臺灣博碩士論文加值系統

病毒性神經壞死 (viral nervous necrosis, VNN) 主要由神經壞死性病毒(nervous necrosis virus, NNV) 感染所引起，此病毒會感染多種魚類之幼魚，並引起魚類大量死亡，造成經濟上重大損失，其中點帶石斑魚受到的影響最為嚴重。根據新加坡學者Jimmy Kwang團隊先前的研究指出，神經壞死性病毒之殼體蛋白會進入魚類細胞株的細胞核中，並且引起細胞凋亡，由此推論神經壞死性病毒其病毒殼體蛋白可能扮演著除了結構蛋白以外的角色，並推測和調節細胞基因有關。為了更進一步了解神經壞死性病毒殼體蛋白是否具有其它功能，我們實驗室先前利用酵母菌雙雜合系統篩選出許多與殼體蛋白有交互作用的宿主蛋白，其中之一是人類RNF2蛋白(human RING finger protein 2, hRNF2)。經過進一步研究，此產物是hRNF2蛋白之選擇性剪接產物(alternative splicing, hRNF2S)。意外地，殼體蛋白會與選擇性剪接產物hRNF2進行交互作用，但與全長的hRNF2卻不會。為了確認此結果，本論文中利用了酵母菌雙雜合系統和GST親和力分析法以確認此交互作用，並發現hRNF2S是透過其N端與殼體蛋白進行交互作用。由於魚類神經壞死性病毒主要感染魚類，故本論文中也利用酵母菌雙雜合系統、GST親和力分析法、共免疫沉澱法以及免疫螢光染色分析證明殼體蛋白同樣會與石斑魚RNF2蛋白 (OsgRNF2) 進行交互作用。然而此交互作用所扮演的生理功能還需未來更進一步的研究。

Viral nervous necrosis (VNN) is a worldwide disease among marine fishes and causes high mortalities at larval stage and considerable economic damage to the aquaculture industry. Nervous necrosis virus (NNV) is one of the major pathogens of VNN diseases and infects the larvae of orange-spotted grouper (Epinephelus coiodes) seriously. According to previous studies by Jimmy Kwang, the coat protein of NNV could enter the nucleus and trigger the process of apoptosis, implying that coat protein was not only a structural protein but also a potential protein involved in cellular genes regulation. To further study the functions of coat protein, yeast two-hybrid assay was used to screen for the NNV coat-associated proteins using the human fetal brain cDNA library. An alternative splicing form of human Ring finger protein 2 (hRNF2S) was identified. Surprisingly, NNV coat protein interacted with hRNF2S, but not with the full length hRNF2. In this thesis, yeast two-hybrid assay and GST-pull down assay were performed to further confirm the interaction and map the interaction domain between NNV coat and hRNF2 S / hRNF2 FL. NNV coat protein mainly interacts with the N-terminal region of hRNF2S, especially the RING finger domain of RNF2. Since NNV infects fishes naturally, the interaction between NNV coat and fish orange-spotted grouper (Osg) RNF2 was examined both in vitro and in vivo by yeast two-hybrid assay, GST pull-down assay, co-immunoprecipitation assay and immunoflurorescence assay. Thus, the biological function of this interaction will be further studied in the future.

