SARS冠狀病毒(SARS-CoV)包含四個結構蛋白：膜蛋白(M)、核鞘蛋白(N)、套膜蛋白(E)和棘蛋白(S)。許多研究指出，M和N共同表現時會藉由M的C端與N產生交互作用形成類病毒粒子(VLPs)。而我們之前研究發現，單獨表現M就能組裝形成membrane-enveloped vesicles釋出到細胞外。本篇研究欲探討M序列中M-M和M-N交互作用的位置。經由定位突變(site-directed mutagenesis)建構突變的M質體，將突變的M質體單獨表現或與N共同表現。觀察VLPs釋出之能力；經由pull down assay分析M-M以及M-N交互作用之胺基酸序列，更進一步探討定位突變後的M的聚合程度並觀察細胞內分佈的情形。結果顯示，M的N端(HA-M)不影響M的組裝和M-N形成VLP，然而，在M的C端末接上FLAG以及L218L219取代為Ala明顯地影響與N的交互作用。M失去N-linked醣基化(N4Q)，不影響VLPs的產生；M定位突變質體W19A、W91A、Y94A、F95A以及F95L皆顯著地降低VLPs的釋出。進一步探討發現，釋出能力有缺陷的M不影響M-M和M-N交互作用之能力，釋出能力有缺陷的M雖然可產生聚合作用，但部分的能力遭受影響。觀察M在細胞內分佈位置，結果發現破壞M醣化作用以及釋出能力有缺陷的M在細胞內分佈位置與原生型相近，分佈於細胞核周圍和細胞膜上。因此，我們認為釋出能力有缺陷的M雖然不會影響M-M、M-N交互作用能力以及細胞內分佈的位置，但定位突變的位置對於M自我組裝(self-assembly)的能力極為重要。

Severe acute respiratory syndrome coronavirus (SARS-CoV) encodes four structural proteins i.e. membrane (M), nucleocapsid (N), envelope (E) and spike (S). Coronavirus M is necessary for virus-like particles (VLPs) formation. A number of studies have shown that co-expression of M and N can generate substantial amoutns of VLPs and that the M carboxyl-terminal region is important for M-N interaction. Our previous study, however, demonsated that the M can self-assemble and release from cells. We aim to map the domain functionally involved in M-M self-association and M-N interaction. A panel of M mutatns was constructed by site-directed mutagenesis, and each of the resutant M mutants was transiently expressed or co-expressed with N. The release efficiency of VLPs was determined by Western blot. The M-M association and M-N interaction domain was further determined by pull-down assay. The oligomerization and localization of secretion-defective M mutants were analyzed. The data showed that HA tagged at the amino-terminus of M does not significantly affect M self-assembly or M plus N VLP formation. However, substitutions or FLAG tagged at the very carboxyl-terminus of M markedly impaired M-N interaction. Blocking glycosylation of M (N4Q) has no major impacts on M self-assembly and VLP release. Alanine substitution in either W19 or W91 significantly reduced VLP production. The release efficiency of VLPs was also markedly reduced when Y94 and F95 were replaced with alanine or leucine residues. However, the secretion-defective M can release into medium when co-expressed with wild-type M. In addition, GST pull-down assays suggest that these secretion-defective M mutants can interact with N protein. The localization of glycosylation-deficient M and secretion-defective M was similar to wild-type M in that they localized in the plasma membrane and perinuclear areas. Velocity sedimentation analysis suggests that multimerization of most secretion-defective was impaired to a certain extent. These results suggest that although the substitution mutations in M have no detrimental effects on M-M or M-N interaction but they significantly affect M self-assembly and release.

