請用此 Handle URI 來引用此文件：
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/101748完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 潘建源 | zh_TW |
| dc.contributor.advisor | Chien-Yuan Pan | en |
| dc.contributor.author | 郭昕 | zh_TW |
| dc.contributor.author | Hsin Kuo | en |
| dc.date.accessioned | 2026-03-04T16:15:30Z | - |
| dc.date.available | 2026-03-05 | - |
| dc.date.copyright | 2026-03-04 | - |
| dc.date.issued | 2026 | - |
| dc.date.submitted | 2026-02-24 | - |
| dc.identifier.citation | Ali, A., Avalos, R. T., Ponimaskin, E., & Nayak, D. P. (2000). Influenza virus assembly: effect of influenza virus glycoproteins on the membrane association of M1 protein. Journal of virology, 74(18), 8709-8719.
Benton, D. J., Gamblin, S. J., Rosenthal, P. B., & Skehel, J. J. (2020). Structural transitions in influenza haemagglutinin at membrane fusion pH. Nature, 583(7814), 150-153. Bhattacharjee, C., & Mukhopadhyay, A. (2022). Generation of fluorescent HCV pseudoparticles to study early viral entry events-involvement of Rab1a in HCV entry. VirusDisease, 33(2), 172-184. Bogdanow, B., Wang, X., Eichelbaum, K., Sadewasser, A., Husic, I., Paki, K., Budt, M., Hergeselle, M., Vetter, B., & Hou, J. (2019). The dynamic proteome of influenza A virus infection identifies M segment splicing as a host range determinant. Nature Communications, 10(1), 5518. Bonazza, S., & Courtney, D. G. (2025). Influenza A virus RNA localisation and the interceding trafficking pathways of the host cell. PLoS Pathogens, 21(4), e1013090. Bonomini, A., Mercorelli, B., & Loregian, A. (2025). Antiviral strategies against influenza virus: An update on approved and innovative therapeutic approaches. Cellular and Molecular Life Sciences, 82(1), 75. Bouvier, N. M., & Palese, P. (2008). The biology of influenza viruses. Vaccine, 26, D49-D53. Branon, T. C., Bosch, J. A., Sanchez, A. D., Udeshi, N. D., Svinkina, T., Carr, S. A., Feldman, J. L., Perrimon, N., & Ting, A. Y. (2018). Efficient proximity labeling in living cells and organisms with TurboID. Nature biotechnology, 36(9), 880-887. Brass, A. L., Huang, I.-C., Benita, Y., John, S. P., Krishnan, M. N., Feeley, E. M., Ryan, B. J., Weyer, J. L., Van Der Weyden, L., & Fikrig, E. (2009). The IFITM proteins mediate cellular resistance to influenza A H1N1 virus, West Nile virus, and dengue virus. Cell, 139(7), 1243-1254. Carter, T., & Iqbal, M. (2024). The influenza a virus replication cycle: a comprehensive review. Viruses, 16(2), 316. Chaimayo, C., Hayashi, T., Underwood, A., Hodges, E., & Takimoto, T. (2017). Selective incorporation of vRNP into influenza A virions determined by its specific interaction with M1 protein. Virology, 505, 23-32. Cheah, J. S., & Yamada, S. (2017). A simple elution strategy for biotinylated proteins bound to streptavidin conjugated beads using excess biotin and heat. Biochemical and biophysical research communications, 493(4), 1522-1527. Chen, B. J., Leser, G. P., Jackson, D., & Lamb, R. A. (2008). The influenza virus M2 protein cytoplasmic tail interacts with the M1 protein and influences virus assembly at the site of virus budding. Journal of virology, 82(20), 10059-10070. Coombs, K. M., Berard, A., Xu, W., Krokhin, O., Meng, X., Cortens, J. P., Kobasa, D., Wilkins, J., & Brown, E. G. (2010). Quantitative proteomic analyses of influenza virus-infected cultured human lung cells. Journal of virology, 84(20), 10888-10906. Dahmani, I., Ludwig, K., & Chiantia, S. (2019). Influenza