以二次元電泳分析比對非毒性與毒性 H6N1 禽流感病毒株，發現毒性株 HA1 蛋白 質群點之等點電較非毒性株大，此差異源自於 HA1 之胺基酸序列差異，而毒性株之 HA1 較呈鹼性，可能和病毒之組織趨性有關。
利用免疫染色法，清楚發現兩株病毒之 HA1 各有六種等電點異構物。研究證實這 些異構物並非由蛋白質磷酸化所造成，而是與 HA1 之醣質相關。使用 lectin blot 發 現，唾液酸 (sialic acid) 可能會以 α 2-3 之方式與 HA1 醣質之半乳糖 (galactose) 鍵結 後，實驗進一步以醣晶片、HPLC，配合 MALDI MS/MS 建立 HA1 之醣質表現圖譜。
結果發現， HA1 之醣質皆無唾液酸修飾。特殊的是，HA1 大部分之醣質為 m/z 1867 之 未知 X-glycan。X-glycan 為 m/z 1579 之 high mannose type 之醣質再加上一個 m/z 287 未知之物質 X。比較各 HA1 異構物之醣質圖譜發現，等電點越低之 HA1 異構物其 X-glycan 比例越高。因此推論若 X 為酸性物質，則 HA1 之 X-glycan 可能為形成 HA1 多種等電點異構物的原因之一；若 X 為中性物質，則表示越酸性之 HA1 異構物，其 high mannose type 之醣質比例越高。而 HA1 形成多種異構物，則可能和流感病毒之宿 主辨識、組織趨性及逃脫宿主免疫系統有關。
第六章 參考文獻
1. Lupiani, B. & Reddy, S.M. The history of avian influenza. Comp Immunol Microbiol Infect Dis (2008).
2. Webster, R.G., Bean, W.J., Gorman, O.T., Chambers, T.M. & Kawaoka, Y. Evolution and ecology of influenza A viruses. Microbiol Rev 56, 152-79 (1992).
3. Alexander, D.J. Newcastle disease and other avian paramyxoviruses. Rev Sci Tech 19, 443-62 (2000).
4. Gendon Iu, Z. [Influenza pandemic: hypotheses and facts]. Zh Mikrobiol Epidemiol Immunobiol, 109-18 (2008).
5. Basler, C.F. & Aguilar, P.V. Progress in identifying virulence determinants of the 1918 H1N1 and the Southeast Asian H5N1 influenza A viruses. Antiviral Res 79, 166-78 (2008).
6. Kawaoka, Y., Krauss, S. & Webster, R.G. Avian-to-human transmission of the PB1 gene of influenza A viruses in the 1957 and 1968 pandemics. J Virol 63, 4603-8 (1989).
7. Perkins, L.E. & Swayne, D.E. Varied pathogenicity of a Hong Kong-origin H5N1 avian influenza virus in four passerine species and budgerigars. Vet Pathol 40, 14-24 (2003).
8. Perkins, L.E. & Swayne, D.E. Comparative susceptibility of selected avian and mammalian species to a Hong Kong-origin H5N1 high-pathogenicity avian influenza virus. Avian Dis 47, 956-67 (2003).
9. Kaiser, J. A one-size-fits-all flu vaccine? Science 312, 380-2 (2006).
10. Zebedee, S.L. & Lamb, R.A. Influenza A virus M2 protein: monoclonal antibody restriction of virus growth and detection of M2 in virions. J Virol 62, 2762-72 (1988).
11. Cornelia Schroeder1, H.H., Elisabeth Möncke-Buchner and Tse-I Lin. The influenza virus ion channel and maturation cofactor M2 is a cholesterol-binding protein European Biophysics Journal 34, 52-66 (2005).
12. Brown, E.G. Influenza virus genetics. Biomed Pharmacother 54, 196-209 (2000).
13. Naffakh, N., Tomoiu, A., Rameix-Welti, M.A. & van der Werf, S. Host restriction of avian influenza viruses at the level of the ribonucleoproteins. Annu Rev Microbiol 62, 403-24 (2008).