目 錄
誌謝……………………………………………………………………. i
中文摘要……………………………………………………………… ii
英文摘要……………………………………………………………… iv
目錄………………………………………………………………… vi
第一章 序論
SARS冠狀病毒的發現及起源……………………………………… 1
SARS冠狀病毒的分類學…………………………………………… 3
SARS冠狀病毒的基因結構………………………………………… 4
冠狀病毒生活史…………………………………………………… 5
SARS冠狀病毒結構及其結構蛋白………………………………… 5
實驗動機與設計…………………………………………………… 12
第二章 材料與方法
研究材料…………………………………………………………… 13
實驗方法…………………………………………………………… 17
第三章 實驗結果…………………………………………………… 28
第四章 討論………………………………………………………… 36
參考文獻……………………………………………………………… 43
圖表說明……………………………………………………………… 54
附錄………………………………………………………………… 70

1. World Health Organization. Summary of probable SARS cases with onset of illness from 1 November 2002 to 31 July 2003 Based on data as of the 31 December 2003.
2. Antia, R., R. R. Regoes, J. C. Koella, and C. T. Bergstrom. 2003. The role of evolution in the emergence of infectious diseases. Nature 426:658-61.
3. Arpin, N., and P. J. Talbot. 1990. Molecular characterization of the 229E strain of human coronavirus. Adv Exp Med Biol 276:73-80.
4. Baudoux, P., C. Carrat, L. Besnardeau, B. Charley, and H. Laude. 1998. Coronavirus pseudoparticles formed with recombinant M and E proteins induce alpha interferon synthesis by leukocytes. J Virol 72:8636-43.
5. Bos, E. C., W. Luytjes, H. V. van der Meulen, H. K. Koerten, and W. J. Spaan. 1996. The production of recombinant infectious DI-particles of a murine coronavirus in the absence of helper virus. Virology 218:52-60.
6. Bosch, B. J., R. van der Zee, C. A. de Haan, and P. J. Rottier. 2003. The coronavirus spike protein is a class I virus fusion protein: structural and functional characterization of the fusion core complex. J Virol 77:8801-11.
7. Braaten, D., and J. Luban. 2001. Cyclophilin A regulates HIV-1 infectivity, as demonstrated by gene targeting in human T cells. Embo J 20:1300-9.
8. Chan, C. M., C. W. Ma, W. Y. Chan, and H. Y. Chan. 2007. The SARS-Coronavirus Membrane protein induces apoptosis through modulating the Akt survival pathway. Arch Biochem Biophys 459:197-207.
9. Charley, B., and H. Laude. 1988. Induction of alpha interferon by transmissible gastroenteritis coronavirus: role of transmembrane glycoprotein E1. J Virol 62:8-11.
10. Corse, E., and C. E. Machamer. 2002. The cytoplasmic tail of infectious bronchitis virus E protein directs Golgi targeting. J Virol 76:1273-84.
11. Corse, E., and C. E. Machamer. 2000. Infectious bronchitis virus E protein is targeted to the Golgi complex and directs release of virus-like particles. J Virol 74:4319-26.
12. Cyranoski, D., and A. Abbott. 2003. Virus detectives seek source of SARS in China's wild animals. Nature 423:467.
13. de Haan, C. A., M. de Wit, L. Kuo, C. Montalto-Morrison, B. L. Haagmans, S. R. Weiss, P. S. Masters, and P. J. Rottier. 2003. The glycosylation status of the murine hepatitis coronavirus M protein affects the interferogenic capacity of the virus in vitro and its ability to replicate in the liver but not the brain. Virology 312:395-406.
14. de Haan, C. A., L. Kuo, P. S. Masters, H. Vennema, and P. J. Rottier. 1998. Coronavirus particle assembly: primary structure requirements of the membrane protein. J Virol 72:6838-50.
15. de Haan, C. A., M. Smeets, F. Vernooij, H. Vennema, and P. J. Rottier. 1999. Mapping of the coronavirus membrane protein domains involved in interaction with the spike protein. J Virol 73:7441-52.
16. de Haan, C. A., H. Vennema, and P. J. Rottier. 2000. Assembly of the coronavirus envelope: homotypic interactions between the M proteins. J Virol 74:4967-78.
