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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳美如(Mei-Ru Chen) | |
| dc.contributor.author | Hung-Chun Liu | en |
| dc.contributor.author | 劉泓均 | zh_TW |
| dc.date.accessioned | 2021-07-11T15:40:42Z | - |
| dc.date.available | 2025-08-17 | |
| dc.date.copyright | 2020-09-10 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-08-17 | |
| dc.identifier.citation | Alber, F., S. Dokudovskaya, L.M. Veenhoff, W. Zhang, J. Kipper, D. Devos, A. Suprapto, O. Karni-Schmidt, R. Williams, and B.T. Chait. 2007. The molecular architecture of the nuclear pore complex. Nature. 450:695-701. Arii, J., M. Watanabe, F. Maeda, N. Tokai-Nishizumi, T. Chihara, M. Miura, Y. Maruzuru, N. Koyanagi, A. Kato, and Y. Kawaguchi. 2018. ESCRT-III mediates budding across the inner nuclear membrane and regulates its integrity. Nat Commun. 9:3379. Bailer, S.M. 2017. Venture from the Interior-Herpesvirus pUL31 Escorts Capsids from Nucleoplasmic Replication Compartments to Sites of Primary Envelopment at the Inner Nuclear Membrane. Cells. 6. Bell, N.M., and A.M. Lever. 2013. HIV Gag polyprotein: processing and early viral particle assembly. Trends Microbiol. 21:136-144. Bigalke, J.M., and E.E. Heldwein. 2015. Structural basis of membrane budding by the nuclear egress complex of herpesviruses. Embo j. 34:2921-2936. Braunagel, S.C., S.T. Williamson, S. Saksena, Z. Zhong, W.K. Russell, D.H. Russell, and M.D. Summers. 2004. Trafficking of ODV-E66 is mediated via a sorting motif and other viral proteins: facilitated trafficking to the inner nuclear membrane. Proc Natl Acad Sci U S A. 101:8372-8377. Chang, Y., C.H. Tung, Y.T. Huang, J. Lu, J.Y. Chen, and C.H. Tsai. 1999. Requirement for cell-to-cell contact in Epstein-Barr virus infection of nasopharyngeal carcinoma cells and keratinocytes. J Virol. 73:8857-8866. Christ, L., C. Raiborg, E.M. Wenzel, C. Campsteijn, and H. Stenmark. 2017. Cellular Functions and Molecular Mechanisms of the ESCRT Membrane-Scission Machinery. Trends Biochem Sci. 42:42-56. Crawford, D.H., A. Rickinson, and I. Johannessen. 2014. Cancer virus: the story of Epstein-Barr virus. Oxford University Press. Dai, Y.C., Y.T. Liao, Y.T. Juan, Y.Y. Cheng, M.T. Su, Y.Z. Su, H.C. Liu, C.H. Tsai, C.P. Lee, and M.R. Chen. 2020. The Novel Nuclear Targeting and BFRF1-Interacting Domains of BFLF2 Are Essential for Efficient Epstein-Barr Virus Virion Release. J Virol. 94. Davies, B.A., I.F. Azmi, J. Payne, A. Shestakova, B.F. Horazdovsky, M. Babst, and D.J. Katzmann. 2010. Coordination of substrate binding and ATP hydrolysis in Vps4-mediated ESCRT-III disassembly. Molecular biology of the cell. 21:3396-3408. De Magistris, P., and W. Antonin. 2018. The Dynamic Nature of the Nuclear Envelope. Curr Biol. 28:R487-r497. De Martel, C., J. Ferlay, S. Franceschi, J. Vignat, F. Bray, D. Forman, and M. Plummer. 2012. Global burden of cancers attributable to infections in 2008: a review and synthetic analysis. The lancet oncology. 13:607-615. Dechat, T., S.A. Adam, P. Taimen, T. Shimi, and R.D. Goldman. 2010. Nuclear lamins. Cold Spring Harbor perspectives in biology. 2:a000547. Fauquet, C.M., M.A. Mayo, J. Maniloff, U. Desselberger, and L.A. Ball. 2005. Virus taxonomy: VIIIth report of the International Committee on Taxonomy of Viruses. Academic Press. Funk, C., M. Ott, V. Raschbichler, C.H. Nagel, A. Binz, B. Sodeik, R. Bauerfeind, and S.M. Bailer. 2015. The Herpes Simplex Virus Protein pUL31 Escorts Nucleocapsids to Sites of Nuclear Egress, a Process Coordinated by Its N-Terminal Domain. PLoS Pathog. 