Abstract (Chinese version)………………………………………………………………I
Abstract………………………………………………………………………………II
Index……………………………………………………………………………………III
Chapter I: Introduction…………………………………………………………………1
1. Nervous Necrosis Virus (NNV)………………………………………………1
1.1 Characteristic of NNV……………………………………………………1
1.2 Clinical signs of NNV infection…………………………………………3
1.3 Epidemiology……………………………………………………………3
1.4 The diagnosis of NNV………………………………………………4
1.5 NNV coat protein………………………………………………………4
2. Polycomb group (PcG) protein………………………………………………6
2.1 Ring Finger Protein 2 (RNF2)…………………………………………7
3. Ubquitination………………………………………………………………10
4. Motive………………………………………………………………………12
Chapter II: Materials and methods……………………………………………………13
1. Experimental materials……………………………………………………13
1.1 Biomaterial…………………………………………………………13
1.2 Cell culture medium…..………………………………………………14
1.3 Chemical reagents……………………………………………………14
1.4 Enzyme………………………………………………………………14
1.5 Antibodies……………………………………………………………14
2. Experimental methods………………………………………………………16
2.1 Plasmids construction………………………………………………16
2.2 Isolation of plasmids DNA……………………………………………23
2.3 Competent cells preparation and plasmids transformation…………24
2.4 Yeast Transformation………………………………………………25
2.5 Yeast two-hybrids assay……………………………………………26
2.6 Protein expression systems of E. coli…………………………………26
2.7 Western blot…………………………………………………………28
2.8 GST pull-down assay…………………………………………………29
2.9 Cell culture…………………………………………………………...30
2.10 Cell Transfection………………………………………………………30
2.11 Co-immunoprecipitation assay………………………………………31
2.12 Immunofluorescence assay……………………………………………31
Chapter III: Results……………………………………………………………………33
1. Mapping of the NNV coat interacting domain of hRNF2………………33
2. Mapping of the NNV coat interacting domain of OsgRNF2………………35
3. Interaction between NNV coat and OsgRNF2 in vivo…………………37
4. Subcellular localization of NNV coat protein and OsgRNF2…………………37
Chapter IV: Discussion………………………………………………………………40
1. The RNF2 amino acids 94 to 137 might be important for interaction with NNV coat…………………………………………………………………39
2. The last C-terminus 30 amino acids and other regions of RNF2 might play a role in regulating the interaction with NNV coat…………………………39
3. Yeast cells expressing GAL4-AD-RNF2 only could grow on selection plates even in the absence of GAL4-BD-NNV coat………………………40
4. The results were inconsistent in verifying the NNV coat interacting domain of TAF7 by yeast two-hybrid assay and GST pull down assay……………41
5. N-terminal region of OsgRNF2 could form a dimer……………………42
6. Both C-terminal region of hRNF2 and OsgRNF2 may have the post-translational modification………………………………………………42
7. Does the hRNF2S or OsgRNF2 have the ubiquitin E3 ligase activity ?......43
8. The biological significance of the interaction between NNV coat and RNF2 proteins……………………………………………………………………43
Chapter V: References………………………………………………………………45
Chapter VI: Tables and figures………………………………………………………57
Chapter VII: Appendix………………………………………………………………69

1.Amerik, A. Y., and M. Hochstrasser. 2004. Mechanism and function of deubiquitinating enzymes. Biochim Biophys Acta 1695:189-207.
2.Arimoto M., M. K., Mizuta Y., et al. 1992. Detection of striped jack nervous necrosis virus (SJNNV) by enzyme-linked immunosorbent assay (ELISA). Fish Pathol 27:191-195.
3.Balkhi, M. Y., K. A. Fitzgerald, and P. M. Pitha. 2008. Functional regulation of MyD88-activated interferon regulatory factor 5 by K63-linked polyubiquitination. Mol Cell Biol 28:7296-308.
4.Ball L. A., a. J. K. L. 1999. Reverse genetics of nodaviruses. Adv. Virus. Res. 53:229-244.
5.Ball L. A., H. D. A., Johnson J. E., Ruechert R. R., and Scotti P. D. 2000. Family Nodaviridae.In:Virus Taxonomy. Seventh Report of the International Committee on Taxonomy of Viruses (ed. by M.H.V. Van Regenmortel, C.M. Fauquet, D.H.Z. Bishop, E.B. Carstens, M.K. Estes, S.M. Lemon, J. Miniloff, M.A. Mayo, D.J. McGeoach, C.R. Pring.
6.Ben-Saadon, R., D. Zaaroor, T. Ziv, and A. Ciechanover. 2006. The polycomb protein Ring1B generates self atypical mixed ubiquitin chains required for its in vitro histone H2A ligase activity. Mol Cell 24:701-11.
7.Boyer, L. A., K. Plath, J. Zeitlinger, T. Brambrink, L. A. Medeiros, T. I. Lee, S. S. Levine, M. Wernig, A. Tajonar, M. K. Ray, G. W. Bell, A. P. Otte, M. Vidal, D. K. Gifford, R. A. Young, and R. Jaenisch. 2006. Polycomb complexes repress developmental regulators in murine embryonic stem cells. Nature 441:349-53.
