gen Virol 79 ( Pt 7): 1677-1685.
McMinn, P., K. Lindsay, D. Perera, H. M. Chan, K. P. Chan and M. J. Cardosa (2001). "Phylogenetic analysis of enterovirus 71 strains isolated during linked epidemics in Malaysia, Singapore, and Western Australia." J Virol 75(16): 7732-7738.
Melnick, J. L. (1984). "Enterovirus type 71 infections: a varied clinical pattern sometimes mimicking paralytic poliomyelitis." Rev Infect Dis 6 Suppl 2: S387-390.
Moscufo, N., A. G. Yafal, A. Rogove, J. Hogle and M. Chow (1993). "A mutation in VP4 defines a new step in the late stages of cell entry by poliovirus." J Virol 67(8): 5075-5078.
Nishimura, Y., M. Shimojima, Y. Tano, T. Miyamura, T. Wakita and H. Shimizu (2009). "Human P-selectin glycoprotein ligand-1 is a functional receptor for enterovirus 71." Nat Med 15(7): 794-797.
Oberste, M. S., S. Penaranda, K. Maher and M. A. Pallansch (2004). "Complete genome sequences of all members of the species Human enterovirus A." Journal of General Virology 85: 1597–1607.
Ooi, M. H., S. C. Wong, Y. Podin, W. Akin, S. del Sel, A. Mohan, C. H. Chieng, D. Perera, D. Clear, D. Wong, E. Blake, J. Cardosa and T. Solomon (2007). "Human enterovirus 71 disease in Sarawak, Malaysia: a prospective clinical, virological, and molecular epidemiological study." Clin Infect Dis 44(5): 646-656.
Phan Van Tu, Nguyen Thi Thanh Thao, David Perera, Truong Khanh Huu, Nguyen Thi Kim Tien, Tang Chi Thuong, Ooi Mong How, Mary Jane Cardosa and P. C. McMinn (2007). "Epidemiologic and Virologic Investigation of Hand, Foot, and Mouth Disease, Southern Vietnam, 2005." Emerging Infectious Diseases 13(11): 1733-1741.
Reed, J. C., B. Molter, C. D. Geary, J. McNevin, J. McElrath, S. Giri, K. C. Klein and J. R. Lingappa (2012). "HIV-1 Gag co-opts a cellular complex containing DDX6, a helicase that facilitates capsid assembly." J Cell Biol 198(3): 439-456.
Schmidt, N. J., E. H. Lennette and H. H. Ho (1974). "An apparently new enterovirus isolated from patients with disease of the central nervous system." J Infect Dis 129(3): 304-309.
Shih, S. R., V. Stollar and M. L. Li (2011). "Host factors in enterovirus 71 replication." J Virol 85(19): 9658-9666.
Solomon, T., P. Lewthwaite, D. Perera, M. J. Cardosa, P. McMinn and M. H. Ooi (2010). "Virology, epidemiology, pathogenesis, and control of enterovirus 71." Lancet Infect Dis 10(11): 778-790.
Tange, T. O., T. H. Jensen and J. Kjems (1996). "In vitro interaction between human immunodeficiency virus type 1 Rev protein and splicing factor ASF/SF2-associated protein, p32." J Biol Chem 271(17): 10066-10072.
Tayyari, F., D. Marchant, T. J. Moraes, W. Duan, P. Mastrangelo and R. G. Hegele (2011). "Identification of nucleolin as a cellular receptor for human respiratory syncytial virus." Nat Med 17(9): 1132-1135.
van Leeuwen, H. C. and P. O'Hare (2001). "Retargeting of the mitochondrial protein p32/gC1Qr to a cytoplasmic compartment and the cell surface." J Cell Sci 114(Pt 11): 2115-2123.
Waggoner, S. N., C. H. Hall and Y. S. Hahn (2007). "HCV core protein interaction with gC1q receptor inhibits Th1 differentiation of CD4+ T cells via suppression of dendritic cell IL-12 production." J Leukoc Biol 82(6): 1407-1419.
Wang, Y., J. E. Finan, J. M. Middeldorp and S. D. Hayward (1997). "P32/TAP, a cellular protein that interacts with EBNA-1 of Epstein-Barr virus." Virology 236(1): 18-29.
Watthanasurorot, A., P. Jiravanichpaisal, I. Soderhall and K. Soderhall (2010). "A gC1qR prevents white spot syndrome virus replication in the freshwater crayfish Pacifastacus leniusculus." J Virol 84(20): 10844-10851.
Weng, T.-Y., L.-C. Chen, H.-W. Shyu, S.-H. Chen, J.-R. Wang, C.-K. Yu, H.-Y. Lei and T.-M. Yeh (2005). "Lactoferrin inhibits enterovirus 71 infection by binding to VP1 protein and host cells." Antiviral Research 67(1): 31-37.
West, A. P., G. S. Shadel and S. Ghosh (2011). "Mitochondria in innate immune responses." Nat Rev Immunol 11(6): 389-402.
Xu, L., N. Xiao, F. Liu, H. Ren and J. Gu (2009). "Inhibition of RIG-I and MDA5-dependent antiviral response by gC1qR at mitochondria." Proc Natl Acad Sci U S A 106(5): 1530-1535.
Yamayoshi, S., Y. Yamashita, J. Li, N. Hanagata, T. Minowa, T. Takemura and S. Koike (2009). "Scavenger receptor B2 is a cellular receptor for enterovirus 71." Nat Med 15(7): 798-801.
Yang, S. L., Y. T. Chou, C. N. Wu and M. S. Ho (2011). "Annexin II binds to capsid protein VP1 of enterovirus 71 and enhances viral infectivity." J Virol 85(22): 11809-11820.
Yao, L., X. Yan, H. Dong, D. R. Nelson, C. Liu and X. Li (2011). "Expression of an IRF-3 fusion protein and mouse estrogen receptor, inhibits hepatitis C viral replication in RIG-I-deficient Huh 7.5 cells." Virol J 8: 445.
Yao, Z. Q., A. Eisen-Vandervelde, S. Ray and Y. S. Hahn (2003). "HCV core/gC1qR interaction arrests T cell cycle progression through stabilization of the cell cycle inhibitor p27Kip1." Virology 314(1): 271-282.
Yeo, W. M. and V. T. Chow (2007). "The VP1 structural protein of enterovirus 71 interacts with human ornithine decarboxylase and gene trap ankyrin repeat." Microb Pathog 42(4): 129-137.
論文全文使用權限
  • 同意授權校內瀏覽/列印電子全文服務,於2018-08-05起公開。
  • 同意授權校外瀏覽/列印電子全文服務,於2018-08-05起公開。


  • 如您有疑問,請聯絡圖書館
    聯絡電話:(06)2757575#65773
    聯絡E-mail:etds@email.ncku.edu.tw