A型流感病毒可以感染人類，鳥類及多種哺乳類動物。它在20世紀有四次突發變種成多型人體互相感染的病原。最近更在亞洲及歐洲持續發生H5N1禽流感，其可能致死的潛在危險，已引起重度的關切，擔心它再度造成全球性流感的大流行。A型流感病毒的基因組可分為八段，每段均被核蓋蛋白(nucleoprotein)包裹著，擁有正黏液病毒科 (Orthomyxoviridae family) 的遺傳性質。藉由核蓋蛋白的寡聚合作用，其與核糖核酸 (RNA) 的鍵結作用，再加上與病毒的核糖核酸聚合酶複合體 (vRNA-dependant RNA polymerase complex : PA, PB1, and PB2) 的鍵結，最後形成病毒的核糖核蛋白複合體 (ribonucleoprotein complex, RNP) 。在核蓋蛋白的氨基酸序列上，做點突變 : E339A或R416A，可使其蛋白質之間 (NP-NP contacts) 的寡聚合作用所需的鹽橋不再作用，因而破壞核蓋蛋白的寡聚合反應，使得原本會呈現大分子聚合物 (Oligomer) 的核蓋蛋白變成單體分子 (Monomer) ! 因此，吾人先選殖出所需的野生型核蓋蛋白，再利用大腸桿菌和pET15b質體的表現系統，成功的取得水溶性且原態 (native) 的核蓋蛋白，並將核蓋蛋白經過單點突變 (E339A或R416A) 後，根據快速蛋白質液相層析的結果發現，此兩個點突變確實都能在一定程度上破壞聚合作用所需的鹽橋，使核蓋蛋白呈現單體分子!此外，吾人也藉由OD260/OD280的比值，得知這些核蓋蛋白能與去氧核糖核酸 (DNA) 有鍵結作用且無法利用高鹽溶液和去氧核糖核酸水解酶 (DNase I) 去除之。另外，結果也發現，野生型的核蓋蛋白能保有且維持多分子聚合態，然而，兩個突變型核蓋蛋白之多分子聚合態並非動力平衡，而是隨著時間趨向形成單體分子! 藉此，吾人得以計算出一定條件下，突變型核蓋蛋白之單體分子形成的平均速率以及其多分子聚合態可能的變換模式。

Influenza A virus can infect humans, birds, and several kinds of mammals. In the 20th century, 4 variants of the virus have arised as human transmissible pathogens. Moreover, the outbreak of H5N1 avian influenza, was recently reported in Europe and Asia. The virus has generated serious concern for its high death rates and the epidemics all over the world. The genome of influenza A virus is separated into 8 RNP segements which are coated with nucleoproteins, a common genetic property of Orthomyxoviridae family. The oligomerization and RNA-binding characteristics of nucleoproteins indicate that they can interact with vRNA-dependant RNA polymerase complex (PA, PB1, and PB2) and vRNA to form ribonucleoprotein complex, RNP. According to previous literatures, the leading factor of oligomerization in NP-NP contacts, salt-bridge between one nucleoprotein E339 and another one R416, can be broken by single point mutation (E339A or R416A), leading to the nucleoprotein oligomers switch to the monomers. Thus, we have created the same mutations on nucleocapsid protein (NP of H1N1 and H3N2), and then obtained soluble and native nucleoproteins to perform FPLC. The FPLC patterns show that the two mutations can indeed break the salt bridge of NP-NP contacts and disrupt the original oligomerization behavior of wild-type NP to form monomeric form. In addition, the nucleoprotein will bind to the DNA during purification process, which is monitored from the OD260/OD280 ratio. However, the DNA can not be dissociated or hydrolyzed completely by using high-salt buffer and DNase I. Additionally, the FPLC patterns show that the oligomerization state of the wild-type NP is retained and maintained. However, the two mutant NPs (NP-E339A and NP-R416A) are not, indicating that the mutant NP polymers will turn into NP monomers over time. Finally, we calculated the average rate of the monomer formation and characterized their possible conversion mode.

References

Akarsu, H., Burmeister, W. P., Petosa, C., Petit, I., Muller, C. W., Ruigrok, R. W., and Baudin, F. (2003). Crystal structure of the M1 protein-binding domain of the influenza A virus nuclear export protein (NEP/NS2). Embo J 22, 4646-4655.