8.Bracken, A. P., D. Pasini, M. Capra, E. Prosperini, E. Colli, and K. Helin. 2003. EZH2 is downstream of the pRB-E2F pathway, essential for proliferation and amplified in cancer. EMBO J 22:5323-35.
9.Breiling, A., B. M. Turner, M. E. Bianchi, and V. Orlando. 2001. General transcription factors bind promoters repressed by Polycomb group proteins. Nature 412:651-5.
10.Brown, J. L., C. Fritsch, J. Mueller, and J. A. Kassis. 2003. The Drosophila pho-like gene encodes a YY1-related DNA binding protein that is redundant with pleiohomeotic in homeotic gene silencing. Development 130:285-94.
11.Cales, C., M. Roman-Trufero, L. Pavon, I. Serrano, T. Melgar, M. Endoh, C. Perez, H. Koseki, and M. Vidal. 2008. Inactivation of the polycomb group protein Ring1B unveils an antiproliferative role in hematopoietic cell expansion and cooperation with tumorigenesis associated with Ink4a deletion. Mol Cell Biol 28:1018-28.
12.Cao, R., Y. Tsukada, and Y. Zhang. 2005. Role of Bmi-1 and Ring1A in H2A ubiquitylation and Hox gene silencing. Mol Cell 20:845-54.
13.Cao, R., L. Wang, H. Wang, L. Xia, H. Erdjument-Bromage, P. Tempst, R. S. Jones, and Y. Zhang. 2002. Role of histone H3 lysine 27 methylation in Polycomb-group silencing. Science 298:1039-43.
14.Chau, V., J. W. Tobias, A. Bachmair, D. Marriott, D. J. Ecker, D. K. Gonda, and A. Varshavsky. 1989. A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein. Science 243:1576-83.
15.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 385:444-54.
16.Chi, S. C., S. C. Lin, H. M. Su, and W. W. Hu. 1999. Temperature effect on nervous necrosis virus infection in grouper cell line and in grouper larvae. Virus Res 63:107-14.
17.Chi, S. C., J. R. Shieh, and S. J. Lin. 2003. Genetic and antigenic analysis of betanodaviruses isolated from aquatic organisms in Taiwan. Dis Aquat Organ 55:221-8.
18.Choi, D., S. J. Lee, S. Hong, I. H. Kim, and S. Kang. 2008. Prohibitin interacts with RNF2 and regulates E2F1 function via dual pathways. Oncogene 27:1716-25.
19.Cyril Dominguez, G. E. F., and Rolf Boelens. 2004. Biological Introduction:RING domain proteins. Contribution to Handbook of Metalloproteins 3:338-351.
20.Czypionka, A., O. R. de los Panos, M. G. Mateu, F. N. Barrera, E. Hurtado-Gomez, J. Gomez, M. Vidal, and J. L. Neira. 2007. The isolated C-terminal domain of Ring1B is a dimer made of stable, well-structured monomers. Biochemistry 46:12764-76.
21.Dalla Valle, L., V. Toffolo, M. Lamprecht, C. Maltese, G. Bovo, P. Belvedere, and L. Colombo. 2005. Development of a sensitive and quantitative diagnostic assay for fish nervous necrosis virus based on two-target real-time PCR. Vet Microbiol 110:167-79.
22.de Napoles, M., J. E. Mermoud, R. Wakao, Y. A. Tang, M. Endoh, R. Appanah, T. B. Nesterova, J. Silva, A. P. Otte, M. Vidal, H. Koseki, and N. Brockdorff. 2004. Polycomb group proteins Ring1A/B link ubiquitylation of histone H2A to heritable gene silencing and X inactivation. Dev Cell 7:663-76.
23.del Mar Lorente, M., C. Marcos-Gutierrez, C. Perez, J. Schoorlemmer, A. Ramirez, T. Magin, and M. Vidal. 2000. Loss- and gain-of-function mutations show a polycomb group function for Ring1A in mice. Development 127:5093-100.
