臺灣博碩士論文加值系統

神經性壞死病毒（NNV）是一種魚類病毒，導致幼魚大量死亡，並在東南亞石斑魚水產養殖業造成重大經濟損失。NNV造成的疾病的治療和控制是困難的，主要因為病毒致病的分子基礎仍未完全了解。先前已經證明，病毒鞘蛋白（CP）定位於魚細胞中的核仁，表明可能有控制宿主的基因表現的作用。採用PCR site-directed mutagenesis方法來刪除或突變位於CP上的核仁定位序列（NoLS），從而產生突變CP NoLSDel和29AlaMut。共聚焦顯微鏡顯示NNVCP的核仁定位在轉染後3小時和24小時在轉染NoLSDel的GK細胞被完全阻斷，然而用29AlaMut轉染的細胞在3小時和24小時仍顯示29％和100％定位於核仁外周。從用野生型或突變型CP轉染的細胞收集RNA樣品並測序用於轉錄體學分析，並且揭示NNVCP核仁定位的調節作用在引入NNVCP後3小時後很早發生。與細胞蛋白表達，蛋白水解，細胞週期控制和核輸入相關的基因受NNVCP核仁定位的影響最大。這些基因在病毒感染期間也受到影響。另外，熒光素酶活性也在NNVCP轉染的細胞中上調。制定阻斷NNVCP的核仁定位或其下游效應的治療或預防策略將有助於緩解NNV帶來的水產養殖業的經濟壓力。

Nervous necrosis virus (NNV) is a devastating fish virus causing mass mortality in juvenile fish and causing significant economic losses in the grouper aquaculture industry in South East Asia. Treatment and control of the disease is difficult primarily because the molecular basis of pathogenicity of the virus is still not fully understood. It has previously been demonstrated that the viral capsid protein (CP) localizes to the nucleolus in fish cells signifying some role it may have in controlling gene expression of the host. We employed a PCR site-directed mutagenesis method to delete or mutate a nucleolar localization sequence (NoLS) located on the capsid protein thus generating the mutant capsids NoLSDel and 29AlaMut. Confocal microscopy showed that nucleolar localization of the NNVCP was completely blocked in GK cells transfected with NoLSDel at 3 hours and 24 hours post transfection, however the cells transfected with 29AlaMut still showed 29% and 100% localization to the nucleolar periphery at 3 hours and 24 hours post transfection, respectively. RNA samples were collected from cells transfected with the wild type or mutant capsids and sequenced for transcriptomic analysis, and it was revealed that the regulatory effects of NNVCP nucleolar localization occur quite early after at 3 hours after introduction of the NNVCP. Genes related with cell protein expression, proteolysis, cell cycle control and nuclear import were most influenced by NNVCP nucleolar localization. These genes were also affected during viral infection. Interestingly, luciferase activity was also upregulated in cells primed with the NNVCP. Development of therapeutic or prophylactic strategies that disrupt nucleolar localization of the NNVCP or its downstream effects would help alleviate the economic pressures on the aquaculture industry brought about by NNV.

1. Introduction 1
2. Literature Review 5
2.1 Nervous Necrosis Virus 5
2.2 Nervous Necrosis Virus Capsid Protein 6
2.3 Nucleolus 8
2.4 Viruses Targeting the Nucleolus 9
3. Methodology 12
3.1 PCR Site-directed mutagenesis and cloning of NNVCP NoLS variants 12
3.2 Cell culture, transfections and viral infection 14
3.3 SDS-PAGE and western blotting 14
3.4 Confocal microscopy and immunofluorescensce assay 15
3.5 RNA extraction 16
3.6 RNA sequencing, quantification and transcriptome assembly 17
3.7 cDNA synthesis 18
3.8 Real-time PCR 19
3.9 Luciferase assay 20
4. Results 21
4.1 Site-Directed PCR Mutagenesis Produced NoLS-Mutated and NoLS-Deleted Nervous Necrosis Virus Capsid Proteins 21
4.2 Complete Nucleolar Localization of the Nervous Necrosis Virus Capsid Protein is Blocked by Deletions and Mutations of the NoLS 22
4.3 Transcriptomics and qRT-PCR Analyses Reveal Several DEGs Associated with Nucleolar Localization of the Nervous Necrosis Virus Capsid Protein 23
4.4 Highlighted Genes Were Influenced by NNV in Virally Infected GK Cells 26
4.5 Luciferase Expression is Upregulated in GK Cells Transfected with Nervous Necrosis Virus Capsid Protein 27
5. Discussion 28
6. Figures and Tables 35
7. Appendix 51
8. References 54




 

Boisvert, F. M., S. van Koningsbruggen, J. Navascues and A. I. Lamond (2007). "The multifunctional nucleolus." Nat Rev Mol Cell Biol 8(7): 574-585.