17. DeDiego, M. L., E. Alvarez, F. Almazan, M. T. Rejas, E. Lamirande, A. Roberts, W. J. Shieh, S. R. Zaki, K. Subbarao, and L. Enjuanes. 2007. A severe acute respiratory syndrome coronavirus that lacks the E gene is attenuated in vitro and in vivo. J Virol 81:1701-13.
18. Dougherty, D. A. 1996. Cation-pi interactions in chemistry and biology: a new view of benzene, Phe, Tyr, and Trp. Science 271:163-8.
19. 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.
20. Eickmann, M., S. Becker, H. D. Klenk, H. W. Doerr, K. Stadler, S. Censini, S. Guidotti, V. Masignani, M. Scarselli, M. Mora, C. Donati, J. H. Han, H. C. Song, S. Abrignani, A. Covacci, and R. Rappuoli. 2003. Phylogeny of the SARS coronavirus. Science 302:1504-5.
21. Escors, D., J. Ortego, and L. Enjuanes. 2001. The membrane M protein of the transmissible gastroenteritis coronavirus binds to the internal core through the carboxy-terminus. Adv Exp Med Biol 494:589-93.
22. Escors, D., J. Ortego, H. Laude, and L. Enjuanes. 2001. The membrane M protein carboxy terminus binds to transmissible gastroenteritis coronavirus core and contributes to core stability. J Virol 75:1312-24.
23. Fang, X., L. Ye, K. A. Timani, S. Li, Y. Zen, M. Zhao, H. Zheng, and Z. Wu. 2005. Peptide domain involved in the interaction between membrane protein and nucleocapsid protein of SARS-associated coronavirus. J Biochem Mol Biol 38:381-5.
24. Fang, X., L. B. Ye, Y. Zhang, B. Li, S. Li, L. Kong, Y. Wang, H. Zheng, W. Wang, and Z. Wu. 2006. Nucleocapsid amino acids 211 to 254, in particular, tetrad glutamines, are essential for the interaction between the nucleocapsid and membrane proteins of SARS-associated coronavirus. J Microbiol 44:577-80.
25. Fouchier, R. A., T. Kuiken, M. Schutten, G. van Amerongen, G. J. van Doornum, B. G. van den Hoogen, M. Peiris, W. Lim, K. Stohr, and A. D. Osterhaus. 2003. Aetiology: Koch's postulates fulfilled for SARS virus. Nature 423:240.
26. Gamble, T. R., F. F. Vajdos, S. Yoo, D. K. Worthylake, M. Houseweart, W. I. Sundquist, and C. P. Hill. 1996. Crystal structure of human cyclophilin A bound to the amino-terminal domain of HIV-1 capsid. Cell 87:1285-94.
27. Gibbs, A. J., M. J. Gibbs, and J. S. Armstrong. 2004. The phylogeny of SARS coronavirus. Arch Virol 149:621-4.
28. Godet, M., J. Grosclaude, B. Delmas, and H. Laude. 1994. Major receptor-binding and neutralization determinants are located within the same domain of the transmissible gastroenteritis virus (coronavirus) spike protein. J Virol 68:8008-16.
29. Groneberg, D. A., L. Zhang, T. Welte, P. Zabel, and K. F. Chung. 2003. Severe acute respiratory syndrome: global initiatives for disease diagnosis. Qjm 96:845-52.
30. 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.
31. Hatakeyama, S., Y. Matsuoka, H. Ueshiba, N. Komatsu, K. Itoh, S. Shichijo, T. Kanai, M. Fukushi, I. Ishida, T. Kirikae, T. Sasazuki, and T. Miyoshi-Akiyama. 2008. Dissection and identification of regions required to form pseudoparticles by the interaction between the nucleocapsid (N) and membrane (M) proteins of SARS coronavirus. Virology 380:99-108.
32. He, J. F., G. W. Peng, H. Z. Zheng, H. M. Luo, W. J. Liang, L. H. Li, R. N. Guo, and Z. H. Deng. 2003. [An epidemiological study on the index cases of severe acute respiratory syndrome occurred in different cities among Guangdong province]. Zhonghua Liu Xing Bing Xue Za Zhi 24:347-9.