A matrix protein M1 induces lipid membrane deformation via protein multimerization. Bioscience reports, 39(8), BSR20191024. Dou, D., Revol, R., Östbye, H., Wang, H., & Daniels, R. (2018). Influenza A virus cell entry, replication, virion assembly and movement. Frontiers in immunology, 9, 1581. Elegheert, J., Behiels, E., Bishop, B., Scott, S., Woolley, R. E., Griffiths, S. C., Byrne, E. F., Chang, V. T., Stuart, D. I., & Jones, E. Y. (2018). Lentiviral transduction of mammalian cells for fast, scalable and high-level production of soluble and membrane proteins. Nature protocols, 13(12), 2991-3017. Finkelshtein, D., Werman, A., Novick, D., Barak, S., & Rubinstein, M. (2013). LDL receptor and its family members serve as the cellular receptors for vesicular stomatitis virus. Proceedings of the National Academy of Sciences, 110(18), 7306-7311. Flesch, F. M., Yu, J. W., Lemmon, M. A., & Burger, K. N. (2005). Membrane activity of the phospholipase C-δ1 pleckstrin homology (PH) domain. Biochemical Journal, 389(2), 435-441. Green, N. (1963). Avidin. 1. The use of [14C] biotin for kinetic studies and for assay. Biochemical Journal, 89(3), 585. Gyurkovska, V., Murtazina, R., Zhao, S. F., Shikano, S., Okamoto, Y., & Segev, N. (2023). Dual function of Rab1A in secretion and autophagy: hypervariable domain dependence. Life Science Alliance, 6(5). Haas, K. M., McGregor, M. J., Bouhaddou, M., Polacco, B. J., Kim, E.-Y., Nguyen, T. T., Newton, B. W., Urbanowski, M., Kim, H., & Williams, M. A. (2023). Proteomic and genetic analyses of influenza A viruses identify pan-viral host targets. Nature Communications, 14(1), 6030. Han, J., Perez, J. T., Chen, C., Li, Y., Benitez, A., Kandasamy, M., Lee, Y., Andrade, J., & Manicassamy, B. (2018). Genome-wide CRISPR/Cas9 screen identifies host factors essential for influenza virus replication. Cell reports, 23(2), 596-607. Hao, L., Sakurai, A., Watanabe, T., Sorensen, E., Nidom, C. A., Newton, M. A., Ahlquist, P., & Kawaoka, Y. (2008). Drosophila RNAi screen identifies host genes important for influenza virus replication. Nature, 454(7206), 890-893. Hayden, F. (2009). Developing new antiviral agents for influenza treatment: what does the future hold? Clinical Infectious Diseases, 48(Supplement_1), S3-S13. Heaton, N. S., Moshkina, N., Fenouil, R., Gardner, T. J., Aguirre, S., Shah, P. S., Zhao, N., Manganaro, L., Hultquist, J. F., & Noel, J. (2016). Targeting viral proteostasis limits influenza virus, HIV, and dengue virus infection. Immunity, 44(1), 46-58. Hussain, M., Galvin, H. D., Haw, T. Y., Nutsford, A. N., & Husain, M. (2017). Drug resistance in influenza A virus: the epidemiology and management. Infection and drug resistance, 121-134. Hutagalung, A. H., & Novick, P. J. (2011). Role of Rab GTPases in membrane traffic and cell physiology. Physiological reviews, 91(1), 119-149. Hutchinson, E. C., Charles, P. D., Hester, S. S., Thomas, B., Trudgian, D., Martínez-Alonso, M., & Fodor, E. (2014). Conserved and host-specific features of influenza virion architecture. Nature Communications, 5(1), 4816. Jiang, L., Chen, H., & Li, C. (2023). Advances in deciphering the interactions between viral proteins of influenza A virus and host cellular proteins. Cell Insight, 2(2), 100079. Jowers, T. P., Featherstone, R. J., Reynolds, D. K., Brown, H. K., James, J., Prescott, A., Haga, I. R., & Beard, P. M. (2015). RAB1A promotes Vaccinia virus replication by facilitating the production of intracellular enveloped virions. Virology, 475, 66-73. König, R., Stertz, S., Zhou, Y., Inoue, A., Hoffmann, H.