14. Sidorenko, Y. & Reichl, U. Structured model of influenza virus replication in MDCK
15. Chen, W. et al. A novel influenza A virus mitochondrial protein that induces cell death.
Nat Med 7, 1306-12 (2001).
16. Portela, A., Zurcher, T., Nieto, A. & Ortin, J. Replication of orthomyxoviruses. Adv Virus Res 54, 319-48 (1999).
17. Mikulasova, A., Vareckova, E. & Fodor, E. Transcription and replication of the influenza a virus genome. Acta Virol 44, 273-82 (2000).
18. Bouvier, N.M. & Palese, P. The biology of influenza viruses. Vaccine 26 Suppl 4, D49-53 (2008).
19. Neumann, G., Hughes, M.T. & Kawaoka, Y. Influenza A virus NS2 protein mediates vRNP nuclear export through NES-independent interaction with hCRM1. Embo J 19, 6751-8 (2000).
20. Nayak, D.P., Hui, E.K. & Barman, S. Assembly and budding of influenza virus. Virus Res 106, 147-65 (2004).
21. Webster, R.G. & Rott, R. Influenza virus A pathogenicity: the pivotal role of hemagglutinin. Cell 50, 665-6 (1987).
22. Gong, J., Xu, W. & Zhang, J. Structure and functions of influenza virus neuraminidase.
Curr Med Chem 14, 113-22 (2007).
23. Pinto, L.H. & Lamb, R.A. The M2 proton channels of influenza A and B viruses. J Biol Chem 281, 8997-9000 (2006).
24. Hale, B.G., Randall, R.E., Ortin, J. & Jackson, D. The multifunctional NS1 protein of influenza A viruses. J Gen Virol 89, 2359-76 (2008).
25. Boulo, S., Akarsu, H., Ruigrok, R.W. & Baudin, F. Nuclear traffic of influenza virus proteins and ribonucleoprotein complexes. Virus Res 124, 12-21 (2007).
26. Couceiro, J.N., Paulson, J.C. & Baum, L.G. Influenza virus strains selectively recognize sialyloligosaccharides on human respiratory epithelium; the role of the host cell in selection of hemagglutinin receptor specificity. Virus Res 29, 155-65 (1993).
27. Matrosovich, M.N., Matrosovich, T.Y., Gray, T., Roberts, N.A. & Klenk, H.D.
Human and avian influenza viruses target different cell types in cultures of human airway epithelium. Proc Natl Acad Sci U S A 101, 4620-4 (2004).
28. Sieczkarski, S.B. & Whittaker, G.R. Dissecting virus entry via endocytosis. J Gen Virol 83, 1535-45 (2002).
29. Sieczkarski, S.B. & Whittaker, G.R. Influenza virus can enter and infect cells in the absence of clathrin-mediated endocytosis. J Virol 76, 10455-64 (2002).
30. Cros, J.F. & Palese, P. Trafficking of viral genomic RNA into and out of the nucleus:
influenza, Thogoto and Borna disease viruses. Virus Res 95, 3-12 (2003).
31. Li, X. & Palese, P. Characterization of the polyadenylation signal of influenza virus RNA. J Virol 68, 1245-9 (1994).
32. Luo, G.X., Luytjes, W., Enami, M. & Palese, P. The polyadenylation signal of
influenza virus RNA involves a stretch of uridines followed by the RNA duplex of the panhandle structure. J Virol 65, 2861-7 (1991).
33. Robertson, J.S., Schubert, M. & Lazzarini, R.A. Polyadenylation sites for influenza virus mRNA. J Virol 38, 157-63 (1981).
34. Krug, R.M. Priming of influenza viral RNA transcription by capped heterologous RNAs. Curr Top Microbiol Immunol 93, 125-49 (1981).
35. Park, Y.W. & Katze, M.G. Translational control by influenza virus. Identification of cis-acting sequences and trans-acting factors which may regulate selective viral mRNA translation. J Biol Chem 270, 28433-9 (1995).
36. Portela, A. & Digard, P. The influenza virus nucleoprotein: a multifunctional RNA-binding protein pivotal to virus replication. J Gen Virol 83, 723-34 (2002).
