日本腦炎病毒分類於黃質病毒科、黃質病毒屬，其套膜上的兩種醣蛋白prM和E上分別帶有一個N-linked glycosylation的轉譯後修飾。 日本腦炎病毒的prM和E蛋白形成的複合物對於未成熟病毒顆粒及次病毒顆粒 (subviral particles) 的生合成是很重要的步驟，且prM 與E上之N-linked glycan對於病毒顆粒的形成與其表面性質有很大的影響。 根據前人之研究，在昆蟲細胞中，透過重組桿狀病毒 (recombinant baculovirus) 共同表現其他黃質病毒之prM和E，能得到類似於病毒顆粒的重組次病毒顆粒 (recombinant subviral particles, RSPs)。 因此，為了研究N-linked glycan是否影響JEV之RSPs的形成，我們以點突變的方法破壞prM和E上的醣化位置，並藉由重組桿狀病毒在Sf9昆蟲細胞中表現這些蛋白質。 這些在昆蟲細胞中表現之蛋白質，以PNGaseF確認其醣化之狀態後，發現在昆蟲細胞中JEV之prM和E確實經過醣化修飾，但經過突變的prM(dg) 和E(dg) 則無。 在昆蟲細胞中共同表現prM(dg)/E或prM/E(dg) 時， RSPs的分泌量分別只有wild-type之50% 和40%。 然而，當prM(dg)/E(dg) 在昆蟲細胞中共同表現時， RSPs更減到wild-type之30%。 可見prM和E的N-linked glycosylation對於RSPs的形成有相當大的影響。 為了探討以上的現象，我們進一步針對prM和E的醣化狀態對於prM-E之交互作用、以及對於其本身在昆蟲細胞中的穩定性等與RSPs形成有關之因素進行研究，發現以上兩者與N-linked glycosylation的存在與否無明顯的關係。 從上述的結果看來，N-linked glycosylation確實會影響RSPs的形成，其影響機制仍尚未確定，但已知並非影響prM與E在細胞中之穩定性、以及prM-E之間的交互作用。

Japanese encephalitis is the most important cause of epidemic encephalitis worldwide, especially in eastern and southern Asia. In recent years, JE is spreading geographically and become a more concerned issue. The pathogen of JE, Japanese encephalitis virus (JEV) has two membrane glycoprotein prM and E, which each has one N-linked glycan. The formation of JEV prM-E complex is an important step for the biogenesis of immature virions and subviral particles. The N-linked glycans of JEV prM and E are crucial to viral replication and subviral particles formation. Co-expressing JEV prM and E proteins in insect cells infected by recombinant baculoviruses have been shown to produce recombinant subviral particles (RSPs) similar to virions. In order to analyze how the N-linked glycans affect the process of RSPs biosynthesis, wild-type and glycosylation-mutated prM and E proteins was expressed in Sf9 cells. By treating with the glycosidase PNGase F, it was confirmed that the wild-type JEV prM and E were N-linked glycosylated, while the glycosylation-mutants, prM(dg) and E(dg), were not. In the co-infection of prM(dg)/E and prM/E(dg), the secretion of RSPs was respectively reduced to 50% and 40% of the wild-type level. In the absence of N-linked glycans on both prM and E, the secretion of RSPs was more seriously decreased to 30% of the wild-type level. Furthermore, the N-linked glycans showed no significant relation to the stability, interaction of prM and E proteins, and helped the formation and the secretion of RSPs in some other uncertain mechanisms.

Ali, A., Avalos, R. T., Ponimaskin, E., and Nayak, D. P. (2000). Influenza virus assembly: effect of influenza virus glycoproteins on the membrane association of M1 protein. J Virol 74(18), 8709-19.
Allison, S. L., Stadler, K., Mandl, C. W., Kunz, C., and Heinz, F. X. (1995). Synthesis and secretion of recombinant tick-borne encephalitis virus protein E in soluble and particulate form. J Virol 69(9), 5816-20.
Arar, C., Carpentier, V., Le Caer, J. P., Monsigny, M., Legrand, A., and Roche, A. C. (1995). ERGIC-53, a membrane protein of the endoplasmic reticulum-Golgi intermediate compartment, is identical to MR60, an intracellular mannose-specific lectin of myelomonocytic cells. J Biol Chem 270(8), 3551-3.