-H., Bhattacharyya, S., Alamares, J. G., Tscherne, D. M., Ortigoza, M. B., & Liang, Y. (2010). Human host factors required for influenza virus replication. Nature, 463(7282), 813-817. Karlas, A., Machuy, N., Shin, Y., Pleissner, K.-P., Artarini, A., Heuer, D., Becker, D., Khalil, H., Ogilvie, L. A., & Hess, S. (2010). Genome-wide RNAi screen identifies human host factors crucial for influenza virus replication. Nature, 463(7282), 818-822. Katiyar, H., Arduini, A., Li, Y., & Liang, C. (2024). SARS-CoV-2 assembly: Gaining infectivity and beyond. Viruses, 16(11), 1648. Kawakami, E., Watanabe, T., Fujii, K., Goto, H., Watanabe, S., Noda, T., & Kawaoka, Y. (2011). Strand-specific real-time RT-PCR for distinguishing influenza vRNA, cRNA, and mRNA. Journal of virological methods, 173(1), 1-6. Kim, H., Webster, R. G., & Webby, R. J. (2018). Influenza virus: dealing with a drifting and shifting pathogen. Viral immunology, 31(2), 174-183. Krammer, F., Smith, G. J. D., Fouchier, R. A. M., Peiris, M., Kedzierska, K., Doherty, P. C., Palese, P., Shaw, M. L., Treanor, J., Webster, R. G., & García-Sastre, A. (2018). Influenza. Nature Reviews Disease Primers, 4(1), 3. Kummer, S., Flöttmann, M., Schwanhäusser, B., Sieben, C., Veit, M., Selbach, M., Klipp, E., & Herrmann, A. (2014). Alteration of protein levels during influenza virus H1N1 infection in host cells: a proteomic survey of host and virus reveals differential dynamics. PLoS One, 9(4), e94257. Kuo, R.-L., Chen, C.-J., Tam, E.-H., Huang, C.-G., Li, L.-H., Li, Z.-H., Su, P.-C., Liu, H.-P., & Wu, C.-C. (2018). Interactome analysis of NS1 protein encoded by influenza A H7N9 virus reveals an inhibitory role of NS1 in host mRNA maturation. Journal of proteome research, 17(4), 1474-1484. Li, B., Clohisey, S. M., Chia, B. S., Wang, B., Cui, A., Eisenhaure, T., Schweitzer, L. D., Hoover, P., Parkinson, N. J., & Nachshon, A. (2020). Genome-wide CRISPR screen identifies host dependency factors for influenza A virus infection. Nature Communications, 11(1), 164. Lin JiHui, L. J., Wang ChengBao, W. C., Liang WuLong, L. W., Zhang Jing, Z. J., Zhang LongXiang, Z. L., Lv HuiFang, L. H., Dong Wang, D. W., & Zhang YanMing, Z. Y. (2018). Rab1A is required for assembly of classical swine fever virus particle. Madsen, J. J., Grime, J. M., Rossman, J. S., & Voth, G. A. (2018). Entropic forces drive clustering and spatial localization of influenza A M2 during viral budding. Proceedings of the National Academy of Sciences, 115(37), E8595-E8603. Mair, C. M., Ludwig, K., Herrmann, A., & Sieben, C. (2014). Receptor binding and pH stability—How influenza A virus hemagglutinin affects host-specific virus infection. Biochimica et Biophysica Acta (BBA)-Biomembranes, 1838(4), 1153-1168. Martell, J. D., Deerinck, T. J., Sancak, Y., Poulos, T. L., Mootha, V. K., Sosinsky, G. E., Ellisman, M. H., & Ting, A. Y. (2012). Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy. Nature biotechnology, 30(11), 1143-1148. Martyna, A., Bahsoun, B., Badham, M. D., Srinivasan, S., Howard, M. J., & Rossman, J. S. (2017). Membrane remodeling by the M2 amphipathic helix drives influenza virus membrane scission. Scientific Reports, 7(1), 44695. Mayer, D., Molawi, K., Martínez-Sobrido, L., Ghanem, A., Thomas, S., Baginsky, S., Grossmann, J., García-Sastre, A., & Schwemmle, M. (2007). Identification of cellular interaction partners of the influenza virus ribonucleoprotein complex and polymerase complex using proteomic-based approaches. Journal of proteome research, 6(2), 672-682. McAuley, J. L., Gilbertson, B. P., Trifkovic, S., Brown, L. E., & McKimm-Breschkin, J. L. (2019). Influenza virus neuraminidase structure and functions. Frontiers in microbiology, 10, 39. Mellacheruvu, D., Wright, Z., Couzens, A. L., Lambert, J.