37. Subbarao, K. & Katz, J. Avian influenza viruses infecting humans. Cell Mol Life Sci 57, 1770-84 (2000).
38. Gamblin, S.J. et al. The structure and receptor binding properties of the 1918 influenza hemagglutinin. Science 303, 1838-42 (2004).
39. Rogers, G.N. & D'Souza, B.L. Receptor binding properties of human and animal H1 influenza virus isolates. Virology 173, 317-22 (1989).
40. Connor, R.J., Kawaoka, Y., Webster, R.G. & Paulson, J.C. Receptor specificity in human, avian, and equine H2 and H3 influenza virus isolates. Virology 205, 17-23 (1994).
41. Matrosovich, M., Zhou, N., Kawaoka, Y. & Webster, R. The surface glycoproteins of H5 influenza viruses isolated from humans, chickens, and wild aquatic birds have distinguishable properties. J Virol 73, 1146-55 (1999).
42. Ito, T. et al. Molecular basis for the generation in pigs of influenza A viruses with pandemic potential. J Virol 72, 7367-73 (1998).
43. Bean, W.J. et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol 66, 1129-38 (1992).
44. Parrish, C.R. & Kawaoka, Y. The origins of new pandemic viruses: the acquisition of new host ranges by canine parvovirus and influenza A viruses. Annu Rev Microbiol 59, 553-86 (2005).
45. Recker, M., Pybus, O.G., Nee, S. & Gupta, S. The generation of influenza outbreaks by a network of host immune responses against a limited set of antigenic types. Proc Natl Acad Sci U S A 104, 7711-6 (2007).
46. Rott, R. The pathogenic determinant of influenza virus. Vet Microbiol 33, 303-10 (1992).
47. Horimoto, T. & Kawaoka, Y. Direct reverse transcriptase PCR to determine virulence potential of influenza A viruses in birds. J Clin Microbiol 33, 748-51 (1995).
novel influenza hemagglutinin, H15: criteria for determination of influenza A subtypes.
Virology 217, 508-16 (1996).
49. Hebart, H. et al. CMV infection after allogeneic bone marrow transplantation is associated with the occurrence of various autoantibodies and monoclonal gammopathies. Br J Haematol 95, 138-44 (1996).
50. Keil, W., Niemann, H., Schwarz, R.T. & Klenk, H.D. Carbohydrates of influenza virus. V. Oligosaccharides attached to individual glycosylation sites of the hemagglutinin of fowl plague virus. Virology 133, 77-91 (1984).
51. Skehel, J.J. & Wiley, D.C. Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annu Rev Biochem 69, 531-69 (2000).
52. David J. Vigerust, a.V.L.S. Virus glycosylation: role in virulence and immune interactions Trends in Microbiology 15, 211-218 (2007).
53. Perdue, M.L. & Suarez, D.L. Structural features of the avian influenza virus hemagglutinin that influence virulence. Vet Microbiol 74, 77-86 (2000).
54. Baigent, S.J. & McCauley, J.W. Glycosylation of haemagglutinin and stalk-length of neuraminidase combine to regulate the growth of avian influenza viruses in tissue culture. Virus Res 79, 177-85 (2001).
55. Abe, Y. et al. Effect of the addition of oligosaccharides on the biological activities and antigenicity of influenza A/H3N2 virus hemagglutinin. J Virol 78, 9605-11 (2004).
56. Klenk, H.D., Wagner, R., Heuer, D. & Wolff, T. Importance of hemagglutinin glycosylation for the biological functions of influenza virus. Virus Res 82, 73-5 (2002).
57. Wagner, R., Wolff, T., Herwig, A., Pleschka, S. & Klenk, H.D. Interdependence of hemagglutinin glycosylation and neuraminidase as regulators of influenza virus growth: a study by reverse genetics. J Virol 74, 6316-23 (2000).
58. Deshpande, K.L., Fried, V.A., Ando, M. & Webster, R.G. Glycosylation affects cleavage of an H5N2 influenza virus hemagglutinin and regulates virulence. Proc Natl Acad Sci U S A 84, 36-40 (1987).