Breuil, G., J. R. Bonami, J. F. Pepin and Y. Pichot (1991). "Viral infection (picorna-like virus) associated with mass mortalities in hatchery-reared sea-bass (Dicentrarchus labrax) larvae and juveniles." Aquaculture 97(2): 109-116.
Chen, L. J., Y. C. Su and J. R. Hong (2009). "Betanodavirus non-structural protein B1: A novel anti-necrotic death factor that modulates cell death in early replication cycle in fish cells." Virology 385(2): 444-454.
Chen, N.-C., M. Yoshimura, H.-H. Guan, T.-Y. Wang, Y. Misumi, C.-C. Lin, P. Chuankhayan, A. Nakagawa, S. I. Chan, T. Tsukihara, T.-Y. Chen and C.-J. Chen (2015). "Crystal Structures of a Piscine Betanodavirus: Mechanisms of Capsid Assembly and Viral Infection." PLOS Pathogens 11(10): e1005203.
Chen, R., J. D. Neill, M. K. Estes and B. V. Prasad (2006). "X-ray structure of a native calicivirus: structural insights into antigenic diversity and host specificity." Proc Natl Acad Sci U S A 103(21): 8048-8053.
Chen, S. P., R. H. Peng and P. P. Chiou (2015). "Modulatory effect of CpG oligodeoxynucleotide on a DNA vaccine against nervous necrosis virus in orange-spotted grouper (Epinephelus coioides)." Fish Shellfish Immunol 45(2): 919-926.
Clark, E., B. Nava and M. Caputi (2017). "Tat is a multifunctional viral protein that modulates cellular gene expression and functions." Oncotarget 8(16): 27569-27581.
den Boon, J. A., A. Diaz and P. Ahlquist (2010). "Cytoplasmic viral replication complexes." Cell host & microbe 8(1): 77-85.
Di Bacco, A., J. Ouyang, H.-Y. Lee, A. Catic, H. Ploegh and G. Gill (2006). "The SUMO-Specific Protease SENP5 Is Required for Cell Division." Molecular and Cellular Biology 26(12): 4489-4498.
Doan, Q. K., M. Vandeputte, B. Chatain, T. Morin and F. Allal (2017). "Viral encephalopathy and retinopathy in aquaculture: a review." Journal of Fish Diseases 40(5): 717-742.
Glazebrook, J. S., M. P. Heasman and S. W. de Beer (1990). "Picorna-like viral particles associated with mass mortalities in larval barramundi, Lates calcarifer Bloch." Journal of Fish Diseases 13(3): 245-249.
Gorlich, D. and U. Kutay (1999). "Transport between the cell nucleus and the cytoplasm." Annu Rev Cell Dev Biol 15: 607-660.
Gouzil, J., A. Fablet, E. Lara, G. Caignard, M. Cochet, C. Kundlacz, M. Palmarini, M. Varela, E. Breard, C. Sailleau, C. Viarouge, M. Coulpier, S. Zientara and D. Vitour (2016). "Nonstructural Protein NSs of Schmallenberg Virus Is Targeted to the Nucleolus and Induces Nucleolar Disorganization." Journal of virology 91(1): e01263-01216.