Baudin, F., Bach, C., Cusack, S., and Ruigrok, R. W. (1994). Structure of influenza virus RNP. I. Influenza virus nucleoprotein melts secondary structure in panhandle RNA and exposes the bases to the solvent. Embo J 13, 3158-3165.
Biswas, S. K., Boutz, P. L., and Nayak, D. P. (1998). Influenza virus nucleoprotein interacts with influenza virus polymerase proteins. J Virol 72, 5493-5501.
Blumberg, B. M., Giorgi, C., and Kolakofsky, D. (1983). N protein of vesicular stomatitis virus selectively encapsidates leader RNA in vitro. Cell 32, 559-567.
Boulo, S., Akarsu, H., Ruigrok, R. W., and Baudin, F. (2007). Nuclear traffic of influenza virus proteins and ribonucleoprotein complexes. Virus Res 124, 12-21.
Calza, L., Manfredi, R., and Chiodo, F. (2005). [H5N1 avian influenza]. Recenti Prog Med 96, 523-534.
Chang, S. C., Cheng, Y. Y., and Shih, S. R. (2006). Avian influenza virus: the threat of a pandemic. Chang Gung Med J 29, 130-134.
Chen, W., Calvo, P. A., Malide, D., Gibbs, J., Schubert, U., Bacik, I., Basta, S., O'Neill, R., Schickli, J., Palese, P., et al. (2001). A novel influenza A virus mitochondrial protein that induces cell death. Nat Med 7, 1306-1312.
Colman, P. M., Varghese, J. N., and Laver, W. G. (1983). Structure of the catalytic and antigenic sites in influenza virus neuraminidase. Nature 303, 41-44.
Compans, R. W., Content, J., and Duesberg, P. H. (1972). Structure of the ribonucleoprotein of influenza virus. J Virol 10, 795-800.
Crescenzo-Chaigne, B., Naffakh, N., and van der Werf, S. (1999). Comparative analysis of the ability of the polymerase complexes of influenza viruses type A, B and C to assemble into functional RNPs that allow expression and replication of heterotypic model RNA templates in vivo. Virology 265, 342-353.
Cros, J. F., and Palese, P. (2003). Trafficking of viral genomic RNA into and out of the nucleus: influenza, Thogoto and Borna disease viruses. Virus Res 95, 3-12.
Davey, J., Dimmock, N. J., and Colman, A. (1985). Identification of the sequence responsible for the nuclear accumulation of the influenza virus nucleoprotein in Xenopus oocytes. Cell 40, 667-675.
Duesberg, P. H. (1969). Distinct subunits of the ribonucleoprotein of influenza virus. J Mol Biol 42, 485-499.
Elton, D., Medcalf, E., Bishop, K., and Digard, P. (1999a). Oligomerization of the influenza virus nucleoprotein: identification of positive and negative sequence elements. Virology 260, 190-200.
Elton, D., Medcalf, L., Bishop, K., Harrison, D., and Digard, P. (1999b). Identification of amino acid residues of influenza virus nucleoprotein essential for RNA binding. J Virol 73, 7357-7367.
Enserink, M. (2005). Avian influenza. Pandemic influenza: global update. Science 309, 370-371.
Epstein, S. L., Kong, W. P., Misplon, J. A., Lo, C. Y., Tumpey, T. M., Xu, L., and Nabel, G. J. (2005). Protection against multiple influenza A subtypes by vaccination with highly conserved nucleoprotein. Vaccine 23, 5404-5410.
Gamblin, S. J., Haire, L. F., Russell, R. J., Stevens, D. J., Xiao, B., Ha, Y., Vasisht, N., Steinhauer, D. A., Daniels, R. S., Elliot, A., et al. (2004). The structure and receptor binding properties of the 1918 influenza hemagglutinin. Science 303, 1838-1842.
Honda, A., Ueda, K., Nagata, K., and Ishihama, A. (1988). RNA polymerase of influenza virus: role of NP in RNA chain elongation. J Biochem (Tokyo) 104, 1021-1026.
Horimoto, T., and Kawaoka, Y. (2001). Pandemic threat posed by avian influenza A viruses. Clin Microbiol Rev 14, 129-149.
Iseni, F., Barge, A., Baudin, F., Blondel, D., and Ruigrok, R. W. (1998). Characterization of rabies virus nucleocapsids and recombinant nucleocapsid-like structures. J Gen Virol 79 ( Pt 12), 2909-2919.