24.Dietrich, N., A. P. Bracken, E. Trinh, C. K. Schjerling, H. Koseki, J. Rappsilber, K. Helin, and K. H. Hansen. 2007. Bypass of senescence by the polycomb group protein CBX8 through direct binding to the INK4A-ARF locus. EMBO J 26:1637-48.
25.Fenner, B. J., R. Thiagarajan, H. K. Chua, and J. Kwang. 2006. Betanodavirus B2 is an RNA interference antagonist that facilitates intracellular viral RNA accumulation. J Virol 80:85-94.
26.Francis, N. J., and R. E. Kingston. 2001. Mechanisms of transcriptional memory. Nat Rev Mol Cell Biol 2:409-21.
27.Francis, N. J., R. E. Kingston, and C. L. Woodcock. 2004. Chromatin compaction by a polycomb group protein complex. Science 306:1574-7.
28.Frerichs, G. N., H. D. Rodger, and Z. Peric. 1996. Cell culture isolation of piscine neuropathy nodavirus from juvenile sea bass, Dicentrarchus labrax. J Gen Virol 77 ( Pt 9):2067-71.
29.Fukuda Y., N. H. D., Furuhashi M. & Nakai T. 1996. Mass mortality of cultured sevenband grouper, Epinephelus septemfasciatus, associated with viral nervous necrosis. . Fish Pathol. 31:165-170.
30.Glazebrook, J. S., M. P. Heasman, and S. W. D. Beer. 1990. Picorna-like viral particles associated with mass mortalities in larval barramundi, Lates calcarifer Bloch. J. Fish Dis. 13:245-249.
31.Guo, Y. X., K. Dallmann, and J. Kwang. 2003. Identification of nucleolus localization signal of betanodavirus GGNNV protein alpha. Virology 306:225-35.
32.Haupt, Y., Alexander, W. S., Barri, G., Klinken, S. P. & Adams, J. M. 1991. Novel zinc finger gene implicated as myc collaborator by retrovirally accelerated lymphomagenesis in E mu-myc transgenic mice. Cell 65:753-763.
33.Heard, E. 2005. Delving into the diversity of facultative heterochromatin: the epigenetics of the inactive X chromosome. Curr Opin Genet Dev 15:482-9.
34.Hershko, A., and A. Ciechanover. 1998. The ubiquitin system. Annu Rev Biochem 67:425-79.
35.Hicke, L. 2001. Protein regulation by monoubiquitin. Nat Rev Mol Cell Biol 2:195-201.
36.Hoege, C., B. Pfander, G. L. Moldovan, G. Pyrowolakis, and S. Jentsch. 2002. RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature 419:135-41.
37.Isono, K., Y. Fujimura, J. Shinga, M. Yamaki, O. W. J, Y. Takihara, Y. Murahashi, Y. Takada, Y. Mizutani-Koseki, and H. Koseki. 2005. Mammalian polyhomeotic homologues Phc2 and Phc1 act in synergy to mediate polycomb repression of Hox genes. Mol Cell Biol 25:6694-706.
38.Iwamoto, T., T. Nakai, K. Mori, M. Arimoto, and I. Furusawa. 2000. Cloning of the fish cell line SSN-1 for piscine nodaviruses. Dis Aquat Organ 43:81-9.
39.Kirmizis, A., S. M. Bartley, and P. J. Farnham. 2003. Identification of the polycomb group protein SU(Z)12 as a potential molecular target for human cancer therapy. Mol Cancer Ther 2:113-21.
40.Kuzmichev, A., K. Nishioka, H. Erdjument-Bromage, P. Tempst, and D. Reinberg. 2002. Histone methyltransferase activity associated with a human multiprotein complex containing the Enhancer of Zeste protein. Genes Dev 16:2893-905.
41.Lai, Y. S., J. A. John, C. H. Lin, I. C. Guo, S. C. Chen, K. Fang, and C. Y. Chang. 2003. Establishment of cell lines from a tropical grouper, Epinephelus awoara (Temminck & Schlegel), and their susceptibility to grouper irido- and nodaviruses. J Fish Dis 26:31-42.