33. He, R., A. Leeson, M. Ballantine, A. Andonov, L. Baker, F. Dobie, Y. Li, N. Bastien, H. Feldmann, U. Strocher, S. Theriault, T. Cutts, J. Cao, T. F. Booth, F. A. Plummer, S. Tyler, and X. Li. 2004. Characterization of protein-protein interactions between the nucleocapsid protein and membrane protein of the SARS coronavirus. Virus Res 105:121-5.
34. He, Y., Y. Zhou, H. Wu, Z. Kou, S. Liu, and S. Jiang. 2004. Mapping of antigenic sites on the nucleocapsid protein of the severe acute respiratory syndrome coronavirus. J Clin Microbiol 42:5309-14.
35. Ho, Y., P. H. Lin, C. Y. Liu, S. P. Lee, and Y. C. Chao. 2004. Assembly of human severe acute respiratory syndrome coronavirus-like particles. Biochem Biophys Res Commun 318:833-8.
36. Horzinek, M. C. 1999. Molecular evolution of corona- and toroviruses. Adv Exp Med Biol 473:61-72.
37. Hsieh, P. K., S. C. Chang, C. C. Huang, T. T. Lee, C. W. Hsiao, Y. H. Kou, I. Y. Chen, C. K. Chang, T. H. Huang, and M. F. Chang. 2005. Assembly of severe acute respiratory syndrome coronavirus RNA packaging signal into virus-like particles is nucleocapsid dependent. J Virol 79:13848-55.
38. Hsieh, Y. C., H. C. Li, S. C. Chen, and S. Y. Lo. 2008. Interactions between M protein and other structural proteins of severe, acute respiratory syndrome-associated coronavirus. J Biomed Sci 15:707-17.
39. Hu, Y., J. Wen, L. Tang, H. Zhang, X. Zhang, Y. Li, J. Wang, Y. Han, G. Li, J. Shi, X. Tian, F. Jiang, X. Zhao, J. Wang, S. Liu, C. Zeng, J. Wang, and H. Yang. 2003. The M protein of SARS-CoV: basic structural and immunological properties. Genomics Proteomics Bioinformatics 1:118-30.
40. Huang, Q., L. Yu, A. M. Petros, A. Gunasekera, Z. Liu, N. Xu, P. Hajduk, J. Mack, S. W. Fesik, and E. T. Olejniczak. 2004. Structure of the N-terminal RNA-binding domain of the SARS CoV nucleocapsid protein. Biochemistry 43:6059-63.
41. Huang, Y., Z. Y. Yang, W. P. Kong, and G. J. Nabel. 2004. Generation of synthetic severe acute respiratory syndrome coronavirus pseudoparticles: implications for assembly and vaccine production. J Virol 78:12557-65.
42. Jacobse-Geels, H. E., and M. C. Horzinek. 1983. Expression of feline infectious peritonitis coronavirus antigens on the surface of feline macrophage-like cells. J Gen Virol 64 (Pt 9):1859-66.
43. Klumperman, J., J. K. Locker, A. Meijer, M. C. Horzinek, H. J. Geuze, and P. J. Rottier. 1994. Coronavirus M proteins accumulate in the Golgi complex beyond the site of virion budding. J Virol 68:6523-34.
44. Krijnse-Locker, J., M. Ericsson, P. J. Rottier, and G. Griffiths. 1994. Characterization of the budding compartment of mouse hepatitis virus: evidence that transport from the RER to the Golgi complex requires only one vesicular transport step. J Cell Biol 124:55-70.
45. Ksiazek, T. G., D. Erdman, C. S. Goldsmith, S. R. Zaki, T. Peret, S. Emery, S. Tong, C. Urbani, J. A. Comer, W. Lim, P. E. Rollin, S. F. Dowell, A. E. Ling, C. D. Humphrey, W. J. Shieh, J. Guarner, C. D. Paddock, P. Rota, B. Fields, J. DeRisi, J. Y. Yang, N. Cox, J. M. Hughes, J. W. LeDuc, W. J. Bellini, and L. J. Anderson. 2003. A novel coronavirus associated with severe acute respiratory syndrome. N Engl J Med 348:1953-66.