-P., St-Denis, N. A., Li, T., Miteva, Y. V., Hauri, S., Sardiu, M. E., & Low, T. Y. (2013). The CRAPome: a contaminant repository for affinity purification–mass spectrometry data. Nature methods, 10(8), 730-736. Nachmias, D., Sklan, E. H., Ehrlich, M., & Bacharach, E. (2012). Human immunodeficiency virus type 1 envelope proteins traffic toward virion assembly sites via a TBC1D20/Rab1-regulated pathway. Retrovirology, 9(1), 7. Nayak, D. P., Balogun, R. A., Yamada, H., Zhou, Z. H., & Barman, S. (2009). Influenza virus morphogenesis and budding. Virus Research, 143(2), 147-161. Nayak, D. P., Hui, E. K.-W., & Barman, S. (2004). Assembly and budding of influenza virus. Virus Research, 106(2), 147-165. Noda, T., & Kawaoka, Y. (2010). Structure of influenza virus ribonucleoprotein complexes and their packaging into virions. Reviews in medical virology, 20(6), 380-391. O'Neill, R. E., Talon, J., & Palese, P. (1998). The influenza virus NEP (NS2 protein) mediates the nuclear export of viral ribonucleoproteins. The EMBO journal. Paules, C., & Subbarao, K. (2017). Influenza. The Lancet, 390(10095), 697-708. Petrich, A., Dunsing, V., Bobone, S., & Chiantia, S. (2021). Influenza A M2 recruits M1 to the plasma membrane: A fluorescence fluctuation microscopy study. Biophysical Journal, 120(24), 5478-5490. Peukes, J., Xiong, X., Erlendsson, S., Qu, K., Wan, W., Calder, L. J., Schraidt, O., Kummer, S., Freund, S. M., & Kräusslich, H.-G. (2020). The native structure of the assembled matrix protein 1 of influenza A virus. Nature, 587(7834), 495-498. Potter, C. W. (2001). A history of influenza. Journal of Applied Microbiology, 91(4), 572-579. Rees, J. S., Li, X. W., Perrett, S., Lilley, K. S., & Jackson, A. P. (2017). Selective proteomic proximity labeling assay using tyramide (SPPLAT): a quantitative method for the proteomic analysis of localized membrane‐bound protein clusters. Current Protocols in Protein Science, 88(1), 19.27. 11-19.27. 18. Rosário-Ferreira, N., Preto, A. J., Melo, R., Moreira, I. S., & Brito, R. M. (2020). The central role of non-structural protein 1 (NS1) in influenza biology and infection. International Journal of Molecular Sciences, 21(4), 1511. Rossman, J. S., Jing, X., Leser, G. P., Balannik, V., Pinto, L. H., & Lamb, R. A. (2010). Influenza virus m2 ion channel protein is necessary for filamentous virion formation. Journal of virology, 84(10), 5078-5088. Rossman, J. S., Jing, X., Leser, G. P., & Lamb, R. A. (2010). Influenza virus M2 protein mediates ESCRT-independent membrane scission. Cell, 142(6), 902-913. Rossman, J. S., & Lamb, R. A. (2011). Influenza virus assembly and budding. Virology, 411(2), 229-236. Roux, K. J., Kim, D. I., Raida, M., & Burke, B. (2012). A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. Journal of cell biology, 196(6), 801-810. Schmidt, N. W., Mishra, A., Wang, J., DeGrado, W. F., & Wong, G. C. (2013). Influenza virus A M2 protein generates negative Gaussian membrane curvature necessary for budding and scission. Journal of the American Chemical Society, 135(37), 13710-13719. Shao, W., Li, X., Goraya, M. U., Wang, S., & Chen, J.