Guo, Y. X., K. Dallmann and J. Kwang (2003). "Identification of nucleolus localization signal of betanodavirus GGNNV protein alpha." Virology 306(2): 225-235.
Gustin, K. E. and P. Sarnow (2002). "Inhibition of nuclear import and alteration of nuclear pore complex composition by rhinovirus." J Virol 76(17): 8787-8796.
Hiscox, J. A. (2007). "RNA viruses: hijacking the dynamic nucleolus." Nature Reviews Microbiology 5: 119.
Huang, R., G. Zhu, J. Zhang, Y. Lai, Y. Xu, J. He and J. Xie (2017). "Betanodavirus-like particles enter host cells via clathrin-mediated endocytosis in a cholesterol-, pH- and cytoskeleton-dependent manner." Veterinary research 48(1): 8-8.
Iwamoto, T., K. Mise, A. Takeda, Y. Okinaka, K. Mori, M. Arimoto, T. Okuno and T. Nakai (2005). "Characterization of Striped jack nervous necrosis virus subgenomic RNA3 and biological activities of its encoded protein B2." J Gen Virol 86(Pt 10): 2807-2816.
Khan, S., B. C. Fielding, T. H. Tan, C. F. Chou, S. Shen, S. G. Lim, W. Hong and Y. J. Tan (2006). "Over-expression of severe acute respiratory syndrome coronavirus 3b protein induces both apoptosis and necrosis in Vero E6 cells." Virus Res 122(1-2): 20-27.
Kubota, S., T. D. Copeland and R. J. Pomerantz (1999). "Nuclear and nucleolar targeting of human ribosomal protein S25: Common features shared with HIV-1 regulatory proteins." Oncogene 18(7): 1503-1514.
Leung, A. K. and A. I. Lamond (2003). "The dynamics of the nucleolus." Crit Rev Eukaryot Gene Expr 13(1): 39-54.
Li, J., C. Y. Shi, J. Huang, W. G. Geng, S. Q. Wang and Z. D. Su (2014). "Complete Genome Sequence of a Betanodavirus Isolated from Half-Smooth Tongue Sole (Cynoglossus semilaevis)." Genome announcements 2(6): e01312-01314.
Li, M. J., J. Kim, S. Li, J. Zaia, J. K. Yee, J. Anderson, R. Akkina and J. J. Rossi (2005). "Long-term inhibition of HIV-1 infection in primary hematopoietic cells by lentiviral vector delivery of a triple combination of anti-HIV shRNA, anti-CCR5 ribozyme, and a nucleolar-localizing TAR decoy." Mol Ther 12(5): 900-909.
Li, Y. P. (1997). "Protein B23 is an important human factor for the nucleolar localization of the human immunodeficiency virus protein Tat." Journal of virology 71(5): 4098-4102.
Liu, W., C.-H. Hsu, C.-Y. Chang, H.-H. Chen and C.-S. Lin (2006). "Immune response against grouper nervous necrosis virus by vaccination of virus-like particles." Vaccine 24(37): 6282-6287.
Liu, W., C. H. Hsu, Y. R. Hong, S. C. Wu, C. H. Wang, Y. M. Wu, C. B. Chao and C. S. Lin (2005). "Early endocytosis pathways in SSN-1 cells infected by dragon grouper nervous necrosis virus." J Gen Virol 86(Pt 9): 2553-2561.
Louvet, E., H. R. Junera, I. Berthuy and D. Hernandez-Verdun (2006). "Compartmentation of the nucleolar processing proteins in the granular component is a CK2-driven process." Mol Biol Cell 17(6): 2537-2546.
Lu, M.-W., F.-H. Ngou, Y.-M. Chao, Y.-S. Lai, N.-Y. Chen, F.-Y. Lee and P. P. Chiou (2012). "Transcriptome characterization and gene expression of Epinephelus spp in endoplasmic reticulum stress-related pathway during betanodavirus infection in vitro." BMC genomics 13: 651-651.