Back, N. K., Smit, L., De Jong, J. J., Keulen, W., Schutten, M., Goudsmit, J., and Tersmette, M. (1994). An N-glycan within the human immunodeficiency virus type 1 gp120 V3 loop affects virus neutralization. Virology 199(2), 431-8.
Banerjee, K. (1996). Emerging viral infections with special references to India. Indian J. Med. Res. 103, 177-200.
Baumert, T. F., Ito, S., Wong, D. T., and Liang, T. J. (1998). Hepatitis C virus structural proteins assemble into viruslike particles in insect cells. J Virol 72(5), 3827-36.
Beasley, D. W. (2005). Recent advances in the molecular biology of west nile virus. Curr Mol Med 5(8), 835-50.
Beasley, D. W., Whiteman, M. C., Zhang, S., Huang, C. Y., Schneider, B. S., Smith, D. R., Gromowski, G. D., Higgs, S., Kinney, R. M., and Barrett, A. D. (2005). Envelope protein glycosylation status influences mouse neuroinvasion phenotype of genetic lineage 1 West Nile virus strains. J Virol 79(13), 8339-47.
Benjouad, A., Gluckman, J. C., Montagnier, L., and Bahraoui, E. (1993). Specificity of antibodies produced against native or desialylated human immunodeficiency virus type 1 recombinant gp160. J Virol 67(3), 1693-7.
Berger, I., Fitzgerald, D. J., and Richmond, T. J. (2004). Baculovirus expression system for heterologous multiprotein complexes. Nat Biotechnol 22(12), 1583-7.
Bolmstedt, A., Hemming, A., Flodby, P., Berntsson, P., Travis, B., Lin, J. P., Ledbetter, J., Tsu, T., Wigzell, H., Hu, S. L., and et al. (1991). Effects of mutations in glycosylation sites and disulphide bonds on processing, CD4-binding and fusion activity of human immunodeficiency virus envelope glycoproteins. J Gen Virol 72 ( Pt 6), 1269-77.
Botarelli, P., Houlden, B. A., Haigwood, N. L., Servis, C., Montagna, D., and Abrignani, S. (1991). N-glycosylation of HIV-gp120 may constrain recognition by T lymphocytes. J Immunol 147(9), 3128-32.
Choi, S. H., Kim, S. Y., Park, K. J., Kim, Y. J., and Hwang, S. B. (2004). Hepatitis C virus core protein is efficiently released into the culture medium in insect cells. J Biochem Mol Biol 37(6), 735-40.
Corver, J., Ortiz, A., Allison, S. L., Schalich, J., Heinz, F. X., and Wilschut, J. (2000). Membrane fusion activity of tick-borne encephalitis virus and recombinant subviral particles in a liposomal model system. Virology 269(1), 37-46.
Courageot, M. P., Frenkiel, M. P., Dos Santos, C. D., Deubel, V., and Despres, P. (2000). Alpha-glucosidase inhibitors reduce dengue virus production by affecting the initial steps of virion morphogenesis in the endoplasmic reticulum. J Virol 74(1), 564-72.
Elshuber, S., Allison, S. L., Heinz, F. X., and Mandl, C. W. (2003). Cleavage of protein prM is necessary for infection of BHK-21 cells by tick-borne encephalitis virus. J Gen Virol 84(Pt 1), 183-91.
Fausch, S. C., Da Silva, D. M., Eiben, G. L., Le Poole, I. C., and Kast, W. M. (2003). HPV protein/peptide vaccines: from animal models to clinical trials. Front Biosci 8, s81-91.
Ferlenghi, I., Clarke, M., Ruttan, T., Allison, S. L., Schalich, J., Heinz, F. X., Harrison, S. C., Rey, F. A., and Fuller, S. D. (2001). Molecular organization of a recombinant subviral particle from tick-borne encephalitis virus. Mol Cell 7(3), 593-602.
Fiedler, K., Parton, R. G., Kellner, R., Etzold, T., and Simons, K. (1994). VIP36, a novel component of glycolipid rafts and exocytic carrier vesicles in epithelial cells. Embo J 13(7), 1729-40.
Fonseca, B. A., Pincus, S., Shope, R. E., Paoletti, E., and Mason, P. W. (1994). Recombinant vaccinia viruses co-expressing dengue-1 glycoproteins prM and E induce neutralizing antibodies in mice. Vaccine 12(3), 279-85.
