臺灣博碩士論文加值系統

嚴重急性呼吸道症候群(Severe Acute Respiratory Syndrome)是一種新的傳染性疾病，引起此疾病的病原為SARS-CoV，它是一種以前所不曾發現的冠狀病毒，它的基因體和其他的冠狀病毒一樣，從5’端到3’端依序為replicase、spike、envelope、membrane和nucleocapsid，除了replicase之外其他的結構基因都和病毒的組裝有關。Replicase 是一個非結構性蛋白質對於病毒基因體的複製相當重要。在SARS-CoV中以生物資訊的方法預測出9個非結構性蛋白質，分別為ORF3、ORF4、ORF7、ORF8、ORF9、ORF10、ORF11、ORF13和ORF14，他們位在之前所說的這些病毒基因之間。在這九個非結構性蛋白質中，ORF3被認為會在生物體中表現，因為在SARS病人血清中能偵測到抗體反應。而其他八個ORF在活體中的表現和功能則仍待研究。
本論文的目的是在研究ORF11的表現和它的功能。ORF11是一個獨特的基因，在果子狸身上發現類似SARS-CoV的冠狀病毒中並沒有看到此基因存在，所以它有可能和SARS-CoV對人體感染後的嚴重影響有關。ORF11可能會從subgenomic RNA 2.0和2.5上被轉譯出來。從活體外共同轉錄轉譯系統中並無法看到ORF11的訊號，可能是因為被背景雜訊擋住。然而以GST-ORF11融合蛋白質做抗原，以SARS病人的血清做西方墨點法分析時，能看到anti-ORF11的抗體反應，因此推測ORF11可能在活體中被表現。以ORF11-flag的表現質體來觀測ORF11在Vero E6中的位置，得知其表現在細胞質中。本實驗藉由酵母菌雙雜合分析來推測ORF11的功能，以人類的白血球cDNA基因庫來篩選，發現RYBP (Ring1 and YY1 binding protein)是和ORF11相互作用的蛋白質。本實驗也以活體外共同轉錄轉譯系統加上canine microsomal membranes來證明ORF11和RYBP的交互作用。RYBP之前被知道和細胞自裁有關，因此我們推測ORF11可能會和細胞凋零有關。

SARS-CoV is identified as the agent that causes human severe acute respiratory syndrome (SARS). It is a new member of coronaviridae family and shares the similar genome organization as that of other members. The basic order of genes for the family of coronaviridae is replicase, spike, envelope, membrane protein and nucleocapsid, from 5’ to 3’ end. All except replicase are structure genes that are essential for the maturation of virion. The replicase is considered as a nonstructural (NS) protein, which is essential for the replication of viral genome. There are at least nine additional NS proteins potentially derived from open reading frames (ORFs) that scatter among and within the aforementioned viral gene loci. They are, as predicted by bioinformatics, ORFs 3, 4, 7, 8, 9, 10, 11, 13, and 14. Of the nine ORFs, ORF3 protein is indirectly implicated to have been actually expressed in vivo by the detection of anti-ORF3 antibody in the sera of SARS patients. The status of in vivo expression and functions of the remaining 8 ORFs remain to be explored.
The purpose of this thesis is to study the expression and function of ORF11. The ORF11 gene is unique in that it doesn’t have a counterpart in the Civet SARS coronavirus. Thus, it might have unique contributions to the severity of SARS-CoV infection in human. Both viral subgenomic RNAs 2.0 and 2.5 are potential templates for the translation of ORF11. The in vitro transcription-translation coupled assay using both templates have failed to ascertain the presence of ORF11 due to the obscurity caused by the presence of one background band. However, the in vivo expression of ORF11 is indirectly substantiated by the presence of anti-ORF11 antibody in one SARS patient, using GST-ORF11 recombinant protein as the antigen in a Western blot assay. The intracellular localization of ORF11 has been determined to be in the cytoplasm of Vero E6 cell transfected with an ORF11-flag expression vector. The functionality of ORF11 is evaluated through the use of a yeast two-hybrid assay with human leukocyte cDNA library. A RYBP (Ring1 and YY1 binding protein) has been identified as the binding partner of ORF11 protein. This interaction has also been verified in vitro by the binding of both proteins produced in an in vitro transcription-translation coupled system, in the presence of canine microsomal membranes. RYBP is known to be possibly involved in apoptosis, suggesting the potential function of the ORF11.

Abraham, S., T.E. Kienzle, W.E. Lapps, and D.A. Brian. 1990. Sequence and expression analysis of potential nonstructural proteins of 4.9, 4.8, 12.7, and 9.5 kDa encoded between the spike and membrane protein genes of the bovine coronavirus. Virology. 177:488-95.