Lu, M. W., Y. M. Chao, T. C. Guo, N. Santi, O. Evensen, S. K. Kasani, J. R. Hong and J. L. Wu (2008). "The interferon response is involved in nervous necrosis virus acute and persistent infection in zebrafish infection model." Mol Immunol 45(4): 1146-1152.
Lu, M. W., W. Liu and C. S. Lin (2003). "Infection competition against grouper nervous necrosis virus by virus-like particles produced in Escherichia coli." J Gen Virol 84(Pt 6): 1577-1582.
Luo, H. (2016). "Interplay between the virus and the ubiquitin–proteasome system: molecular mechanism of viral pathogenesis." Current Opinion in Virology 17: 1-10.
Lymberopoulos, M. H., A. Bourget, N. Ben Abdeljelil and A. Pearson (2011). "Involvement of the UL24 protein in herpes simplex virus 1-induced dispersal of B23 and in nuclear egress." Virology 412(2): 341-348.
Mai, W., F. Huang, H. Chen, Y. Zhou and Y. Chen (2017). "Nervous necrosis virus capsid protein exploits nucleolar phosphoprotein Nucleophosmin (B23) function for viral replication." Virus Res 230: 1-6.
Matthews, D. A. and M. O. J. Olson (2006). "What is new in the nucleolus?: workshop on the nucleolus: new perspectives." EMBO reports 7(9): 870-873.
Meng, W., S.-C. Han, C.-C. Li, H.-J. Dong and X.-J. Wang (2018). "Multifunctional viral protein γ34.5 manipulates nucleolar protein NOP53 for optimal viral replication of HSV-1." Cell Death & Disease 9(2): 103.
Montanari, M., M. Macaluso, A. Cittadini and A. Giordano (2006). "Role of geminin: from normal control of DNA replication to cancer formation and progression?" Cell Death & Differentiation 13(7): 1052-1056.
Mori, K.-i., T. Nakai, M. Nagahara, K. Muroga, T. Mekuchi and T. Kanno (1991). "A Viral Disease in Hatchery-reared Larvae and Juveniles of Redspotted Grouper." Fish Pathology 26(4): 209-210.
Mori, K., T. Nakai, K. Muroga, M. Arimoto, K. Mushiake and I. Furusawa (1992). "Properties of a new virus belonging to nodaviridae found in larval striped jack (Pseudocaranx dentex) with nervous necrosis." Virology 187(1): 368-371.
Munday, B. L., J. Kwang and N. Moody (2002). "Betanodavirus infections of teleost fish: a review." Journal of Fish Diseases 25(3): 127-142.
Nishizawa, T., K.-i. Mori, M. Furuhashi, T. Nakai, I. Furusawa and K. Muroga (1995). "Comparison of the coat protein genes of five fish nodaviruses, the causative agents of viral nervous necrosis in marine fish." Journal of General Virology 76(7): 1563-1569.
Paladino, P., E. Marcon, J. Greenblatt and L. Frappier (2014). "Identification of Herpesvirus Proteins That Contribute to G₁/S Arrest." Journal of Virology 88(8): 4480-4492.
Passos-Castilho, A. M., C. Marchand and D. Archambault (2018). "B23/nucleophosmin interacts with bovine immunodeficiency virus Rev protein and facilitates viral replication." Virology 515: 158-164.
Rikkonen, M., J. Peranen and L. Kaariainen (1992). "Nuclear and nucleolar targeting signals of Semliki Forest virus nonstructural protein nsP2." Virology 189(2): 462-473.
Salvatore, M., C. F. Basler, J.-P. Parisien, C. M. Horvath, S. Bourmakina, H. Zheng, T. Muster, P. Palese and A. García-Sastre (2002). "Effects of Influenza A Virus NS1 Protein on Protein Expression: the NS1 Protein Enhances Translation and Is Not Required for Shutoff of Host Protein Synthesis." Journal of Virology 76(3): 1206-1212.
