Aurintricarboxylic acid抑制流感病毒A型與腸病毒71型感染機制與腸病毒蛋白酶攻擊宿主免疫系統之研究

Abstract

RNA 病毒由於有高度之變異性，所引發病毒感染會威脅人類生命健康，為有效解決此問題，可經由兩個方向著手。其一為找尋新化學藥物並發展抗病毒藥物對抗多變的病毒；再者是了解病毒與宿主的交互作用，藉此以獲得有效治療方法。本論文以流感病毒為例，建立病毒表面蛋白-神經氨酸酶之高效率冷光分析篩選平台，經篩選超過2000多個結構迥異的化合物，結果發現Aurintricarboxylic acid (ATA)為最具潛力的化合物。ATA具有抑制神經氨酸酶與突變株(H274Y)活性，且有效抑制病毒H1N1、H3N2與H5N1的複製。經由分子嵌合(molecular docking)結果顯示，ATA可結合在酵素的基質結合位置，文獻報導指出ATA具廣泛的抗病毒功效，故我們進一步研究此化合物對抗腸病毒的作用機制。根據腸病毒中和試驗與病毒班結果顯示，ATA可有效抑制腸病毒複製。為確認化合物抑制病毒複製時期，以ATA添加在不同病毒複製週期，結果顯示ATA影響早期病毒複製。分別測試ATA對病毒早期之Internal Ribosome Entry Site (IRES)調控病毒多蛋白轉譯作用、病毒蛋白酶2A與3C之分解作用、對病毒的RNA聚合酶(3D polymerase)之影響，由結果顯示ATA經由抑制3D複製酶活性而抑制病毒複製。本研究另一方向是釐清病毒與宿主免疫系統之交互關係，IFN □/□為目前用於治療抗病毒之細胞激素，然而仍有多種病毒藉由不同策略躲開IFN系統授予的抗病毒機制。腸病毒是臨床上具有重要影響力的病毒，至今仍無有效治療方法，已知第一型干擾素並不能完全有效地抑制腸病毒EV71的複製；此結果更顯示病毒與宿主間可能存在複雜的致病機轉。針對Innate Immunity訊息傳遞鏈-第一型干擾素途徑等訊息傳遞鏈上相關蛋白質研究，並藉生物資訊分析3C病毒蛋白酶的切割受質部位，我們以in vivo及in vitro實驗作佐證，建立有效率研究模式。

RNA viruses have an extremely high mutation rate which causes serious diseases in human. There are two ways to solve this problem. One is to screen the novel compounds for against the RNA viruses, and the other one is to study the relationship between the virus and host cells to deal with emerging challenges. In this study, the RNA virus - influenza virus was used as a target virus. We established a neuraminidase (NA) chemiluminescent assay system for high throughtput enzyme inhibitor screening. After screening approximately 2000 structurally diverse compounds, we found that aurintricarboxylic acid (ATA) was a potent inhibitor for NA of both group-1 and group-2 influenza viruses. ATA was equally potent in inhibiting the NA activity derived from wild-type NA and its H274Y mutant renders NA resistance by oseltamivir. Molecular modeling experiments suggested that ATA binds to NA at substrate binding site. ATA has also been demonstrated to inhibit the replication of several RNA viruses. Furthermore, we identified the effects of ATA on the clinically important RNA virus - EV71 replication and to explore the underlying mechanism. In this study, ATA was found to be an inhibitor for the replication of EV71. Mode-of-action studies revealed that ATA was able to inhibit the RNA-dependent RNA polymerase (RdRp, 3D polymerase) activity of EV71, while neither the internal ribosomal entry site (IRES) mediated translation of viral polyprotein nor the viral 3C protease (3Cpro) activity was affected. Overall, findings in this study suggest that ATA is able to effectively inhibit EV71 replication through interfering with the viral 3D polymerase. IFN-□/β is an antiviral cytokine and currently is used for treating viral infections. Meanwhile, many viruses are known to possess multiple strategies to escape the antiviral mechanism conferred by host’s IFN system. Because there is no proven therapy to treat EV71 infection, our goal is to understand the relationship between the viruses and host immune system. Through a bioinformatics approach, we found that several proteins involved in the IFN-response pathway are susceptible to cleavage by the EV-71 encoded 3C protease. Subsequent in vivo and in vitro cleavage assays will be performed to examine analytical results.