11:e1004957. Gagnon, K.T., L. Li, Y. Chu, B.A. Janowski, and D.R. Corey. 2014. RNAi factors are present and active in human cell nuclei. Cell Rep. 6:211-221. Garrus, J.E., U.K. von Schwedler, O.W. Pornillos, S.G. Morham, K.H. Zavitz, H.E. Wang, D.A. Wettstein, K.M. Stray, M. Côté, R.L. Rich, D.G. Myszka, and W.I. Sundquist. 2001. Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding. Cell. 107:55-65. Gonnella, R., A. Farina, R. Santarelli, S. Raffa, R. Feederle, R. Bei, M. Granato, A. Modesti, L. Frati, H.-J. Delecluse, M.R. Torrisi, A. Angeloni, and A. Faggioni. 2005. Characterization and Intracellular Localization of the Epstein-Barr Virus Protein BFLF2: Interactions with BFRF1 and with the Nuclear Lamina. Journal of Virology. 79:3713-3727. Granato, M., R. Feederle, A. Farina, R. Gonnella, R. Santarelli, B. Hub, A. Faggioni, and H.-J. Delecluse. 2008. Deletion of Epstein-Barr Virus BFLF2 Leads to Impaired Viral DNA Packaging and Primary Egress as Well as to the Production of Defective Viral Particles. Journal of Virology. 82:4042-4051. Hellberg, T., L. Paßvogel, K.S. Schulz, B.G. Klupp, and T.C. Mettenleiter. 2016. Nuclear Egress of Herpesviruses: The Prototypic Vesicular Nucleocytoplasmic Transport. Adv Virus Res. 94:81-140. Hinshaw, J.E., B.O. Carragher, and R.A. Milligan. 1992. Architecture and design of the nuclear pore complex. Cell. 69:1133-1141. Ho, R., and R.A. Hegele. 2019. Complex effects of laminopathy mutations on nuclear structure and function. Clinical genetics. 95:199-209. Jha, H.C., Y. Pei, and E.S. Robertson. 2016. Epstein-Barr Virus: Diseases Linked to Infection and Transformation. Front Microbiol. 7:1602. Kang, M.S., and E. Kieff. 2015. Epstein-Barr virus latent genes. Exp Mol Med. 47:e131. Katta, S.S., C.J. Smoyer, and S.L. Jaspersen. 2014. Destination: inner nuclear membrane. Trends in cell biology. 24:221-229. Katzmann, D.J., M. Babst, and S.D. Emr. 2001. Ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex, ESCRT-I. Cell. 106:145-155. Khan, G., and M.J. Hashim. 2014. Global burden of deaths from Epstein-Barr virus attributable malignancies 1990-2010. Infectious agents and cancer. 9:38. Khmelinskii, A., E. Blaszczak, M. Pantazopoulou, B. Fischer, D.J. Omnus, G. Le Dez, A. Brossard, A. Gunnarsson, J.D. Barry, M. Meurer, D. Kirrmaier, C. Boone, W. Huber, G. Rabut, P.O. Ljungdahl, and M. Knop. 2014. Protein quality control at the inner nuclear membrane. Nature. 516:410-413. Lake, C.M., and L.M. Hutt-Fletcher. 2004. The Epstein-Barr virus BFRF1 and BFLF2 proteins interact and coexpression alters their cellular localization. Virology. 320:99-106. Lange, A., R.E. Mills, C.J. Lange, M. Stewart, S.E. Devine, and A.H. Corbett. 2007. Classical nuclear localization signals: definition, function, and interaction with importin alpha. The Journal of biological chemistry. 282:5101-5105. Lee, C.P., P.T. Liu, H.N. Kung, M.T. Su, H.H. Chua, Y.H. Chang, C.W. Chang, C.H. Tsai, F.T. Liu, and M.R. Chen. 2012. The ESCRT machinery is recruited by the viral BFRF1 protein to the nucleus-associated membrane for the maturation of Epstein-Barr Virus. PLoS pathogens. 8:e1002904. Lim, R.Y., K.S. Ullman, and B. Fahrenkrog. 2008. Biology and biophysics of the nuclear pore complex and its components. International review of cell and molecular biology. 267:299-342. Lin, C.T., W.Y. Chan, W. Chen, H.M. Huang, H.C. Wu, M.M. Hsu, S.M. Chuang, and C.C. Wang. 1993. Characterization of seven newly established nasopharyngeal carcinoma cell lines. Lab Invest. 