59. Zambon, M.C. Epidemiology and pathogenesis of influenza. J Antimicrob Chemother 44 Suppl B, 3-9 (1999).
60. Sauter, N.K. et al. Binding of influenza virus hemagglutinin to analogs of its cell-surface receptor, sialic acid: analysis by proton nuclear magnetic resonance spectroscopy and X-ray crystallography. Biochemistry 31, 9609-21 (1992).
61. Wang, C.W. & Wang, C.H. Experimental selection of virus derivatives with variations in virulence from a single low-pathogenicity H6N1 avian influenza virus field isolate.
Avian Dis 47, 1416-22 (2003).
62. 何杰龍. 建立單株抗體庫以應用於臺灣家禽流行性感冒病毒之蛋白質體學研究.
國立台灣大學微生物與生物化學研究所博士班資格考論文 (2006).
63. Privalsky, M.L. & Penhoet, E.E. Influenza virus proteins: identity, synthesis, and modification analyzed by two-dimensional gel electrophoresis. Proc Natl Acad Sci U S A 75, 3625-9 (1978).
64. Hsu, C.N. & Wang, C.H. Sequence comparison between two quasi strains of H6N1 with different pathogenicity from a single parental isolate. J Microbiol Immunol Infect 39, 292-6 (2006).
65. Philpott, M., Hioe, C., Sheerar, M. & Hinshaw, V.S. Hemagglutinin mutations related to attenuation and altered cell tropism of a virulent avian influenza A virus. J Virol 64, 2941-7 (1990).
66. Bosch, F.X. Studies on the development of the charge heterogeneity of the influenza virus glycoproteins. Arch Virol 83, 311-7 (1985).
67. Horimoto, T. & Kawaoka, Y. Molecular changes in virulent mutants arising from avirulent avian influenza viruses during replication in 14-day-old embryonated eggs.
Virology 206, 755-9 (1995).
68. Gendoo, D.M., El-Hefnawi, M.M., Werner, M. & Siam, R. Correlating novel variable and conserved motifs in the Hemagglutinin protein with significant biological functions. Virol J 5, 91 (2008).
69. Spiro, M.J. & Spiro, R.G. Sulfation of the N-linked oligosaccharides of influenza virus hemagglutinin: temporal relationships and localization of sulfotransferases.
Glycobiology 10, 1235-42 (2000).
70. Lin, H.C. et al. Prediction of tyrosine sulfation sites in animal viruses. Biochem Biophys Res Commun 312, 1154-8 (2003).
71. Klenk, H.D., Caliguiri, L.A. & Choppin, P.W. The proteins of the parainfluenza virus SV5. II. The carbohydrate content and glycoproteins of the virion. Virology 42, 473-81 (1970).
72. Wei, C.J. et al. Comparative efficacy of neutralizing antibodies elicited by recombinant hemagglutinin proteins from avian H5N1 influenza virus. J Virol 82, 6200-8 (2008).