46. Kubo, H., Y. K. Yamada, and F. Taguchi. 1994. Localization of neutralizing epitopes and the receptor-binding site within the amino-terminal 330 amino acids of the murine coronavirus spike protein. J Virol 68:5403-10.
47. Kuiken, T., R. A. Fouchier, M. Schutten, G. F. Rimmelzwaan, G. van Amerongen, D. van Riel, J. D. Laman, T. de Jong, G. van Doornum, W. Lim, A. E. Ling, P. K. Chan, J. S. Tam, M. C. Zambon, R. Gopal, C. Drosten, S. van der Werf, N. Escriou, J. C. Manuguerra, K. Stohr, J. S. Peiris, and A. D. Osterhaus. 2003. Newly discovered coronavirus as the primary cause of severe acute respiratory syndrome. Lancet 362:263-70.
48. Kuo, L., and P. S. Masters. 2002. Genetic evidence for a structural interaction between the carboxy termini of the membrane and nucleocapsid proteins of mouse hepatitis virus. J Virol 76:4987-99.
49. Lai, M. M. 2003. SARS virus: the beginning of the unraveling of a new coronavirus. J Biomed Sci 10:664-75.
50. Laude, H., J. Gelfi, L. Lavenant, and B. Charley. 1992. Single amino acid changes in the viral glycoprotein M affect induction of alpha interferon by the coronavirus transmissible gastroenteritis virus. J Virol 66:743-9.
51. Laviada, M. D., S. P. Videgain, L. Moreno, F. Alonso, L. Enjuanes, and J. M. Escribano. 1990. Expression of swine transmissible gastroenteritis virus envelope antigens on the surface of infected cells: epitopes externally exposed. Virus Res 16:247-54.
52. Lee, Y. N., L. K. Chen, H. C. Ma, H. H. Yang, H. P. Li, and S. Y. Lo. 2005. Thermal aggregation of SARS-CoV membrane protein. J Virol Methods 129:152-61.
53. Li, F., W. Li, M. Farzan, and S. C. Harrison. 2005. Structure of SARS coronavirus spike receptor-binding domain complexed with receptor. Science 309:1864-8.
54. Li, W., M. J. Moore, N. Vasilieva, J. Sui, S. K. Wong, M. A. Berne, M. Somasundaran, J. L. Sullivan, K. Luzuriaga, T. C. Greenough, H. Choe, and M. Farzan. 2003. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 426:450-4.
55. Liao, Y., Q. Yuan, J. Torres, J. P. Tam, and D. X. Liu. 2006. Biochemical and functional characterization of the membrane association and membrane permeabilizing activity of the severe acute respiratory syndrome coronavirus envelope protein. Virology 349:264-75.
56. Lim, K. P., and D. X. Liu. 2001. The missing link in coronavirus assembly. Retention of the avian coronavirus infectious bronchitis virus envelope protein in the pre-Golgi compartments and physical interaction between the envelope and membrane proteins. J Biol Chem 276:17515-23.
57. 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.
58. Liu, X., Y. Shi, P. Li, L. Li, Y. Yi, Q. Ma, and C. Cao. 2004. Profile of antibodies to the nucleocapsid protein of the severe acute respiratory syndrome (SARS)-associated coronavirus in probable SARS patients. Clin Diagn Lab Immunol 11:227-8.
59. Locker, J. K., D. J. Opstelten, M. Ericsson, M. C. Horzinek, and P. J. Rottier. 1995. Oligomerization of a trans-Golgi/trans-Golgi network retained protein occurs in the Golgi complex and may be part of its retention. J Biol Chem 270:8815-21.
60. Luo, C., H. Luo, S. Zheng, C. Gui, L. Yue, C. Yu, T. Sun, P. He, J. Chen, J. Shen, X. Luo, Y. Li, H. Liu, D. Bai, J. Shen, Y. Yang, F. Li, J. Zuo, R. Hilgenfeld, G. Pei, K. Chen, X. Shen, and H. Jiang. 2004. Nucleocapsid protein of SARS coronavirus tightly binds to human cyclophilin A. Biochem Biophys Res Commun 321:557-65.