Anton, I.M., S. Gonzalez, M.J. Bullido, M. Corsin, C. Risco, J.P. Langeveld, and L. Enjuanes. 1996. Cooperation between transmissible gastroenteritis coronavirus (TGEV) structural proteins in the in vitro induction of virus-specific antibodies. Virus Res. 46:111-24.
Bacha, U., J. Barrila, A. Velazquez-Campoy, S.A. Leavitt, and E. Freire. 2004. Identification of Novel Inhibitors of the SARS Coronavirus Main Protease 3CL(pro). Biochemistry. 43:4906-12.
Bischoff, F.R., C. Klebe, J. Kretschmer, A. Wittinghofer, and H. Ponstingl. 1994. RanGAP1 induces GTPase activity of nuclear Ras-related Ran. Proc Natl Acad Sci U S A. 91:2587-91.
Bischoff, F.R., and H. Ponstingl. 1991. Catalysis of guanine nucleotide exchange on Ran by the mitotic regulator RCC1. Nature. 354:80-2.
Chen, X., B. Zhou, M. Li, X. Liang, H. Wang, G. Yang, and X. Le. 2004. Serology of severe acute respiratory syndrome: implications for surveillance and outcome. J Infect Dis. 189:1158-63.
Chinese, S.M.E.C. 2004. Molecular evolution of the SARS coronavirus during the course of the SARS epidemic in China. Science. 303:1666-9.
Cinatl, J., B. Morgenstern, G. Bauer, P. Chandra, H. Rabenau, and H.W. Doerr. 2003. Treatment of SARS with human interferons. Lancet. 362:293-4.
Denti, S., A. Sirri, A. Cheli, L. Rogge, G. Innamorati, S. Putignano, M. Fabbri, R. Pardi, and E. Bianchi. 2004. RanBPM is a phosphoprotein that associates with the plasma membrane and interacts with the integrin LFA-1. J Biol Chem. 279:13027-34.
Dimitrov, D.S. 2003. The secret life of ACE2 as a receptor for the SARS virus. Cell. 115:652-3.
Ding, Y., L. He, Q. Zhang, Z. Huang, X. Che, J. Hou, H. Wang, H. Shen, L. Qiu, Z. Li, J. Geng, J. Cai, H. Han, X. Li, W. Kang, D. Weng, P. Liang, and S. Jiang. 2004. Organ distribution of severe acute respiratory syndrome (SARS) associated coronavirus (SARS-CoV) in SARS patients: implications for pathogenesis and virus transmission pathways. J Pathol. 203:622-30.
Drosten, C., S. Gunther, W. Preiser, S. van der Werf, H.R. Brodt, S. Becker, H. Rabenau, M. Panning, L. Kolesnikova, R.A. Fouchier, A. Berger, A.M. Burguiere, J. Cinatl, M. Eickmann, N. Escriou, K. Grywna, S. Kramme, J.C. Manuguerra, S. Muller, V. Rickerts, M. Sturmer, S. Vieth, H.D. Klenk, A.D. Osterhaus, H. Schmitz, and H.W. Doerr. 2003. Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N Engl J Med. 348:1967-76.
Garcia, E., C. Marcos-Gutierrez, M. del Mar Lorente, J.C. Moreno, and M. Vidal. 1999. RYBP, a new repressor protein that interacts with components of the mammalian Polycomb complex, and with the transcription factor YY1. Embo J. 18:3404-18.
Guan, Y., B.J. Zheng, Y.Q. He, X.L. Liu, Z.X. Zhuang, C.L. Cheung, S.W. Luo, P.H. Li, L.J. Zhang, Y.J. Guan, K.M. Butt, K.L. Wong, K.W. Chan, W. Lim, K.F. Shortridge, K.Y. Yuen, J.S. Peiris, and L.L. Poon. 2003. Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China. Science. 302:276-8.
Hsu, S.C., H.P. Lin, J.C. Wu, K.L. Ko, I.J. Sheen, B.S. Yan, C.K. Chou, and W.J. Syu. 2000. Characterization of a strain-specific monoclonal antibody to hepatitis delta virus antigen. J Virol Methods. 87:53-62.
K. V. Holmes, in Fields Virology, D. M. Knipe, P. M.Howley, Eds. (Lippincott Williams & Wilkins, NewYork, ed. 4, 2001), chap. 36.
Koyama, A.H., T. Fukumori, M. Fujita, H. Irie, and A. Adachi. 2000. Physiological significance of apoptosis in animal virus infection. Microbes Infect. 2:1111-7.
Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227:680-5.
Leung, W.K., K.F. To, P.K. Chan, H.L. Chan, A.K. Wu, N. Lee, K.Y. Yuen, and J.J. Sung. 2003. Enteric involvement of severe acute respiratory syndrome-associated coronavirus infection. Gastroenterology. 125:1011-7.