-L. (2017). Evolution of influenza a virus by mutation and re-assortment. International Journal of Molecular Sciences, 18(8), 1650. Shapira, S. D., Gat-Viks, I., Shum, B. O., Dricot, A., de Grace, M. M., Wu, L., Gupta, P. B., Hao, T., Silver, S. J., & Root, D. E. (2009). A physical and regulatory map of host-influenza interactions reveals pathways in H1N1 infection. Cell, 139(7), 1255-1267. Shaw, M. L., Stone, K. L., Colangelo, C. M., Gulcicek, E. E., & Palese, P. (2008). Cellular proteins in influenza virus particles. PLoS Pathogens, 4(6), e1000085. Shtyrya, Y., Mochalova, L., & Bovin, N. (2009). Influenza virus neuraminidase: structure and function. Acta Naturae (англоязычная версия), 1(2 (2)), 26-32. Simon, P. F. o., McCorrister, S., Hu, P., Chong, P., Silaghi, A., Westmacott, G., Coombs, K. M., & Kobasa, D. (2015). Highly pathogenic H5N1 and novel H7N9 influenza A viruses induce more profound proteomic host responses than seasonal and pandemic H1N1 strains. Journal of proteome research, 14(11), 4511-4523. Singh, S. M., & Murray, D. (2003). Molecular modeling of the membrane targeting of phospholipase C pleckstrin homology domains. Protein science, 12(9), 1934-1953. Spearman, P. (2018). Viral interactions with host cell Rab GTPases. Small GTPases, 9(1-2), 192-201. Szklarczyk, D., Kirsch, R., Koutrouli, M., Nastou, K., Mehryary, F., Hachilif, R., Gable, A. L., Fang, T., Doncheva, N. T., & Pyysalo, S. (2023). The STRING database in 2023: protein–protein association networks and functional enrichment analyses for any sequenced genome of interest. Nucleic acids research, 51(D1), D638-D646. Treanor, J. (2004). Influenza vaccine—outmaneuvering antigenic shift and drift. New England Journal of Medicine, 350(3), 218-220. Tsai, W.-J. (2023). A proximity-labeling proteomic approach to identify the cell membrane-associated factors in the late stage of IAV infection [Masters' Thesis, Naitonal Taiwan University]. Taipei. Vester, D., Rapp, E., Gade, D., Genzel, Y., & Reichl, U. (2009). Quantitative analysis of cellular proteome alterations in human influenza A virus‐infected mammalian cell lines. Proteomics, 9(12), 3316-3327. Várnai, P., & Balla, T. (1998). Visualization of phosphoinositides that bind pleckstrin homology domains: calcium-and agonist-induced dynamic changes and relationship to myo-[3H] inositol-labeled phosphoinositide pools. The Journal of cell biology, 143(2), 501-510. Wang, L., Fu, B., Li, W., Patil, G., Liu, L., Dorf, M. E., & Li, S. (2017). Comparative influenza protein interactomes identify the role of plakophilin 2 in virus restriction. Nature Communications, 8(1), 13876. Watanabe, T., Kawakami, E., Shoemaker, J. E., Lopes, T. J., Matsuoka, Y., Tomita, Y., Kozuka-Hata, H., Gorai, T., Kuwahara, T., & Takeda, E. (2014). Influenza virus-host interactome screen as a platform for antiviral drug development. Cell host & microbe, 16(6), 795-805. Watanabe, T., Watanabe, S., & Kawaoka, Y. (2010). Cellular networks involved in the influenza virus life cycle. Cell host & microbe, 7(6), 427-439. Wiśniewski, J. R. (2019). Filter aided sample preparation–a tutorial. Analytica chimica acta, 1090, 23-30. Yamauchi, Y. (2018). Influenza virus. Springer. Yamayoshi, S., Watanabe, M., Goto, H., & Kawaoka, Y. (2016). Identification of a novel viral protein expressed from the PB2 segment of influenza A virus. Journal of virology, 90(1), 444-456. Yang, X., Li, X., Zhang, Y., Rodriguez-Rodriguez, L., Xiang, M., Wang, H.