問答錄
論文口試問答摘要
口試委員︰
陳水田 博士、張世宗 博士、楊健志 博士、王金和 博士、莊榮輝 博士。
陳水田 老師︰
1. 研究 2838N 和 2838V 致命性的差異時，為何將研究重點放在只有兩個胺基酸不同 之 HA1 而非多個胺基酸差異之 HA2 上面？
答︰
因 HA1 擁有辨識宿主的受體結合位置，為病毒感染宿主的第一個步驟。且比 較 HA1 及 HA2 之胺基酸差異可以發現，HA1 僅僅兩個胺基酸的差異會造成三個 charge 的改變，也可以在 2-DE 上看到差異，因此以 HA1 為研究重點。
2. HA1 只有 329 個胺基酸，為何在 2-DE 上分子量高達 50 kD 以上？
答︰
HA1 為一個高度醣基化的蛋白質，多出來的 1 kD 以上的分子量為 HA1 醣質 的分子量。
3. HA1 六個 pI isoforms 在 2-DE 中左邊異構物的分子量較大之原因？
答︰
在實驗最後發現隨著異構物等電點的不同，其醣基化的型態也不同，因此這些 分子量微小的差異可能來自於醣基化的差異。
4. 未知物 X？
答︰
未知物 X 在實驗過程中發現，它可能不是醣類，而是不尋常的 modification。
5. 位於病毒表面的 HA trimer，其什麼位置位於外側？酸性變成鹼性的胺基酸改變是否 也位於外側，因此影響了功能？
答︰
含有宿主結合位置之 HA1 的球狀頭部，位於病毒外側。在三級結構的預測中 也可發現，2838N 及 2838N 之 HA1 的兩個差異的胺基酸亦位於外側。
張世宗 老師︰
1. 在 HA1 的六個等電點異構物中，哪一個是最原始的型態？
答︰
由 EMBOSS IEP 預測可得知，最鹼性也就是等電點最大的異構物為最原始的型 態，而其餘五個等電點較小的異購物則是可能是後修飾酸化所造成。
2. 為何在唾液酸酶酶切後，M1 會向 pI 較低的位置移動？
答︰
在唾液酸酶酶切後，預測蛋白質會回歸到較鹼性的 pI 質點，但在本實驗中卻無 發獲得預期的實驗結果，這有可能是唾液酸酶內含有不純的物質導致此現象，亦或 是 M1 蛋白質點之 pI 值超過 strip 可分析之範圍。
楊健志 老師︰
1. 就胺基酸序列來看，是否就可以預測出 charge 改變之兩個胺基酸位置位於蛋白質之 內測或外側？
答︰
可以。
楊老師補充︰這種 charge 改變通常會位於蛋白質外側，因為電性的不同會嚴重影響 到蛋白質三級結構。
2. 為什麼這六個異構物的點不是連續的，而是點點分明？
答︰
一般來說，若是醣基化造成異構物的產生可能會出現脫尾的現象，但實驗卻發 現 2838 strain 的 HA1 異構物清楚的分開成六點，這也有可能是因為 X 為中性物 質，也就是 2838 strain 的 HA1 醣質並不帶電，也就是這些 HA1 異構物的成因可 能並非醣基化所造成。
3. 六個異構物和致病力之間是否相關？
答︰
研究發現 HA 之等電點會和病毒的毒性及組織趨性相關，另外，HA1 本身的多 型性可能也會有助於 HA1 與宿主的結合以及逃脫免疫系統。
王金和 老師︰
1. 為什麼在唾液酸酶酶切以後，HA1 蛋白質點消失？
答︰
在 SDS-PAGE 實驗中可發現，HA1 在四小時之後就有被降解的現象，這可能 是因為酵素反應液中混有不純物所致。
2. 為什麼在唾液酸酶酶切以後，在 SDS-PAGE 上可看到斷掉的 HA1，在 2-DE 卻無 法看到？
答︰
因為 HA1 在斷裂後，pI 值就會改變，因此不會在全長的 HA1 下再看到一排 相對應之蛋白質點，另外，這些斷裂的 HA1 也有可能是在 2-DE 的前處理中被 lose 掉。
莊榮輝 老師︰
1. 2838 strain 之 HA1 是有 4 個還是 6 個預測之醣基化位置？
答︰
在 2838 strain 之 HA1 胺基酸序列中可發現，符合 Asn-X-S/T 之序列有 6 處，但其中有兩組 Asn 太過相近，因此推測最有可能的醣基化位置是 4 個，另外 兩處符合之序列，醣基化的可能性較低。
2. 這種 HA1 的等電點異構物是否只單獨存在於雞蛋培養的病毒？
答︰
參考相關研究資料可發現，於細胞內培養之病毒，其 HA1 也會出現等電點異 構物，因此認為此現象不單單只存在於雞蛋繁殖之病毒中。
3. HA1 之 106 & 304 兩處不同之胺基酸位置是否遠離三聚體的連接處？
答︰
在 2838N & 2838V 之 HA1 中，兩處胺基酸的改變，造成了三個電性的差異，
而這些胺基酸在形成三聚體之後，位於 HA 之外側，若再三聚體的連接處則可能會 嚴重影響三聚體之構形。