Kaverin, N. V. (1980). [Genome of influenza virus: organization, function, evolution]. Mol Biol (Mosk) 14, 245-260.
Kawaguchi, A., and Nagata, K. (2006). [Molecular mechanism of replication and transcription of the influenza virus genome and host factors]. Uirusu 56, 99-108.
Kingsbury, D. W., Jones, I. M., and Murti, K. G. (1987). Assembly of influenza ribonucleoprotein in vitro using recombinant nucleoprotein. Virology 156, 396-403.
Klumpp, K., Ruigrok, R. W., and Baudin, F. (1997). Roles of the influenza virus polymerase and nucleoprotein in forming a functional RNP structure. Embo J 16, 1248-1257.
Kobayashi, M., Toyoda, T., Adyshev, D. M., Azuma, Y., and Ishihama, A. (1994). Molecular dissection of influenza virus nucleoprotein: deletion mapping of the RNA binding domain. J Virol 68, 8433-8436.
Kochergin-Nikitskii, K. S., Rudneva, I. A., Timofeeva, T. A., Il'iushina, N. A., Varich, N. L., Vlasova, A. N., Kaverin, N. V., and L'Vov D, K. (2007). [Reassortment and gene interactions in the crossing of low-pathogenic avian influenza H5 virus with human influenza virus]. Vopr Virusol 52, 23-28.
Lamb, R. A., and Choppin, P. W. (1983). The gene structure and replication of influenza virus. Annu Rev Biochem 52, 467-506.
Landman, W. J., and Schrier, C. C. (2004). [Avian influenza: eradication from commercial poultry is still not in sight]. Tijdschr Diergeneeskd 129, 782-796.
Li, R. A., Palese, P., and Krystal, M. (1989). Complementation and analysis of an NP mutant of influenza virus. Virus Res 12, 97-111.
Liu, J., Lynch, P. A., Chien, C. Y., Montelione, G. T., Krug, R. M., and Berman, H. M. (1997). Crystal structure of the unique RNA-binding domain of the influenza virus NS1 protein. Nat Struct Biol 4, 896-899.
Maeda, Y., Horimoto, T., and Kawaoka, Y. (2003). [Classification and genome structure of influenza virus]. Nippon Rinsho 61, 1886-1891.
Mayer, D., Molawi, K., Martinez-Sobrido, L., Ghanem, A., Thomas, S., Baginsky, S., Grossmann, J., Garcia-Sastre, A., and Schwemmle, M. (2007). Identification of cellular interaction partners of the influenza virus ribonucleoprotein complex and polymerase complex using proteomic-based approaches. J Proteome Res 6, 672-682.
Medcalf, L., Poole, E., Elton, D., and Digard, P. (1999). Temperature-sensitive lesions in two influenza A viruses defective for replicative transcription disrupt RNA binding by the nucleoprotein. J Virol 73, 7349-7356.
Mena, I., Jambrina, E., Albo, C., Perales, B., Ortin, J., Arrese, M., Vallejo, D., and Portela, A. (1999). Mutational analysis of influenza A virus nucleoprotein: identification of mutations that affect RNA replication. J Virol 73, 1186-1194.
Neumann, G., Castrucci, M. R., and Kawaoka, Y. (1997). Nuclear import and export of influenza virus nucleoprotein. J Virol 71, 9690-9700.
Noda, T., Sagara, H., Yen, A., Takada, A., Kida, H., Cheng, R. H., and Kawaoka, Y. (2006). Architecture of ribonucleoprotein complexes in influenza A virus particles. Nature 439, 490-492.
Ohba, K., Yoshida, S., Zahidunnabi Dewan, M., Shimura, H., Sakamaki, N., Takeshita, F., Yamamoto, N., and Okuda, K. (2007). Mutant influenza A virus nucleoprotein is preferentially localized in the cytoplasm and its immunization in mice shows higher immunogenicity and cross-reactivity. Vaccine 25, 4291-4300.
Ozawa, M., Fujii, K., Muramoto, Y., Yamada, S., Yamayoshi, S., Takada, A., Goto, H., Horimoto, T., and Kawaoka, Y. (2007). Contributions of two nuclear localization signals of influenza A virus nucleoprotein to viral replication. J Virol 81, 30-41.
Phipps, K. R., and Li, H. (2007). Protein-RNA contacts at crystal packing surfaces. Proteins 67, 121-127.