42.Lam, Y. A., W. Xu, G. N. DeMartino, and R. E. Cohen. 1997. Editing of ubiquitin conjugates by an isopeptidase in the 26S proteasome. Nature 385:737-40.
43.Le Breton A., G. L., Sweetman J. & Olievier F. 1997. Viral nervous necrosis (VNN) associated with mass mortalities in cage-reared sea bass, Dicentrarchus labrax (L.). J. Fish Dis. 20:145-151.
44.Lee, S. J., D. Choi, H. Rhim, and S. Kang. 2005. E3 ubiquitin ligase RNF2 interacts with the S6'' proteasomal ATPase subunit and increases the ATP hydrolysis activity of S6''. Biochem J 389:457-63.
45.Lee, S. J., J. Y. Choi, Y. M. Sung, H. Park, H. Rhim, and S. Kang. 2001. E3 ligase activity of RING finger proteins that interact with Hip-2, a human ubiquitin-conjugating enzyme. FEBS Lett 503:61-4.
46.Lee, T. I., R. G. Jenner, L. A. Boyer, M. G. Guenther, S. S. Levine, R. M. Kumar, B. Chevalier, S. E. Johnstone, M. F. Cole, K. Isono, H. Koseki, T. Fuchikami, K. Abe, H. L. Murray, J. P. Zucker, B. Yuan, G. W. Bell, E. Herbolsheimer, N. M. Hannett, K. Sun, D. T. Odom, A. P. Otte, T. L. Volkert, D. P. Bartel, D. A. Melton, D. K. Gifford, R. Jaenisch, and R. A. Young. 2006. Control of developmental regulators by Polycomb in human embryonic stem cells. Cell 125:301-13.
47.Levine, S. S., I. F. King, and R. E. Kingston. 2004. Division of labor in polycomb group repression. Trends Biochem Sci 29:478-85.
48.Li, Z., R. Cao, M. Wang, M. P. Myers, Y. Zhang, and R. M. Xu. 2006. Structure of a Bmi-1-Ring1B polycomb group ubiquitin ligase complex. J Biol Chem 281:20643-9.
49.Lu, T.-C. 2008. Gene cloning of orange-spotted grouper (Epinephelus coioides) RNF2 and characterization of the interaction between RNF2 protein and the Nervous Necrosis Virus (NNV) coat protein. Master thesis. Institute of Molecular Biology, National Chung Cheng University, Gia-Yi,Taiwan.
50.M. Comps, J. P. a. J. B. 1996. Investigation of fish encephalitis viruses (FEV)expression in marine fishes using DIG-labelled probes. Aquaculture 143:113-121.
51.Mori KI, N. T., Nagahara M, Muroga K, MEKUCHI T, Kanno T. 1991. A viral disease in hatcherry-rared larvae and juveniles of redspotted grouper. Fish Pathol 26:209–210.
52.Muller, J., C. M. Hart, N. J. Francis, M. L. Vargas, A. Sengupta, B. Wild, E. L. Miller, M. B. O''Connor, R. E. Kingston, and J. A. Simon. 2002. Histone methyltransferase activity of a Drosophila Polycomb group repressor complex. Cell 111:197-208.
53.Munday, B. L., J Kwang and N Moody. 2002. Betanodavirus infections of teleost fish: a review. Journal of Fish Disease 25:127-142.
54.Nguyen HD, M. K., Nakai T, and Muroga K. 1997. Tissue distribution of striped jack nervous necrosis virus (SJNNV) in adult striped jack. Diseases of Aquatic Organisms 28:87-91.
55.Nishizawa, T., M. Furuhashi, T. Nagai, T. Nakai, and K. Muroga. 1997. Genomic classification of fish nodaviruses by molecular phylogenetic analysis of the coat protein gene. Appl Environ Microbiol 63:1633-6.
56.Nishizawa, T., K. 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. J Gen Virol 76 ( Pt 7):1563-9.
57.P. Kaesberg, J.-Y. S., J.-P. Wery, B.H. Selling, M.V. Hosur and J.E. Johnson. 1990. Structural homology among four nodaviruses as deduced by sequencing and X-ray crystallography. J Mol Biol 214:423-435.