Liu, S., G. Xiao, Y. Chen, Y. He, J. Niu, C.R. Escalante, H. Xiong, J. Farmar, A.K. Debnath, P. Tien, and S. Jiang. 2004. Interaction between heptad repeat 1 and 2 regions in spike protein of SARS-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors. Lancet. 363:938-47.
Lowy, R.J. 2003. Influenza virus induction of apoptosis by intrinsic and extrinsic mechanisms. Int Rev Immunol. 22:425-49.
M. M. C. Lai, K. V. Holmes, in Fields Virology, D. M.Knipe, P. M. Howley, Eds. (Lippincott Williams &Wilkins, New York, ed. 4, 2001), chap.35.
Marra, M.A., S.J. Jones, C.R. Astell, R.A. Holt, A. Brooks-Wilson, Y.S. Butterfield, J. Khattra, J.K. Asano, S.A. Barber, S.Y. Chan, A. Cloutier, S.M. Coughlin, D. Freeman, N. Girn, O.L. Griffith, S.R. Leach, M. Mayo, H. McDonald, S.B. Montgomery, P.K. Pandoh, A.S. Petrescu, A.G. Robertson, J.E. Schein, A. Siddiqui, D.E. Smailus, J.M. Stott, G.S. Yang, F. Plummer, A. Andonov, H. Artsob, N. Bastien, K. Bernard, T.F. Booth, D. Bowness, M. Czub, M. Drebot, L. Fernando, R. Flick, M. Garbutt, M. Gray, A. Grolla, S. Jones, H. Feldmann, A. Meyers, A. Kabani, Y. Li, S. Normand, U. Stroher, G.A. Tipples, S. Tyler, R. Vogrig, D. Ward, B. Watson, R.C. Brunham, M. Krajden, M. Petric, D.M. Skowronski, C. Upton, and R.L. Roper. 2003. The Genome sequence of the SARS-associated coronavirus. Science. 300:1399-404.
Mizutani, T., S. Fukushi, M. Saijo, I. Kurane, and S. Morikawa. 2004. Phosphorylation of p38 MAPK and its downstream targets in SARS coronavirus-infected cells. Biochem Biophys Res Commun. 319:1228-34.
Nakamura, M., H. Masuda, J. Horii, K. Kuma, N. Yokoyama, T. Ohba, H. Nishitani, T. Miyata, M. Tanaka, and T. Nishimoto. 1998. When overexpressed, a novel centrosomal protein, RanBPM, causes ectopic microtubule nucleation similar to gamma-tubulin. J Cell Biol. 143:1041-52.
Nishimoto, T. 1999. A new role of ran GTPase. Biochem Biophys Res Commun. 262:571-4.
Peiris, J.S., C.M. Chu, V.C. Cheng, K.S. Chan, I.F. Hung, L.L. Poon, K.I. Law, B.S. Tang, T.Y. Hon, C.S. Chan, K.H. Chan, J.S. Ng, B.J. Zheng, W.L. Ng, R.W. Lai, Y. Guan, and K.Y. Yuen. 2003. Clinical progression and viral load in a community outbreak of coronavirus-associated SARS pneumonia: a prospective study. Lancet. 361:1767-72.
Poutanen, S.M., D.E. Low, B. Henry, S. Finkelstein, D. Rose, K. Green, R. Tellier, R. Draker, D. Adachi, M. Ayers, A.K. Chan, D.M. Skowronski, I. Salit, A.E. Simor, A.S. Slutsky, P.W. Doyle, M. Krajden, M. Petric, R.C. Brunham, and A.J. McGeer. 2003. Identification of severe acute respiratory syndrome in Canada. N Engl J Med. 348:1995-2005.
Qinfen, Z., C. Jinming, H. Xiaojun, Z. Huanying, H. Jicheng, F. Ling, L. Kunpeng, and Z. Jingqiang. 2004. The life cycle of SARS coronavirus in Vero E6 cells. J Med Virol. 73:332-7.
Rota, P.A., M.S. Oberste, S.S. Monroe, W.A. Nix, R. Campagnoli, J.P. Icenogle, S. Penaranda, B. Bankamp, K. Maher, M.H. Chen, S. Tong, A. Tamin, L. Lowe, M. Frace, J.L. DeRisi, Q. Chen, D. Wang, D.D. Erdman, T.C. Peret, C. Burns, T.G. Ksiazek, P.E. Rollin, A. Sanchez, S. Liffick, B. Holloway, J. Limor, K. McCaustland, M. Olsen-Rasmussen, R. Fouchier, S. Gunther, A.D. Osterhaus, C. Drosten, M.A. Pallansch, L.J. Anderson, and W.J. Bellini. 2003. Characterization of a novel coronavirus associated with severe acute respiratory syndrome. Science. 300:1394-9.