61. Luo, H., J. Chen, K. Chen, X. Shen, and H. Jiang. 2006. Carboxyl terminus of severe acute respiratory syndrome coronavirus nucleocapsid protein: self-association analysis and nucleic acid binding characterization. Biochemistry 45:11827-35.
62. Luo, H., Q. Chen, J. Chen, K. Chen, X. Shen, and H. Jiang. 2005. The nucleocapsid protein of SARS coronavirus has a high binding affinity to the human cellular heterogeneous nuclear ribonucleoprotein A1. FEBS Lett 579:2623-8.
63. Luo, H., D. Wu, C. Shen, K. Chen, X. Shen, and H. Jiang. 2006. Severe acute respiratory syndrome coronavirus membrane protein interacts with nucleocapsid protein mostly through their carboxyl termini by electrostatic attraction. Int J Biochem Cell Biol 38:589-99.
64. Luo, H., F. Ye, K. Chen, X. Shen, and H. Jiang. 2005. SR-rich motif plays a pivotal role in recombinant SARS coronavirus nucleocapsid protein multimerization. Biochemistry 44:15351-8.
65. 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.
66. Martina, B. E., B. L. Haagmans, T. Kuiken, R. A. Fouchier, G. F. Rimmelzwaan, G. Van Amerongen, J. S. Peiris, W. Lim, and A. D. Osterhaus. 2003. Virology: SARS virus infection of cats and ferrets. Nature 425:915.
67. McBride, C. E., and C. E. Machamer. A single tyrosine in the severe acute respiratory syndrome coronavirus membrane protein cytoplasmic tail is important for efficient interaction with spike protein. J Virol 84:1891-901.
68. McGaughey, G. B., M. Gagne, and A. K. Rappe. 1998. pi-Stacking interactions. Alive and well in proteins. J Biol Chem 273:15458-63.
69. Morikawa, Y., D. J. Hockley, M. V. Nermut, and I. M. Jones. 2000. Roles of matrix, p2, and N-terminal myristoylation in human immunodeficiency virus type 1 Gag assembly. J Virol 74:16-23.
70. Mortola, E., and P. Roy. 2004. Efficient assembly and release of SARS coronavirus-like particles by a heterologous expression system. FEBS Lett 576:174-8.
71. Nal, B., C. Chan, F. Kien, L. Siu, J. Tse, K. Chu, J. Kam, I. Staropoli, B. Crescenzo-Chaigne, N. Escriou, S. van der Werf, K. Y. Yuen, and R. Altmeyer. 2005. Differential maturation and subcellular localization of severe acute respiratory syndrome coronavirus surface proteins S, M and E. J Gen Virol 86:1423-34.
72. Narayanan, K., A. Maeda, J. Maeda, and S. Makino. 2000. Characterization of the coronavirus M protein and nucleocapsid interaction in infected cells. J Virol 74:8127-34.
73. Ng, S. K. 2003. Possible role of an animal vector in the SARS outbreak at Amoy Gardens. Lancet 362:570-2.
74. Niemann, H., B. Boschek, D. Evans, M. Rosing, T. Tamura, and H. D. Klenk. 1982. Post-translational glycosylation of coronavirus glycoprotein E1: inhibition by monensin. Embo J 1:1499-504.
75. Peiris, J. S., S. T. Lai, L. L. Poon, Y. Guan, L. Y. Yam, W. Lim, J. Nicholls, W. K. Yee, W. W. Yan, M. T. Cheung, V. C. Cheng, K. H. Chan, D. N. Tsang, R. W. Yung, T. K. Ng, and K. Y. Yuen. 2003. Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet 361:1319-25.
76. Ponnuswamy, P. K., and M. M. Gromiha. 1994. On the conformational stability of oligonucleotide duplexes and tRNA molecules. J Theor Biol 169:419-32.