58.Paro, R. 1993. Mechanisms of heritable gene repression during development of Drosophila. Curr Opin Cell Biol 5:999-1005.
59.Patnaik, A., V. Chau, and J. W. Wills. 2000. Ubiquitin is part of the retrovirus budding machinery. Proc Natl Acad Sci U S A 97:13069-74.
60.Rao, P. S., A. Satelli, S. Zhang, S. K. Srivastava, K. S. Srivenugopal, and U. S. Rao. 2009. RNF2 is the target for phosphorylation by the p38 MAPK and ERK signaling pathways. Proteomics 9:2776-87.
61.Ringrose, L., and R. Paro. 2004. Epigenetic regulation of cellular memory by the Polycomb and Trithorax group proteins. Annu Rev Genet 38:413-43.
62.Ringrose, L., and R. Paro. 2007. Polycomb/Trithorax response elements and epigenetic memory of cell identity. Development 134:223-32.
63.Roman-Trufero, M., H. R. Mendez-Gomez, C. Perez, A. Hijikata, Y. Fujimura, T. Endo, H. Koseki, C. Vicario-Abejon, and M. Vidal. 2009. Maintenance of undifferentiated state and self-renewal of embryonic neural stem cells by Polycomb protein Ring1B. Stem cells 27:1559-70.
64.Saurin, A. J., Z. Shao, H. Erdjument-Bromage, P. Tempst, and R. E. Kingston. 2001. A Drosophila Polycomb group complex includes Zeste and dTAFII proteins. Nature 412:655-60.
65.Scheffner, M., J. M. Huibregtse, R. D. Vierstra, and P. M. Howley. 1993. The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53. Cell 75:495-505.
66.Schneemann, A., V. Reddy, and J. E. Johnson. 1998. The structure and function of nodavirus particles: a paradigm for understanding chemical biology. Adv Virus Res 50:381-446.
67.Schubert, U., D. E. Ott, E. N. Chertova, R. Welker, U. Tessmer, M. F. Princiotta, J. R. Bennink, H. G. Krausslich, and J. W. Yewdell. 2000. Proteasome inhibition interferes with gag polyprotein processing, release, and maturation of HIV-1 and HIV-2. Proc Natl Acad Sci U S A 97:13057-13062.
68.Shieh, J. R., and S. C. Chi. 2005. Production of monoclonal antibodies against grouper nervous necrosis virus (GNNV) and development of an antigen capture ELISA. Dis Aquat Organ 63:53-60.
69.Shumacher, A., C. Faust, and T. Magnuson. 1996. Positional cloning of a global regulator of anterior-posterior patterning in mice. Nature 383:250-253.
70.Starkey, W. G., R. M. Millar, M. E. Jenkins, J. H. Ireland, K. F. Muir, and R. H. Richards. 2004. Detection of piscine nodaviruses by real-time nucleic acid sequence based amplification (NASBA). Dis Aquat Organ 59:93-100.
71.Stock, J. K., S. Giadrossi, M. Casanova, E. Brookes, M. Vidal, H. Koseki, N. Brockdorff, A. G. Fisher, and A. Pombo. 2007. Ring1-mediated ubiquitination of H2A restrains poised RNA polymerase II at bivalent genes in mouse ES cells. Nat Cell Biol 9:1428-35.
72.Strack, B., A. Calistri, M. A. Accola, G. Palu, and H. G. Gottlinger. 2000. A role for ubiquitin ligase recruitment in retrovirus release. Proc Natl Acad Sci U S A 97:13063-8.
73.Su, Y. C., J. L. Wu, and J. R. Hong. 2009. Betanodavirus non-structural protein B2: A novel necrotic death factor that induces mitochondria-mediated cell death in fish cells. Virology 385:143-54.
74.Suzuki, M., Y. Mizutani-Koseki, Y. Fujimura, H. Miyagishima, T. Kaneko, Y. Takada, T. Akasaka, H. Tanzawa, Y. Takihara, M. Nakano, H. Masumoto, M. Vidal, K. Isono, and H. Koseki. 2002. Involvement of the Polycomb-group gene Ring1B in the specification of the anterior-posterior axis in mice. Development 129:4171-83.
