利用感染性cDNA clones及中和性抗體探討日本腦炎病毒感染之過程

Using infectious cDNA clones and neutralizing antibodies to study the process of Japanese encephalitis virus infection

林信廷(Hsin-Ting Lin)

指導教授 ： 陳世順 ； 李珮瑜

中臺科技大學/健康科學院/醫學生物科技研究所/碩士(2010年)

摘要

先前研究推測日本腦炎病毒感染細胞，是藉由套膜蛋白（envelope protein；E protein）上帶正電荷之胺基酸與細胞膜上帶負電荷之長鏈不分支的葡萄糖胺多醣（glycosaminoglycans；GAGs）作用，而誘導配體（ligand）與接受器（receptor）結合進入感染細胞。然而，套膜蛋白E上與葡萄糖胺多醣結合之分子決定基（molecular determinant）及病毒感染細胞之過程仍未被釐清。因此本實驗室以反轉遺傳學技術（reverse genetic technique）產生一組有關套膜蛋白E基因特定序列突變的病毒株，來進一步探討前述之問題。初步研究結果顯示，以不同種類葡萄糖胺多醣與病毒預先作用而後感染細胞，發現高度硫酸化之heparin不會抑制Arg-Gly-Asp motif（RGD motif）突變株E-D389G與E-D389H病毒對BHK-21及N18細胞之吸附及感染；另將病毒先與heparin或PL1-365作用，在病毒吸附過程中再加入中和性抗體或heparin，能影響野生型病毒株（wild-type）、E-D389G與E-D389H感染細胞。我們也證明PL1-365能抑制野生型及大部分E基因突變病毒株對細胞之附著以及附著後的進入細胞之階段。除此，溶斑減少中和試驗（plaque reduction neutralization test）試驗結果發現，中和性抗體PL1-365無法完全抑制（~56% 抑制）E-S227P突變株感染細胞。根據我們的以上實驗結果推測，日本腦炎病毒感染細胞早期先與高度硫酸化之葡萄糖胺多醣結合，而後再辨識細胞的接受器進而進入細胞。然而，套膜蛋白上RGD motif是否為與葡萄糖胺多醣結合的重要分子決定基，仍需進一步藉由反轉遺傳學技術產生一系列在套膜蛋白E的GAG binding motif基因序列突變的病毒株，才能進一步驗證上述之觀察。

關鍵字

分子決定基 ； RGD motif ； 套膜蛋白 ； 中和性抗體 ； 葡萄糖胺多醣

並列摘要

Previous studies suggest that cell surface glycosaminoglycans (GAGs) may play an important role in Japanese encephalitis virus (JEV) infection by interaction between viral envelope (E) protein and GAGs, which carries positive charge and negative charge, respectively, and lead to the binding of viral ligand and cellular receptor, and then undergo virus entry. Little is known about the molecular determinants of E protein interacted with GAGs and associated with the process of virus infection. By using reverse genetic technique, we had generated a panel of various site-directed E gene mutants from JEV infectious cDNA clones to further explore the aforesaid issues. Herein, we found that highly sulfated heparin did not inhibit the infection of RGD motif mutants E-D389G and E-D389H. Treatment of heparin or neutralizing antibodies (PL1-365) during virus adsorption and virus entry could affect the infection of wild-type and E gene mutants including E-Q52K, E-E138K, E-S227P, E-S275P, E-S331R, E-D389G, and E-D389H. However, PL1-365 did not inhibit E-S227P infection completely. By using pre- and post-attachment neutralization assay, we also demonstrated the inhibition activity of PL1-365 at the attachment and post-attachment steps of virus infection. Taken together, these results suggest that virus binds to highly sulfated form of GAGs on cell surfaces in the early stage of JEV infection, followed by recognizing the cellular receptor to enter cells. However, it needs to generate a series of mutation on GAG binding motif of E protein by reverse genetic technology to further verify whether RGD motif acts as an important molecular determinant interacting with GAGs.

並列關鍵字

Envelope protein ； Glycosaminoglycans ； neutralizing antibodies ； RGD motif ； Molecular determinants

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