Salvetti, A. and A. Greco (2014). "Viruses and the nucleolus: The fatal attraction." Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease 1842(6): 840-847.
Shetty, M., B. Maiti, K. Shivakumar Santhosh, M. N. Venugopal and I. Karunasagar (2012). "Betanodavirus of marine and freshwater fish: distribution, genomic organization, diagnosis and control measures." Indian journal of virology : an official organ of Indian Virological Society 23(2): 114-123.
Souto, S., E. Merour, S. Biacchesi, M. Bremont, J. G. Olveira and I. Bandin (2015). "In vitro and in vivo characterization of molecular determinants of virulence in reassortant betanodavirus." J Gen Virol 96(Pt 6): 1287-1296.
Souto, S., J. G. Olveira, C. P. Dopazo and I. Bandín (2016). "Reassortant betanodavirus infection in turbot (Scophthalmus maximus)." Journal of Fish Diseases 39(11): 1347-1356.
Su, Y.-C., L. Reshi, L.-J. Chen, W.-H. Li, H.-W. Chiu and J.-R. Hong (2018). "Nuclear targeting of the betanodavirus B1 protein via two arginine-rich domains induces G1/S cell cycle arrest mediated by upregulation of p53/p21." Scientific Reports 8(1): 3079.
Surjit, M., B. Liu, V. T. Chow and S. K. Lal (2006). "The nucleocapsid protein of severe acute respiratory syndrome-coronavirus inhibits the activity of cyclin-cyclin-dependent kinase complex and blocks S phase progression in mammalian cells." J Biol Chem 281(16): 10669-10681.
Thiery, R., J. Cozien, C. de Boisseson, S. Kerbart-Boscher and L. Nevarez (2004). "Genomic classification of new betanodavirus isolates by phylogenetic analysis of the coat protein gene suggests a low host-fish species specificity." J Gen Virol 85(Pt 10): 3079-3087.
Toffan, A., V. Panzarin, M. Toson, K. Cecchettin and F. Pascoli (2016). "Water temperature affects pathogenicity of different betanodavirus genotypes in experimentally challenged Dicentrarchus labrax." Dis Aquat Organ 119(3): 231-238.
Toffan, A., F. Pascoli, T. Pretto, V. Panzarin, M. Abbadi, A. Buratin, R. Quartesan, D. Gijón and F. Padrós (2017). "Viral nervous necrosis in gilthead sea bream (Sparus aurata) caused by reassortant betanodavirus RGNNV/SJNNV: an emerging threat for Mediterranean aquaculture." Scientific Reports 7: 46755.
Vermeulen, K., D. R. Van Bockstaele and Z. N. Berneman (2003). "The cell cycle: a review of regulation, deregulation and therapeutic targets in cancer." Cell Proliferation 36(3): 131-149.
Xu, Z. and T. C. Hobman (2012). "The helicase activity of DDX56 is required for its role in assembly of infectious West Nile virus particles." Virology 433(1): 226-235.
Yang, M.-R., S. R. Lee, W. Oh, E.-W. Lee, J.-Y. Yeh, J.-J. Nah, Y.-S. Joo, J. Shin, H.-W. Lee, S. Pyo and J. Song (2008). "West Nile virus capsid protein induces p53-mediated apoptosis via the sequestration of HDM2 to the nucleolus." Cellular Microbiology 10(1): 165-176.
Yang, M. R., S. R. Lee, W. Oh, E. W. Lee, J. Y. Yeh, J. J. Nah, Y. S. Joo, J. Shin, H. W. Lee, S. Pyo and J. Song (2008). "West Nile virus capsid protein induces p53-mediated apoptosis via the sequestration of HDM2 to the nucleolus." Cell Microbiol 10(1): 165-176.
Yoshikoshi, K. and K. Inoue (1990). "Viral nervous necrosis in hatchery-reared larvae and juveniles of Japanese parrotfish, Oplegnathus fasciatus (Temminck & Schlegel)." Journal of Fish Diseases 13(1): 69-77.