Sawa, C., T. Yoshikawa, F. Matsuda-Suzuki, S. Delehouzee, M. Goto, H. Watanabe, J. Sawada, K. Kataoka, and H. Handa. 2002. YEAF1/RYBP and YAF-2 are functionally distinct members of a cofactor family for the YY1 and E4TF1/hGABP transcription factors. J Biol Chem. 277:22484-90.
Schagger, H., and G. von Jagow. 1987. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 166:368-79.
Snijder, E.J., P.J. Bredenbeek, J.C. Dobbe, V. Thiel, J. Ziebuhr, L.L. Poon, Y. Guan, M. Rozanov, W.J. Spaan, and A.E. Gorbalenya. 2003. Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage. J Mol Biol. 331:991-1004.
Stadler, K., V. Masignani, M. Eickmann, S. Becker, S. Abrignani, H.D. Klenk, and R. Rappuoli. 2003. SARS--beginning to understand a new virus. Nat Rev Microbiol. 1:209-18.
Thiel, V., J. Herold, B. Schelle, and S.G. Siddell. 2001. Viral replicase gene products suffice for coronavirus discontinuous transcription. J Virol. 75:6676-81.
Thiel, V., K.A. Ivanov, A. Putics, T. Hertzig, B. Schelle, S. Bayer, B. Weissbrich, E.J. Snijder, H. Rabenau, H.W. Doerr, A.E. Gorbalenya, and J. Ziebuhr. 2003. Mechanisms and enzymes involved in SARS coronavirus genome expression. J Gen Virol. 84:2305-15.
Turner, A.J., J.A. Hiscox, and N.M. Hooper. 2004. ACE2: from vasopeptidase to SARS virus receptor. Trends Pharmacol Sci. 25:291-4.
von Grotthuss, M., L.S. Wyrwicz, and L. Rychlewski. 2003. mRNA cap-1 methyltransferase in the SARS genome. Cell. 113:701-2.
Yan, H., G. Xiao, J. Zhang, Y. Hu, F. Yuan, D.K. Cole, C. Zheng, and G.F. Gao. 2004. SARS coronavirus induces apoptosis in Vero E6 cells. J Med Virol. 73:323-31.
Yu, C.J., Y.C. Chen, C.H. Hsiao, T.C. Kuo, S.C. Chang, C.Y. Lu, W.C. Wei, C.H. Lee, L.M. Huang, M.F. Chang, H.N. Ho, and F.J. Lee. 2004. Identification of a novel protein 3a from severe acute respiratory syndrome coronavirus. FEBS Lett. 565:111-6.
Yu, X.J., C. Luo, J.C. Lin, P. Hao, Y.Y. He, Z.M. Guo, L. Qin, J. Su, B.S. Liu, Y. Huang, P. Nan, C.S. Li, B. Xiong, X.M. Luo, G.P. Zhao, G. Pei, K.X. Chen, X. Shen, J.H. Shen, J.P. Zou, W.Z. He, T.L. Shi, Y. Zhong, H.L. Jiang, and Y.X. Li. 2003. Putative hAPN receptor binding sites in SARS_CoV spike protein. Acta Pharmacol Sin. 24:481-8.
Zeng, F.Y., C.W. Chan, M.N. Chan, J.D. Chen, K.Y. Chow, C.C. Hon, K.H. Hui, J. Li, V.Y. Li, C.Y. Wang, P.Y. Wang, Y. Guan, B. Zheng, L.L. Poon, K.H. Chan, K.Y. Yuen, J.S. Peiris, and F.C. Leung. 2003. The complete genome sequence of severe acute respiratory syndrome coronavirus strain HKU-39849 (HK-39). Exp Biol Med (Maywood). 228:866-73.
Zheng, L., O. Schickling, M.E. Peter, and M.J. Lenardo. 2001. The death effector domain-associated factor plays distinct regulatory roles in the nucleus and cytoplasm. J Biol Chem. 276:31945-52.
Zhong, Y.F., X.M. Gao, S.L. Wang, Z.G. Xie, Y. Ma, W.G. Fang, W.Z. Zou, X.L. Li, Q.Y. Zhang, W. Wang, Z.D. Zhao, and J. Gu. 2003. [Pathologic study of circulating blood leukocytes in severe acute respiratory syndrome]. Zhonghua Yi Xue Za Zhi. 83:2137-41.
Zhu, M.S., Y. Pan, H.Q. Chen, Y. Shen, X.C. Wang, Y.J. Sun, and K.H. Tao. 2004. Induction of SARS-nucleoprotein-specific immune response by use of DNA vaccine. Immunol Lett. 92:237-43.
Zoltick, P.W., J.L. Leibowitz, J. DeVries, C.J. Pachuk, and S.R. Weiss. 1990. Detection of mouse hepatitis virus nonstructural proteins using antisera directed against bacterial viral fusion proteins. Adv Exp Med Biol. 276:291-9.
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