68:716-727. Lye, M.F., M. Sharma, K. El Omari, D.J. Filman, J.P. Schuermann, J.M. Hogle, and D.M. Coen. 2015. Unexpected features and mechanism of heterodimer formation of a herpesvirus nuclear egress complex. Embo j. 34:2937-2952. Maimon, T., N. Elad, I. Dahan, and O. Medalia. 2012. The human nuclear pore complex as revealed by cryo-electron tomography. Structure. 20:998-1006. Marschall, M., S. Häge, M. Conrad, S. Alkhashrom, J. Kicuntod, J. Schweininger, M. Kriegel, J. Lösing, J. Tillmanns, F. Neipel, J. Eichler, Y.A. Muller, and H. Sticht. 2020. Nuclear Egress Complexes of HCMV and Other Herpesviruses: Solving the Puzzle of Sequence Coevolution, Conserved Structures and Subfamily-Spanning Binding Properties. Viruses. 12. Martin-Serrano, J., S.W. Eastman, W. Chung, and P.D. Bieniasz. 2005. HECT ubiquitin ligases link viral and cellular PPXY motifs to the vacuolar protein-sorting pathway. J Cell Biol. 168:89-101. Mettenleiter, T.C. 2002. Herpesvirus Assembly and Egress. Journal of Virology. 76:1537-1547. Morita, E., V. Sandrin, J. McCullough, A. Katsuyama, I. Baci Hamilton, and W.I. Sundquist. 2011. ESCRT-III protein requirements for HIV-1 budding. Cell host microbe. 9:235-242. Morrison, L.A., and G.S. DeLassus. 2011. Breach of the nuclear lamina during assembly of herpes simplex viruses. Nucleus. 2:271-276. Muller, Y.A., S. Häge, S. Alkhashrom, T. Höllriegl, S. Weigert, S. Dolles, K. Hof, S.A. Walzer, C. Egerer-Sieber, M. Conrad, S. Holst, J. Lösing, E. Sonntag, H. Sticht, J. Eichler, and M. Marschall. 2020. High-resolution crystal structures of two prototypical β- and γ-herpesviral nuclear egress complexes unravel the determinants of subfamily specificity. J Biol Chem. 295:3189-3201. Patel, V.P., and C.T. Chu. 2011. Nuclear transport, oxidative stress, and neurodegeneration. Int J Clin Exp Pathol. 4:215-229. Pawliczek, T., and C.M. Crump. 2009a. Herpes simplex virus type 1 production requires a functional ESCRT-III complex but is independent of TSG101 and ALIX expression. Journal of virology. 83:11254-11264. Pawliczek, T., and C.M. Crump. 2009b. Herpes Simplex Virus Type 1 Production Requires a Functional ESCRT-III Complex but Is Independent of TSG101 and ALIX Expression. 83:11254-11264. Reichelt, R., A. Holzenburg, E.L. Buhle, Jr., M. Jarnik, A. Engel, and U. Aebi. 1990. Correlation between structure and mass distribution of the nuclear pore complex and of distinct pore complex components. J Cell Biol. 110:883-894. Reynolds, A.E., B.J. Ryckman, J.D. Baines, Y. Zhou, L. Liang, and R.J. Roller. 2001. U(L)31 and U(L)34 proteins of herpes simplex virus type 1 form a complex that accumulates at the nuclear rim and is required for envelopment of nucleocapsids. J Virol. 75:8803-8817. Roller, R.J., and J.D. Baines. 2017. Herpesvirus Nuclear Egress. Adv Anat Embryol Cell Biol. 223:143-169. Schmidt, O., and D. Teis. 2012. The ESCRT machinery. Curr Biol. 22:R116-120. Scourfield, E.J., and J. Martin-Serrano. 2017. Growing functions of the ESCRT machinery in cell biology and viral replication. Biochem Soc Trans. 45:613-634. Sette, P., V. Dussupt, and F. Bouamr. 2012a. Identification of the HIV-1 NC binding interface in Alix Bro1 reveals a role for RNA. Journal of virology. 86:11608-11615. Sette, P., V. Dussupt, and F. Bouamr. 2012b. Identification of the HIV-1 NC Binding Interface in Alix Bro1 Reveals a Role for RNA. 86:11608-11615. Skepper, J.N., A. Whiteley, H. Browne, and A. Minson. 2001. Herpes simplex virus nucleocapsids mature to progeny virions by an envelopment --> deenvelopment --> reenvelopment pathway. J Virol. 