-Y., & Zheng, X. S. (2016). Rab1 in cell signaling, cancer and other diseases. Oncogene, 35(44), 5699-5704. Ye, Z., Liu, T., Offringa, D. P., McInnis, J., & Levandowski, R. A. (1999). Association of influenza virus matrix protein with ribonucleoproteins. Journal of virology, 73(9), 7467-7473. Yi, C., Cai, C., Cheng, Z., Zhao, Y., Yang, X., Wu, Y., Wang, X., Jin, Z., Xiang, Y., & Jin, M. (2022). Genome-wide CRISPR-Cas9 screening identifies the CYTH2 host gene as a potential therapeutic target of influenza viral infection. Cell reports, 38(13). Zhou, Y., Zhou, B., Pache, L., Chang, M., Khodabakhshi, A. H., Tanaseichuk, O., Benner, C., & Chanda, S. K. (2019). Metascape provides a biologist-oriented resource for the analysis of systems-level datasets. Nature Communications, 10(1), 1523. Zimmer, S. M., & Burke, D. S. (2009). Historical Perspective — Emergence of Influenza A (H1N1) Viruses. New England Journal of Medicine, 361(3), 279-285. | - |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/101748 | - |
| dc.description.abstract | 在A型流感病毒(Influenza A virus, IAV)感染的後期，新合成的病毒結構蛋白與基因組會集合到細胞膜上的脂筏 (lipid raft)處形成子代病毒。與其他包膜病毒不同，IAV不經由常見的內體分選複合體 (endosomal sorting complex required for transport, ESCRT)途徑進行出芽，而是採用一種非ESCRT依賴性的方式進行病毒組裝與出芽。目前關於IAV病毒顆粒形成的模型多著重於病毒蛋白在此過程中的功能，特別是基質蛋白(Matrix proteins)與神經胺酸酶 (Neuraminidase)，而宿主因子(Host Factors)在IAV的組裝與出芽機制中所扮演的角色與作用方式仍不明朗。儘管先前的研究透過許多不同方式提出可能參與IAV生命週期中各個階段的宿主因子，然而在後續進行的驗證中，只有其中少部分的宿主因子被證實對於病毒的組裝或出芽有所貢獻。因此，在這項研究中我們選擇運用以鄰近標記法 (Proximity-Labelling assay, PL assay)為基礎的蛋白質體學方法來辨識特定感染時間點會位在細胞膜周邊、可能與病毒蛋白有相互作用的宿主因子。
鄰近標記法利用特定的催化酶對其周圍半徑大約10奈米內的分子進行標定。而透過將此催化酶與具目標特徵的蛋白質（又稱「誘餌」）融合，就能標定特定亞細胞區域附近的分子。在本研究中，我們將錨定於細胞膜內側的PLCD1作為誘餌，建立了穩定表達其PH結構域與生物素連接酶 (Biotin Ligase) TurboID共同組成的融合蛋白 (PH-PLCD1-GFP-TurboID)的A549穩定細胞株。這個融合蛋白能在添加生物素 (Biotin)後迅速標記活細胞內足夠靠近細胞膜的宿主因子。利用這個方法，我們不僅可以標定本來就位於細胞膜附近的宿主因子，也可以標記那些短暫接近細胞膜或與細胞膜僅有微弱交互作用的因子。在標定後，我們以鏈親和素磁珠 (Streptavidin Magnetic Beads)純化被標定的蛋白質以進行質譜分析。藉此，我們得以鑑定出在IAV感染晚期的細胞膜周圍含量有所改變的宿主因子，進一步揭示其可能的功能，以及病毒與宿主在時間與空間上的交互作用。在未感染與IAV感染的組別中，質譜分析共鑑定出約6000個被生物素標記的蛋白質，我們從中選定了Ras-related proteins Rab-1A (Rab1A)為研究對象。Rab1A在感染後出現不同的分布，且抑制Rab1A表現會導致病毒核蛋白 (Nucleoprotein, NP)在細胞內的基因表現量與病毒產量的改變。我們將進一步研究這些新發現的宿主因子如何影響IAV的生命週期。 | zh_TW |
| dc.description.abstract | During the late stage of influenza A virus (IAV) infection, newly synthesized viral components, including the structural proteins and genomes, are recruited to the lipid raft domains of the cell plasma membrane to produce progeny virions. Unlike many other enveloped viruses, IAV buds from the cell through an endosomal sorting complex required for transport (ESCRT)-independent pathway. Current models of IAV virion formation primarily address the functions and roles of viral proteins, particularly matrix proteins and neuraminidase, in this process. However, the identities and functions of host factors participating in the assembly and budding mechanisms of IAV remain largely unknown. Although various studies had attempted to identify host factors that were involved in individual stages of the IAV life cycle with diverse approaches, only a few candidates were proven to contribute to virus assembly and budding processes. Therefore, in this study, we utilize the proximity-labelling (PL) based proteomic approach to investigate the host-virus interactions occurring near the plasma membrane at the infection times of interest.