77. Reilley, B., M. Van Herp, D. Sermand, and N. Dentico. 2003. SARS and Carlo Urbani. N Engl J Med 348:1951-2.
78. 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.
79. Sal-Man, N., D. Gerber, I. Bloch, and Y. Shai. 2007. Specificity in transmembrane helix-helix interactions mediated by aromatic residues. J Biol Chem 282:19753-61.
80. Satija, N., and S. K. Lal. 2007. The molecular biology of SARS coronavirus. Ann N Y Acad Sci 1102:26-38.
81. Shi, S. T., P. Huang, H. P. Li, and M. M. Lai. 2000. Heterogeneous nuclear ribonucleoprotein A1 regulates RNA synthesis of a cytoplasmic virus. Embo J 19:4701-11.
82. Shi, Y., Y. Yi, P. Li, T. Kuang, L. Li, M. Dong, Q. Ma, and C. Cao. 2003. Diagnosis of severe acute respiratory syndrome (SARS) by detection of SARS coronavirus nucleocapsid antibodies in an antigen-capturing enzyme-linked immunosorbent assay. J Clin Microbiol 41:5781-2.
83. Siu, Y. L., K. T. Teoh, J. Lo, C. M. Chan, F. Kien, N. Escriou, S. W. Tsao, J. M. Nicholls, R. Altmeyer, J. S. Peiris, R. Bruzzone, and B. Nal. 2008. The M, E, and N structural proteins of the severe acute respiratory syndrome coronavirus are required for efficient assembly, trafficking, and release of virus-like particles. J Virol 82:11318-30.
84. 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.
85. Spiga, O., A. Bernini, A. Ciutti, S. Chiellini, N. Menciassi, F. Finetti, V. Causarono, F. Anselmi, F. Prischi, and N. Niccolai. 2003. Molecular modelling of S1 and S2 subunits of SARS coronavirus spike glycoprotein. Biochem Biophys Res Commun 310:78-83.
86. 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.
87. Stern, D. F., and B. M. Sefton. 1982. Coronavirus proteins: structure and function of the oligosaccharides of the avian infectious bronchitis virus glycoproteins. J Virol 44:804-12.
88. Surjit, M., B. Liu, P. Kumar, V. T. Chow, and S. K. Lal. 2004. The nucleocapsid protein of the SARS coronavirus is capable of self-association through a C-terminal 209 amino acid interaction domain. Biochem Biophys Res Commun 317:1030-6.
89. Tan, Y. J., S. G. Lim, and W. Hong. 2005. Characterization of viral proteins encoded by the SARS-coronavirus genome. Antiviral Res 65:69-78.
90. 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.
91. To, L. T., S. Bernard, and I. Lantier. 1991. Fixed-cell immunoperoxidase technique for the study of surface antigens induced by the coronavirus of transmissible gastroenteritis (TGEV). Vet Microbiol 29:361-8.
92. Tooze, J., S. Tooze, and G. Warren. 1984. Replication of coronavirus MHV-A59 in sac- cells: determination of the first site of budding of progeny virions. Eur J Cell Biol 33:281-93.
93. Tooze, J., and S. A. Tooze. 1985. Infection of AtT20 murine pituitary tumour cells by mouse hepatitis virus strain A59: virus budding is restricted to the Golgi region. Eur J Cell Biol 37:203-12.
94. Torres, J., J. Wang, K. Parthasarathy, and D. X. Liu. 2005. The transmembrane oligomers of coronavirus protein E. Biophys J 88:1283-90.
95. Tripet, B., M. W. Howard, M. Jobling, R. K. Holmes, K. V. Holmes, and R. S. Hodges. 2004. Structural characterization of the SARS-coronavirus spike S fusion protein core. J Biol Chem 279:20836-49.
96. Tseng, Y. T., S. M. Wang, K. J. Huang, A. I. Lee, C. C. Chiang, and C. T. Wang. Self-assembly of severe acute respiratory syndrome coronavirus membrane protein. J Biol Chem 285:12862-72.