75:5697-5702. Sosa, B.A., U. Kutay, and T.U. Schwartz. 2013. Structural insights into LINC complexes. Current opinion in structural biology. 23:285-291. Stewart, C.L., K.J. Roux, and B. Burke. 2007. Blurring the boundary: the nuclear envelope extends its reach. Science. 318:1408-1412. Streck, N.T., J. Carmichael, and N.J. Buchkovich. 2018. Nonenvelopment Role for the ESCRT-III Complex during Human Cytomegalovirus Infection. J Virol. 92. Sundquist, W.I., and H.G. Kräusslich. 2012. HIV-1 assembly, budding, and maturation. Cold Spring Harb Perspect Med. 2:a006924. Tapley, E.C., N. Ly, and D.A. Starr. 2011. Multiple mechanisms actively target the SUN protein UNC-84 to the inner nuclear membrane. Mol Biol Cell. 22:1739-1752. Ungricht, R., and U. Kutay. 2017. Mechanisms and functions of nuclear envelope remodelling. Nature Reviews molecular cell biology. 18:229. Vietri, M., M. Radulovic, and H. Stenmark. 2020. The many functions of ESCRTs. Nat Rev Mol Cell Biol. 21:25-42. Walzer, S.A., C. Egerer-Sieber, H. Sticht, M. Sevvana, K. Hohl, J. Milbradt, Y.A. Muller, and M. Marschall. 2015. Crystal Structure of the Human Cytomegalovirus pUL50-pUL53 Core Nuclear Egress Complex Provides Insight into a Unique Assembly Scaffold for Virus-Host Protein Interactions. J Biol Chem. 290:27452-27458. Wente, S.R. 2000. Gatekeepers of the nucleus. Science. 288:1374-1377. Wente, S.R., and M.P. Rout. 2010. The nuclear pore complex and nuclear transport. Cold Spring Harb Perspect Biol. 2:a000562. Whitley, R.J. 1996. Herpesviruses. In Medical Microbiology. S. Baron, editor. University of Texas Medical Branch at Galveston Copyright © 1996, The University of Texas Medical Branch at Galveston., Galveston (TX). Young, L.S., L.F. Yap, and P.G. Murray. 2016. Epstein-Barr virus: more than 50 years old and still providing surprises. Nat Rev Cancer. 16:789-802. Zeev-Ben-Mordehai, T., M. Weberruß, M. Lorenz, J. Cheleski, T. Hellberg, C. Whittle, K. El Omari, D. Vasishtan, K.C. Dent, K. Harlos, K. Franzke, C. Hagen, B.G. Klupp, W. Antonin, T.C. Mettenleiter, and K. Grünewald. 2015. Crystal Structure of the Herpesvirus Nuclear Egress Complex Provides Insights into Inner Nuclear Membrane Remodeling. Cell Rep. 13:2645-2652. Zhai, Q., R.D. Fisher, H.Y. Chung, D.G. Myszka, W.I. Sundquist, and C.P. Hill. 2008. Structural and functional studies of ALIX interactions with YPX(n)L late domains of HIV-1 and EIAV. Nat Struct Mol Biol. 15:43-49. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/79061 | - |
| dc.description.abstract | 中文摘要 當EB病毒在細胞核內完成溶裂期複製(lytic replication)與衣殼化(encapsidation) 之後，會透過出核複合體(Nuclear egress complex) BFRF1/BFLF2的幫助運送到細胞質中，完成後續病毒顆粒的成熟以及釋出步驟。在先前實驗室的研究中發現BFRF1可以透過吸引Alix的方式，藉由ESCRT模組(Endosomal sorting complex required for transport machinery)幫助病毒的出核。在單獨表現BFRF1時，BFRF1就能夠由核膜上產生囊泡(vesicles)釋放到細胞質中。先前實驗室研究發現BFRF1可以透過兩個區域與Alix進行交互作用，分別是Late domain 1 (LD1)區域與Alix的Bro區域可以進行交互作用，另外也可以透過EBV specific region (ESR)區域與Alix的proline rich region (PRR)進行交互作用。在本篇研究中發現ESRBFRF1-PRRAlix之間的交互作用會因為核酸酶的作用而降低，顯示ESR-PRR之間的交互作用可能有核酸的參與。另外在共軛焦顯微鏡觀察不同變異的BFRF1分子在細胞中的分布位置時發現ESR區域的刪除會使得BFRF1分子分散在細胞質中。更進一步使用細胞離拆 (cell fractionation) 分離細胞核與細胞質, 發現缺少ESR區域會使得BFRF1分布在細胞質中，因此我們推測ESR區域對BFRF1入核是重要的。利用內輸蛋白-β (importin β)的抑制劑Importazole (IPZ) 處理細胞之後發現BFRF1仍能夠與內膜蛋白emerin產生共位的現象，這個結果推測BFRF1的入核過程不需要內輸蛋白-β的幫助。為了探討BFRF1入核的可能途徑，我們更發現GFP-Alix在BFRF1共同表現的情況下，會分佈至細胞核中。我們推測BFRF1的入核機制可能需要Alix以及ESCRT模組的幫助。最後我們發現在帶有EB病毒的上皮細胞NA中表現CHMP1以及CHMP2等ESCRT-III分子的顯性抑制突變(dominant negative mutants)會導致釋出的病毒量減少。未來仍需進一步釐清不同ESCRT-III分子在EB病毒的成熟過程中所扮演的角色。 | zh_TW |