The PL assay employs specific engineered enzymes that will label biomolecules within its 10-nm radius with certain substrates. By fusing the aforementioned enzyme with the protein possessing features of interest, also known as the “bait”, the labeling can be performed within the vicinity of the subcellular compartment where the bait is located. In this research, we fused the biotin ligase TurboID with the PH domain of PLCD1, which is located at the inner side of plasma membrane, and established a A549 cell line that stably expresses the PH-PLCD1-GFP-TurboID fusion protein. This construct enables prompt labeling of the nearby host factors after the addition of exogenous biotin. With this approach, we can not only label host factors abundant at the plasma membrane, but also those loosely or transiently associated with it. The biotinylated proteins were then purified by the magnetic streptavidin beads and further analyzed via mass spectrometry. This strategy enabled us to identify host proteins with altered abundance at the vicinity of plasma membrane during different stages of IAV infection, implying their potential functional roles in infection events, and study the spatiotemporal-specific virus-host interactome. Using mass spectrometry, we identified 6478 biotinylated proteins across mock and four IAV-infected groups. These were subjected to further data filtering to select high-confidence candidates. Among these candidates, Ras-related proteins Rab-1A (Rab1A) were selected for further examination. Rab1A exhibited altered subcellular distributions upon the infection and the knockdown of Rab1A resulted in changes in viral nucleoprotein (NP) gene expression as well as virus production. How these novel host factors influence the replication cycle of IAV will be further investigated. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2026-03-04T16:15:30Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2026-03-04T16:15:30Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 口試委員審定書 i
謝辭 ii 摘要 iv Abstract vi Table of Contents ix List of Figures xiv List of Tables xv Ⅰ. Introduction 1 1. Overview of the study: towards the host factors engaged in influenza virus assembly and budding at the plasma membrane 1 2. Influenza virus 3 2.1. Overview of influenza virus 4 2.2 Structure and protein functions of IAV 6 2.3 The life cycle of IAV 8 2.4 Approaches for identifying host factors involved in IAV life cycle 10 3. Proximity-labeling assay (PL assay) 14 4. Pilot study 16 5. Overview of the thesis 18 Ⅱ. Materials and methods 21 1. Cell and virus 21 2. DNA transfection of cells 22 2.1 Liposome-based transfection 22 2.2 Electroporation 22 2.3 Non-loposomal polymer-based transfection 22 3. Transformation, colony PCR and plasmid DNA purification 23 4. Pseudotyped lentivirus particle preparation and stable cell lines generation 24 5. Virus infection 25 6. Immunofluorescence assay (IFA) 26 7. Immunoblot analysis 26 8. Proximity labeling assay (PL assay) 27 9. Measurement of protein concentration 27 9.1 QubitTM Protein BR Assay 28 9.2 PierceTM 660 nM Protein Assay 28 9.3 PierceTM BCA Protein Assay 28 10. Purification of biotinylated proteins 28 11. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) 29 12. Data processing for data‐independent acquisition mass spectrometry 31 13. Bioinformatics and Statistics 32 14. Gene knockdown by siRNA 32 15. Total RNA extraction from infected cell and supernatant 32 16. Reverse transcription 33 17. Real-time polymerase chain reaction (qPCR) 33 18. Hemagglutination (HA) assay 34 19. Immunoprecipitation 34 20. Statistical analysis. 