Pleschka, S., Jaskunas, R., Engelhardt, O. G., Zurcher, T., Palese, P., and Garcia-Sastre, A. (1996). A plasmid-based reverse genetics system for influenza A virus. J Virol 70, 4188-4192.
Pons, M. W., Schulze, I. T., Hirst, G. K., and Hauser, R. (1969). Isolation and characterization of the ribonucleoprotein of influenza virus. Virology 39, 250-259.
Portela, A., and Digard, P. (2002). The influenza virus nucleoprotein: a multifunctional RNA-binding protein pivotal to virus replication. J Gen Virol 83, 723-734.
Prokudina-Kantorovich, E. N., and Semenova, N. P. (1996). Intracellular oligomerization of influenza virus nucleoprotein. Virology 223, 51-56.
Reid, A. H., Taubenberger, J. K., and Fanning, T. G. (2004). Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol 2, 909-914.
Ruigrok, R. W., and Baudin, F. (1995). Structure of influenza virus ribonucleoprotein particles. II. Purified RNA-free influenza virus ribonucleoprotein forms structures that are indistinguishable from the intact influenza virus ribonucleoprotein particles. J Gen Virol 76 ( Pt 4), 1009-1014.
Semenova, N. P., Chumakov, V. M., Rudneva, I. A., and Prokudina, E. N. (1998). [Role of certain factors in intracellular oligomerization of influenza virus nucleoproteins]. Vopr Virusol 43, 24-29.
Sha, B., and Luo, M. (1997). Structure of a bifunctional membrane-RNA binding protein, influenza virus matrix protein M1. Nat Struct Biol 4, 239-244.
Spicuzza, L., Spicuzza, A., La Rosa, M., Polosa, R., and Di Maria, G. (2007). New and emerging infectious diseases. Allergy Asthma Proc 28, 28-34.
Stevens, J., Corper, A. L., Basler, C. F., Taubenberger, J. K., Palese, P., and Wilson, I. A. (2004). Structure of the uncleaved human H1 hemagglutinin from the extinct 1918 influenza virus. Science 303, 1866-1870.
Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dubendorff, J. W. (1990). Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185, 60-89.
Vreede, F. T., and Brownlee, G. G. (2007). Influenza virion-derived viral ribonucleoproteins synthesize both mRNA and cRNA in vitro. J Virol 81, 2196-2204.
Vreede, F. T., Jung, T. E., and Brownlee, G. G. (2004). Model suggesting that replication of influenza virus is regulated by stabilization of replicative intermediates. J Virol 78, 9568-9572.
Winter, G., and Fields, S. (1981). The structure of the gene encoding the nucleoprotein of human influenza virus A/PR/8/34. Virology 114, 423-428.
Wong, S. S., and Yuen, K. Y. (2006). Avian influenza virus infections in humans. Chest 129, 156-168.
Wu, R., Hu, S., Xiao, Y., Li, Z., Shi, D., and Bi, D. (2007). Development of Indirect Enzyme-linked Immunosorbent Assay with Nucleoprotein as Antigen for Detection and Quantification of Antibodies against Avian Influenza Virus. Vet Res Commun 31, 631-641.
Yamanaka, K., Ishihama, A., and Nagata, K. (1990). Reconstitution of influenza virus RNA-nucleoprotein complexes structurally resembling native viral ribonucleoprotein cores. J Biol Chem 265, 11151-11155.
Ye, Q., Krug, R. M., and Tao, Y. J. (2006). The mechanism by which influenza A virus nucleoprotein forms oligomers and binds RNA. Nature 444, 1078-1082.
Yu, H., Zhang, G. H., Hua, R. H., Zhang, Q., Liu, T. Q., Liao, M., and Tong, G. Z. (2007). Isolation and genetic analysis of human origin H1N1 and H3N2 influenza viruses from pigs in China. Biochem Biophys Res Commun 356, 91-96.
Zebedee, S. L., and Lamb, R. A. (1989). Nucleotide sequences of influenza A virus RNA segment 7: a comparison of five isolates. Nucleic Acids Res 17, 2870.
Zhao, H., Ekstrom, M., and Garoff, H. (1998). The M1 and NP proteins of influenza A virus form homo- but not heterooligomeric complexes when coexpressed in BHK-21 cells. J Gen Virol 79 ( Pt 10), 2435-2446.