| dc.description.abstract | Abstract After Epstein-Barr virus (EBV) lytic DNA replication and encapsidation in the nucleus, nucleocapsids are transported into the cytoplasm through a nuclear egress complex (NEC) BFRF1/BFLF2 mediated mechanism for virion maturation and release. Previously, we demonstrated that BFRF1 is able to interact with Alix, which is an adaptor protein of endosomal sorting complex required for transport (ESCRT), to recruit the ESCRT machinery for viral nuclear egress. By expression alone, BFRF1 is able to induce nuclear envelope-derived cytoplasmic vesicles. Two domains of BFRF1 were mapped to interact with Alix, namely late domain 1 (LD1) interacts with BCK1-like resistance to osmotic shock protein 1 (Bro1) domain of Alix and EBV specific region (ESR) interacts with proline rich region (PRR) of Alix. In this study, we demonstrate that the interaction between ESRBFRF1 and PRRAlix decreased after nuclease treatment, suggesting this interaction is mediated by nucleic acids. In confocal microscopy, we found deletion of ESR domain (BFRF1 ESR) caused BFRF1 to diffuse in the cytoplasm. Furthermore, BFRF1 ESR was fractionated into the cytoplasmic fraction, suggesting ESR domain is required for the nuclear targeting of BFRF1. In the presence of importin β inhibitor Importazole (IPZ), BFRF1 still co-localizes with emerin, which is the inner nuclear membrane (INM) marker. It suggests that the nuclear membrane targeting mechanism of BFRF1 is independent of importin β. In addition, we found GFP-Alix is redistributed into the nucleus with BFRF1 co-expression. It suggests that the nuclear targeting pathway of BFRF1 may require Alix and ESCRT machinery. Finally, we showed that expression of dominant negative CHMP1 or CHMP2 in EBV positive epithelial cells NA leads to the decrease of virion secretion. The exact roles of these ESCRT-III components in EBV maturation process need to be further characterized. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-11T15:40:42Z (GMT). No. of bitstreams: 1 U0001-1708202018420200.pdf: 46500638 bytes, checksum: 4e2377bb2c182a67d8d4393ba49ada7e (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | Contents 口試委員審定書 誌謝 中文摘要 Abstract Contents 1. Introduction---------------------------------------------------------------------------------1 1.1 Epstein-Barr Virus (EBV)--------------------------------------------------------------1 1.1.1 The classification and the associated diseases of EBV-----------------------1 1.1.2 The life cycle of EBV------------------------------------------------------------2 1.2 The nucleocytoplasmic transport mechanism----------------------------------------3 1.2.1 The structure of nuclear envelope (NE)----------------------------------------3 1.2.2 Nuclear import and export-------------------------------------------------------4 1.2.3 The hypothetic models for inner nuclear membrane protein targeting----6 1.3 Nuclear egress of herpesviruses-------------------------------------------------------7 1.3.1 The nuclear egress process of herpesviruses--------------------------------7 1.3.2 Nuclear egress complex of herpes viruses----------------------------------8 1.3.3 The structure of nuclear egress complex------------------------------------9 1.3.4 EBV inner nuclear membrane protein BFRF1 and its interaction with BFLF2—10 1.3.5 The differences of nuclear egress between EBV and other herpesviruses---------11 1.4 The Endosomal Sorting Complex Required for transport (ESCRT) machinery--------11 