Fournillier, A., Wychowski, C., Boucreux, D., Baumert, T. F., Meunier, J. C., Jacobs, D., Muguet, S., Depla, E., and Inchauspe, G. (2001). Induction of hepatitis C virus E1 envelope protein-specific immune response can be enhanced by mutation of N-glycosylation sites. J Virol 75(24), 12088-97.
Fullekrug, J., Scheiffele, P., and Simons, K. (1999). VIP36 localisation to the early secretory pathway. J Cell Sci 112 ( Pt 17), 2813-21.
Garcea, R. L., and Gissmann, L. (2004). Virus-like particles as vaccines and vessels for the delivery of small molecules. Curr Opin Biotechnol 15(6), 513-7.
Girard, C., Ravallec, M., Mariller, M., Bossy, J. P., Cahour, A., Lopez-Ferber, M., Devauchelle, G., Inchauspe, G., and Duonor-Cerutti, M. (2004). Effect of the 5' non-translated region on self-assembly of hepatitis C virus genotype 1a structural proteins produced in insect cells. J Gen Virol 85(Pt 12), 3659-70.
Gomez-Puertas, P., Albo, C., Perez-Pastrana, E., Vivo, A., and Portela, A. (2000). Influenza virus matrix protein is the major driving force in virus budding. J Virol 74(24), 11538-47.
Goto, A., Yoshii, K., Obara, M., Ueki, T., Mizutani, T., Kariwa, H., and Takashima, I. (2005). Role of the N-linked glycans of the prM and E envelope proteins in tick-borne encephalitis virus particle secretion. Vaccine 23(23), 3043-52.
Guirakhoo, F., Heinz, F. X., and Kunz, C. (1989). Epitope model of tick-borne encephalitis virus envelope glycoprotein E: analysis of structural properties, role of carbohydrate side chain, and conformational changes occurring at acidic pH. Virology 169(1), 90-9.
Hanna, S. L., Pierson, T. C., Sanchez, M. D., Ahmed, A. A., Murtadha, M. M., and Doms, R. W. (2005). N-linked glycosylation of west nile virus envelope proteins influences particle assembly and infectivity. J Virol 79(21), 13262-74.
Hara-Kuge, S., Ohkura, T., Seko, A., and Yamashita, K. (1999). Vesicular-integral membrane protein, VIP36, recognizes high-mannose type glycans containing alpha1-->2 mannosyl residues in MDCK cells. Glycobiology 9(8), 833-9.
Harper, D. M., Franco, E. L., Wheeler, C., Ferris, D. G., Jenkins, D., Schuind, A., Zahaf, T., Innis, B., Naud, P., De Carvalho, N. S., Roteli-Martins, C. M., Teixeira, J., Blatter, M. M., Korn, A. P., Quint, W., and Dubin, G. (2004). Efficacy of a bivalent L1 virus-like particle vaccine in prevention of infection with human papillomavirus types 16 and 18 in young women: a randomised controlled trial. Lancet 364(9447), 1757-65.
Hauri, H., Appenzeller, C., Kuhn, F., and Nufer, O. (2000). Lectins and traffic in the secretory pathway. FEBS Lett 476(1-2), 32-7.
Helenius, A., and Aebi, M. (2001). Intracellular functions of N-linked glycans. Science 291(5512), 2364-9.
Huppa, J. B., and Ploegh, H. L. (1998). The eS-Sence of -SH in the ER. Cell 92(2), 145-8.
Imperiali, B., and O'Connor, S. E. (1999). Effect of N-linked glycosylation on glycopeptide and glycoprotein structure. Curr Opin Chem Biol 3(6), 643-9.
Jeong, S. H., Qiao, M., Nascimbeni, M., Hu, Z., Rehermann, B., Murthy, K., and Liang, T. J. (2004). Immunization with hepatitis C virus-like particles induces humoral and cellular immune responses in nonhuman primates. J Virol 78(13), 6995-7003.
Kojima, A., Yasuda, A., Asanuma, H., Ishikawa, T., Takamizawa, A., Yasui, K., and Kurata, T. (2003). Stable high-producer cell clone expressing virus-like particles of the Japanese encephalitis virus e protein for a second-generation subunit vaccine. J Virol 77(16), 8745-55.