97. Vennema, H., G. J. Godeke, J. W. Rossen, W. F. Voorhout, M. C. Horzinek, D. J. Opstelten, and P. J. Rottier. 1996. Nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes. Embo J 15:2020-8.
98. Voss, D., A. Kern, E. Traggiai, M. Eickmann, K. Stadler, A. Lanzavecchia, and S. Becker. 2006. Characterization of severe acute respiratory syndrome coronavirus membrane protein. FEBS Lett 580:968-73.
99. Voss, D., S. Pfefferle, C. Drosten, L. Stevermann, E. Traggiai, A. Lanzavecchia, and S. Becker. 2009. Studies on membrane topology, N-glycosylation and functionality of SARS-CoV membrane protein. Virol J 6:79.
100. Wang, J., J. Wen, J. Li, J. Yin, Q. Zhu, H. Wang, Y. Yang, E. Qin, B. You, W. Li, X. Li, S. Huang, R. Yang, X. Zhang, L. Yang, T. Zhang, Y. Yin, X. Cui, X. Tang, L. Wang, B. He, L. Ma, T. Lei, C. Zeng, J. Fang, J. Yu, J. Wang, H. Yang, M. B. West, A. Bhatnagar, Y. Lu, N. Xu, and S. Liu. 2003. Assessment of immunoreactive synthetic peptides from the structural proteins of severe acute respiratory syndrome coronavirus. Clin Chem 49:1989-96.
101. Waters, M. L. 2002. Aromatic interactions in model systems. Curr Opin Chem Biol 6:736-41.
102. Weisz, O. A., A. M. Swift, and C. E. Machamer. 1993. Oligomerization of a membrane protein correlates with its retention in the Golgi complex. J Cell Biol 122:1185-96.
103. Wong, S. K., W. Li, M. J. Moore, H. Choe, and M. Farzan. 2004. A 193-amino acid fragment of the SARS coronavirus S protein efficiently binds angiotensin-converting enzyme 2. J Biol Chem 279:3197-201.
104. Wu, X. D., B. Shang, R. F. Yang, H. Yu, Z. H. Ma, X. Shen, Y. Y. Ji, Y. Lin, Y. D. Wu, G. M. Lin, L. Tian, X. Q. Gan, S. Yang, W. H. Jiang, E. H. Dai, X. Y. Wang, H. L. Jiang, Y. H. Xie, X. L. Zhu, G. Pei, L. Li, J. R. Wu, and B. Sun. 2004. The spike protein of severe acute respiratory syndrome (SARS) is cleaved in virus infected Vero-E6 cells. Cell Res 14:400-6.
105. Xu, Y., Y. Liu, Z. Lou, L. Qin, X. Li, Z. Bai, H. Pang, P. Tien, G. F. Gao, and Z. Rao. 2004. Structural basis for coronavirus-mediated membrane fusion. Crystal structure of mouse hepatitis virus spike protein fusion core. J Biol Chem 279:30514-22.
106. Xu, Y., Z. Lou, Y. Liu, H. Pang, P. Tien, G. F. Gao, and Z. Rao. 2004. Crystal structure of severe acute respiratory syndrome coronavirus spike protein fusion core. J Biol Chem 279:49414-9.
107. Yu, I. M., C. L. Gustafson, J. Diao, J. W. Burgner, 2nd, Z. Li, J. Zhang, and J. Chen. 2005. Recombinant severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein forms a dimer through its C-terminal domain. J Biol Chem 280:23280-6.
108. Yuan, Q., Y. Liao, J. Torres, J. P. Tam, and D. X. Liu. 2006. Biochemical evidence for the presence of mixed membrane topologies of the severe acute respiratory syndrome coronavirus envelope protein expressed in mammalian cells. FEBS Lett 580:3192-200.
109. Zhu, G., and H. W. Chen. 2004. Monophyletic relationship between severe acute respiratory syndrome coronavirus and group 2 coronaviruses. J Infect Dis 189:1676-8.