1.4.1 The ESCRT machinery-------------------------------------------------------11 1.4.2 The cellular function of ESCRT machinery--------------------------------13 1.4.3 The regulation of ESCRT machinery during viral budding -------------13 1.4.4 The role of ESCRT machinery in the replication of Herpes viruses----14 1.4.5 The relationship between ESCRT adaptor protein Alix and BFRF1----15 1.5 Specific aims of this study------------------------------------------------------------15 2. Material and Methods--------------------------------------------------------------------17 2.1 Cell culture------------------------------------------------------------------------------17 2.2 Transfection----------------------------------------------------------------------------18 2.3 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis-------------------------------------------------------------------18 2.4 GST pull down assay------------------------------------------------------------------19 2.5 Indirect immunofluorescence assay-------------------------------------------------20 2.6 Cellular fractionation------------------------------------------------------------------21 2.6.1 To separate the cellular fractionation through mechanical force----------21 2.6.2 To separate the ER membrane into the cytoplasmic fractionation by detergent-containing hypotonic lysis buffer----------------------------------------22 2.7 Extraction of intracellular EBV DNA-----------------------------------------------22 2.8 Extraction of extracellular EBV DNA-----------------------------------------------23 2.9 Quantitative real-time PCR (qPCR) analysis---------------------------------------23 3. Results---------------------------------------------------------------------------------------25 3.1 The interaction between BFRF1 ESR domain and Alix PRR domain may be nucleic acid dependent----------------------------------------------------------------25 3.2 To determine the nuclear targeting mechanism of BFRF1------------------------27 3.2.1 ESR domain is required for BFRF1 INM targeting-------------------------27 3.2.2 To separate the ER membrane into the cytoplasmic fraction--------------29 3.2.3 HA-BFRF1 ΔESR is separated into the cytoplasmic fraction after fractionation ----------------------------------------------------------------------------30 3.2.4 The INM targeting process of BFRF1 may be independent of importin mediated nuclear import --------------------------------------------------------------31 3.2.5 Alix depletion may affect the nuclear targeting process of BFRF1-------32 3.2.6 GFP-Alix was redistributed into the nucleus and the cytoplasm with BFRF1 co-transfection----------------------------------------------------------------34 3.3 The subcellular localization of endogenous CHMP4B and VPS4 after BFRF1. overexpression-------------------------------------------------------------------------34 3.4 Screening of the ESCRT-III components participated in EBV maturation -----35 3.4.1 The reactivation time course of NA cells-------------------------------------36 3.4.2 CHMP1 and CHMP2 may participate in EBV maturation process ------37 4. Discussion----------------------------------------------------------------------------------38 4.1 BFRF1 ESR domain interacts with Alix PRR domain in a RNA dependent manner----------------------------------------------------------------------------------38 4.2 ESR domain is required for BFRF1 INM targeting-------------------------------39 4.3 BFRF1 targets to inner nuclear