Konishi, E., Fujii, A., and Mason, P. W. (2001). Generation and characterization of a mammalian cell line continuously expressing Japanese encephalitis virus subviral particles. J Virol 75(5), 2204-12.
Konishi, E., and Mason, P. W. (1993). Proper maturation of the Japanese encephalitis virus envelope glycoprotein requires cosynthesis with the premembrane protein. J Virol 67(3), 1672-5.
Konishi, E., Pincus, S., Paoletti, E., Shope, R. E., Burrage, T., and Mason, P. W. (1992). Mice immunized with a subviral particle containing the Japanese encephalitis virus prM/M and E proteins are protected from lethal JEV infection. Virology 188(2), 714-20.
Koutsky, L. A., Ault, K. A., Wheeler, C. M., Brown, D. R., Barr, E., Alvarez, F. B., Chiacchierini, L. M., and Jansen, K. U. (2002). A controlled trial of a human papillomavirus type 16 vaccine. N Engl J Med 347(21), 1645-51.
Kwong, P. D., Wyatt, R., Robinson, J., Sweet, R. W., Sodroski, J., and Hendrickson, W. A. (1998). Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature 393(6686), 648-59.
Latham, T., and Galarza, J. M. (2001). Formation of wild-type and chimeric influenza virus-like particles following simultaneous expression of only four structural proteins. J Virol 75(13), 6154-65.
Lee, J., Park, J. S., Moon, J. Y., Kim, K. Y., and Moon, H. M. (2003). The influence of glycosylation on secretion, stability, and immunogenicity of recombinant HBV pre-S antigen synthesized in Saccharomyces cerevisiae. Biochem Biophys Res Commun 303(2), 427-32.
Lin, C. W., and Wu, S. C. (2003). A functional epitope determinant on domain III of the Japanese encephalitis virus envelope protein interacted with neutralizing-antibody combining sites. J Virol 77(4), 2600-6.
Lin, Y. J., and Wu, S. C. (2005). Histidine at residue 99 and the transmembrane region of the precursor membrane prM protein are important for the prM-E heterodimeric complex formation of Japanese encephalitis virus. J Virol 79(13), 8535-44.
Maranga, L., Cruz, P. E., Aunins, J. G., and Carrondo, M. J. (2002). Production of core and virus-like particles with baculovirus infected insect cells. Adv Biochem Eng Biotechnol 74, 183-206.
Modis, Y., Ogata, S., Clements, D., and Harrison, S. C. (2003). A ligand-binding pocket in the dengue virus envelope glycoprotein. Proc Natl Acad Sci U S A 100(12), 6986-91.
Mortola, E., and Roy, P. (2004). Efficient assembly and release of SARS coronavirus-like particles by a heterologous expression system. FEBS Lett 576(1-2), 174-8.
Mukhopadhyay, S., Kuhn, R. J., and Rossmann, M. G. (2005). A structural perspective of the flavivirus life cycle. Nat Rev Microbiol 3(1), 13-22.
Nichols, W. C., Seligsohn, U., Zivelin, A., Terry, V. H., Hertel, C. E., Wheatley, M. A., Moussalli, M. J., Hauri, H. P., Ciavarella, N., Kaufman, R. J., and Ginsburg, D. (1998). Mutations in the ER-Golgi intermediate compartment protein ERGIC-53 cause combined deficiency of coagulation factors V and VIII. Cell 93(1), 61-70.
Noad, R., and Roy, P. (2003). Virus-like particles as immunogens. Trends Microbiol 11(9), 438-44.
Op De Beeck, A., Molenkamp, R., Caron, M., Ben Younes, A., Bredenbeek, P., and Dubuisson, J. (2003). Role of the transmembrane domains of prM and E proteins in the formation of yellow fever virus envelope. J Virol 77(2), 813-20.
Petry, H., Goldmann, C., Ast, O., and Luke, W. (2003). The use of virus-like particles for gene transfer. Curr Opin Mol Ther 5(5), 524-8.
Pincus, S., Mason, P. W., Konishi, E., Fonseca, B. A., Shope, R. E., Rice, C. M., and Paoletti, E. (1992). Recombinant vaccinia virus producing the prM and E proteins of yellow fever virus protects mice from lethal yellow fever encephalitis. Virology 187(1), 290-7.