35 Ⅲ. Results 36 1. Construction of GFP-TurboID and PH-PLCD1-GFP(truncated)TurboID stable cell lines 36 1.1 Construction of GFP-TurboID and PH-PLCD1-GFP-(truncated)TurboID transfer plasmids 38 1.2 Establishment of GFP-TurboID and PH-PLCD1-GFP-(truncated)TurboID stable cell lines 39 2. Validation of stable cell lines 40 2.1 Subcellular localization of fusion protein in the stable cell lines 40 2.2 The impact of IAV infection on fusion protein localization and biotinylation in the stable cell lines 41 2.3 The impact of IAV infection on the expression level, molecular weight and the biotinylation profile of the fusion proteins in the stable cell lines 41 3. Mass spectrometry (MS)-based proteomics across infection time points 42 3.1 Sample preparation for MS-based proteomics 42 3.2 Analysis of the label-free quantitative mass spectrometry results 44 3.3 Analysis of proteins in cluster 1 49 4. Rab1A might potentially involve in IAV life cycle 51 4.1 Subcellular localization of Rab1A changes upon IAV infection 51 4.2 Rab1A depletion results in lower viral particle production 53 4.3 The interaction between Rab1A and IAV viral proteins 55 Ⅳ. Discussion 57 1. Data filtering of the identified proteins in the datasets 57 1.1 Unexpected challenges in background subtraction 57 1.2 Filtering proteins detected at a single timepoint in the PH-PLCD1-GFP-TurboID dataset 58 1.3 Establishing a rational filtering strategy using the GFP-TurboID and PH-PLCD1-GFP-(truncated)TurboID datasets 59 2. The interaction between Rab1A and influenza A virus components 60 3. The fluctuation in virus production in Rab1A knockdown cells 65 Ⅴ. References 66 Ⅵ. Figures 74 Ⅶ. Tables 91 | - |
| dc.language.iso | en | - |
| dc.subject | A型流感病毒 | - |
| dc.subject | 鄰近標定蛋白質體學 | - |
| dc.subject | 細胞膜 | - |
| dc.subject | 宿主因子 | - |
| dc.subject | Rab1A蛋白 | - |
| dc.subject | Influenza A virus | - |
| dc.subject | proximity labeling proteomics | - |
| dc.subject | plasma membrane | - |
| dc.subject | host factors | - |
| dc.subject | Rab1A | - |
| dc.title | 以鄰近標記蛋白質體學探究流感病毒形成時在細胞膜上的病毒—宿主交互作用之動態 | zh_TW |
| dc.title | Utilizing Proximity-Labeling Proteomics to Explore the Spatio-temporal Specific Influenza Virus-Host Interactome in the Virion Formation at the Plasma Membrane | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 114-1 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.coadvisor | 王宜萱 | zh_TW |
| dc.contributor.coadvisor | I-Hsuan Wang | en |
| dc.contributor.oralexamcommittee | 阮雪芬;張淑媛;蔡松智 | zh_TW |
| dc.contributor.oralexamcommittee | Hsueh-Fen Juan;Sui-Yuan Chang;Kevin Tsai | en |
| dc.subject.keyword | A型流感病毒,鄰近標定蛋白質體學細胞膜宿主因子Rab1A蛋白 | zh_TW |
| dc.subject.keyword | Influenza A virus,proximity labeling proteomicsplasma membranehost factorsRab1A | en |
| dc.relation.page | 100 | - |
| dc.identifier.doi | 10.6342/NTU202600659 | - |
| dc.rights.note | 同意授權(限校園內公開) | - |
| dc.date.accepted | 2026-02-24 | - |
| dc.contributor.author-college | 生命科學院 | - |
| dc.contributor.author-dept | 生命科學系 | - |
| dc.date.embargo-lift | 2026-03-05 | - |
| 顯示於系所單位： | 生命科學系 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-114-1.pdf 授權僅限NTU校內IP使用（校園外請利用VPN校外連線服務） | 3.9 MB | Adobe PDF |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。