75.Tan, C., B. Huang, S. F. Chang, G. H. Ngoh, B. Munday, S. C. Chen, and J. Kwang. 2001. Determination of the complete nucleotide sequences of RNA1 and RNA2 from greasy grouper (Epinephelus tauvina) nervous necrosis virus, Singapore strain. J Gen Virol 82:647-53.
76.Tan, J. M., E. S. Wong, D. S. Kirkpatrick, O. Pletnikova, H. S. Ko, S. P. Tay, M. W. Ho, J. Troncoso, S. P. Gygi, M. K. Lee, V. L. Dawson, T. M. Dawson, and K. L. Lim. 2008. Lysine 63-linked ubiquitination promotes the formation and autophagic clearance of protein inclusions associated with neurodegenerative diseases. Hum Mol Genet 17:431-9.
77.van der Stoop, P., E. A. Boutsma, D. Hulsman, S. Noback, M. Heimerikx, R. M. Kerkhoven, J. W. Voncken, L. F. Wessels, and M. van Lohuizen. 2008. Ubiquitin E3 ligase Ring1b/Rnf2 of polycomb repressive complex 1 contributes to stable maintenance of mouse embryonic stem cells. PLoS ONE 3:e2235.
78.van Lohuizen, M., S. Verbeek, B. Scheijen, E. Wientjens, H. van der Gulden, and A. Berns. 1991. Identification of cooperating oncogenes in E mu-myc transgenic mice by provirus tagging. Cell 65:737-52.
79.Visser, H. P., M. J. Gunster, H. C. Kluin-Nelemans, E. M. Manders, F. M. Raaphorst, C. J. Meijer, R. Willemze, and A. P. Otte. 2001. The Polycomb group protein EZH2 is upregulated in proliferating, cultured human mantle cell lymphoma. Br J Haematol 112:950-8.
80.von Arnim, A. G. 2001. A hitchhiker''s guide to the proteasome. Sci STKE 2001:PE2.
81.Voncken, J. W., B. A. Roelen, M. Roefs, S. de Vries, E. Verhoeven, S. Marino, J. Deschamps, and M. van Lohuizen. 2003. Rnf2 (Ring1b) deficiency causes gastrulation arrest and cell cycle inhibition. Proc Natl Acad Sci U S A 100:2468-73.
82.Wang, H., L. Wang, H. Erdjument-Bromage, M. Vidal, P. Tempst, R. S. Jones, and Y. Zhang. 2004. Role of histone H2A ubiquitination in Polycomb silencing. Nature 431:873-8.
83.Wolf, D. H., and W. Hilt. 2004. The proteasome: a proteolytic nanomachine of cell regulation and waste disposal. Biochim Biophys Acta 1695:19-31.
84.Wong, B. R., F. Parlati, K. Qu, S. Demo, T. Pray, J. Huang, D. G. Payan, and M. K. Bennett. 2003. Drug discovery in the ubiquitin regulatory pathway. Drug Discov Today 8:746-54.
85.Wong, C. K., Z. Chen, K. L. So, D. Li, and P. Li. 2007. Polycomb group protein RING1B is a direct substrate of Caspases-3 and -9. Biochim Biophys Acta 1773:844-52.
86.Wu, H. C., C. S. Chiu, J. L. Wu, H. Y. Gong, M. C. Chen, M. W. Lu, and J. R. Hong. 2008. Zebrafish anti-apoptotic protein zfBcl-xL can block betanodavirus protein alpha-induced mitochondria-mediated secondary necrosis cell death. Fish Shellfish Immunol 24:436-49.
87.Yoshikoshi, K., and K. Inoue. 1990. Viral nervous necrosis in hatchery-reared larve and juveniles of Japanese parrotfish, Oplegnathus fasciatus (Temminck & Schlegel). J Fish Dis 13:69-77.
88.Zhu, Q., J. Yao, G. Wani, J. Chen, Q. E. Wang, and A. A. Wani. 2004. The ubiquitin-proteasome pathway is required for the function of the viral VP16 transcriptional activation domain. FEBS Lett 556:19-25.