Popova, R., and Zhang, X. (2002). The spike but not the hemagglutinin/esterase protein of bovine coronavirus is necessary and sufficient for viral infection. Virology 294(1), 222-36.
Pratap, J., Rajamohan, G., and Dikshit, K. L. (2000). Characteristics of glycosylated streptokinase secreted from Pichia pastoris: enhanced resistance of SK to proteolysis by glycosylation. Appl Microbiol Biotechnol 53(4), 469-75.
Pushko, P., Tumpey, T. M., Bu, F., Knell, J., Robinson, R., and Smith, G. (2005). Influenza virus-like particles comprised of the HA, NA, and M1 proteins of H9N2 influenza virus induce protective immune responses in BALB/c mice. Vaccine 23(50), 5751-9.
Rey, F. A., Heinz, F. X., Mandl, C., Kunz, C., and Harrison, S. C. (1995). The envelope glycoprotein from tick-borne encephalitis virus at 2 A resolution. Nature 375(6529), 291-8.
Schalich, J., Allison, S. L., Stiasny, K., Mandl, C. W., Kunz, C., and Heinz, F. X. (1996). Recombinant subviral particles from tick-borne encephalitis virus are fusogenic and provide a model system for studying flavivirus envelope glycoprotein functions. J Virol 70(7), 4549-57.
Sodora, D. L., Cohen, G. H., Muggeridge, M. I., and Eisenberg, R. J. (1991). Absence of asparagine-linked oligosaccharides from glycoprotein D of herpes simplex virus type 1 results in a structurally altered but biologically active protein. J Virol 65(8), 4424-31.
Spiro, R. G., Zhu, Q., Bhoyroo, V., and Soling, H. D. (1996). Definition of the lectin-like properties of the molecular chaperone, calreticulin, and demonstration of its copurification with endomannosidase from rat liver Golgi. J Biol Chem 271(19), 11588-94.
Stadler, K., Allison, S. L., Schalich, J., and Heinz, F. X. (1997). Proteolytic activation of tick-borne encephalitis virus by furin. J Virol 71(11), 8475-81.
Stanley, M. A. (2003). Progress in prophylactic and therapeutic vaccines for human papillomavirus infection. Expert Rev Vaccines 2(3), 381-9.
Vorndam, V., Mathews, J. H., Barrett, A. D., Roehrig, J. T., and Trent, D. W. (1993). Molecular and biological characterization of a non-glycosylated isolate of St Louis encephalitis virus. J Gen Virol 74 ( Pt 12), 2653-60.
Ware, F. E., Vassilakos, A., Peterson, P. A., Jackson, M. R., Lehrman, M. A., and Williams, D. B. (1995). The molecular chaperone calnexin binds Glc1Man9GlcNAc2 oligosaccharide as an initial step in recognizing unfolded glycoproteins. J Biol Chem 270(9), 4697-704.
Winkler, G., Heinz, F. X., and Kunz, C. (1987). Studies on the glycosylation of flavivirus E proteins and the role of carbohydrate in antigenic structure. Virology 159(2), 237-43.
Wormald, M. R., and Dwek, R. A. (1999). Glycoproteins: glycan presentation and protein-fold stability. Structure 7(7), R155-60.
Wu, S. C., and Lee, S. C. (2001). Complete nucleotide sequence and cell-line multiplication pattern of the attenuated variant CH2195LA of Japanese encephalitis virus. Virus Res 73(1), 91-102.
Wu, S. C., Lian, W. C., Hsu, L. C., and Liau, M. Y. (1997). Japanese encephalitis virus antigenic variants with characteristic differences in neutralization resistance and mouse virulence. Virus Res 51(2), 173-81.
Wu, S. F., Lee, C. J., Liao, C. L., Dwek, R. A., Zitzmann, N., and Lin, Y. L. (2002). Antiviral effects of an iminosugar derivative on flavivirus infections. J Virol 76(8), 3596-604.
Xiang, J., Wunschmann, S., George, S. L., Klinzman, D., Schmidt, W. N., LaBrecque, D. R., and Stapleton, J. T. (2002). Recombinant hepatitis C virus-like particles expressed by baculovirus: utility in cell-binding and antibody detection assays. J Med Virol 68(4), 537-43.
Yamshchikov, V. F., and Compans, R. W. (1993). Regulation of the late events in flavivirus protein processing and maturation. Virology 192(1), 38-51.