membrane through an importin-β independent mechanism------------------------------------------------------------------------------40 4.4 The INM targeting process of BFRF1 may be mediated by Alix----------------40 4.5 The recruitment of ESCRT components after BFRF1 overexpression----------42 4.6 The possible ESCRT-III components participating in EBV maturation---------43 5. Figures--------------------------------------------------------------------------------------48 Figure 1. The interaction between BFRF1 ESR and Alix PRR is mediated by nucleic acids----------------------------------------------------------------------------------------------48 Figure 2. ESR domain is required for BFRF1-mediated vesicle formation-------------50 Figure 3. Comparison of the fractionation protocols---------------------------------------52 Figure 4. HA-BFRF1 ΔESR was separated into the cytoplasmic fraction after fractionation-------------------------------------------------------------------------------------54 Figure 5. BFRF1 colocalized with emerin on the nuclear rim after IPZ treatment-----56 Figure 6. Alix knock down may affect BFRF1 nuclear targeting-------------------------58 Figure 7. GFP-Alix re-distributed into the nucleus with HA-BFRF1 co-expression---60 Figure 8. The subcellular distribution of CHMP4 and VPS4 after HA-BFRF1 transfection--------------------------------------------------------------------------------------62 Figure 9. The time course of EBV replication and release in NA cells-------------------64 Figure 10. CHMP1 and CHMP2 may participate in EBV maturation process----------66 6. Supplementary figures and tables--------------------------------------------------------67 Supplementary Figure 1. The interaction between BFRF1 and PRR domain of Alix is defective after nuclease treatment-------------------------------------------------------------67 Supplementary Figure 2. Knockdown of Alix expression abolished BFRF1 induced vesicle formation--------------------------------------------------------------------------------68 Table 1. Plasmids used in this study-----------------------------------------------------------69 Table 2. Primers---------------------------------------------------------------------------------70 Table 3. Primary antibodies--------------------------------------------------------------------71 Table 4. Secondary antibodies-----------------------------------------------------------------72 7. Reference-----------------------------------------------------------------------------------73 | |
| dc.language.iso | zh-TW | |
| dc.subject | ESCRT | zh_TW |
| dc.subject | EB病毒 | zh_TW |
| dc.subject | BFRF1 | zh_TW |
| dc.subject | 入核機制 | zh_TW |
| dc.subject | nuclear targeting | en |
| dc.subject | EBV | en |
| dc.subject | ESCRT | en |
| dc.subject | BFRF1 | en |
| dc.title | 探討EB病毒BFRF1蛋白質功能區域以及入核機制之研究 | zh_TW |
| dc.title | Characterization of the functional domains and the nuclear targeting mechanism of EBV BFRF1 | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 陳炳宏(Ping-Hung Chen),劉雅雯(Ya-Wen Liu),李重霈(Chung-Pei Lee) | |
| dc.subject.keyword | EB病毒,BFRF1,入核機制,ESCRT, | zh_TW |
| dc.subject.keyword | EBV,BFRF1,nuclear targeting,ESCRT, | en |
| dc.relation.page | 78 | |
| dc.identifier.doi | 10.6342/NTU202003837 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2020-08-18 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 微生物學研究所 | zh_TW |
| dc.date.embargo-lift | 2025-08-17 | - |
| 顯示於系所單位： | 微生物學科所 | |
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