嚴重急性呼吸道症候群冠狀病毒之ORF6蛋白拮抗第一型干擾素功能之分子機轉; Molecular Mechanism of the type I interferon antagonist function by SARS Coronavirus ORF6 protein

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Title:	嚴重急性呼吸道症候群冠狀病毒之ORF6蛋白拮抗第一型干擾素功能之分子機轉;Molecular Mechanism of the type I interferon antagonist function by SARS Coronavirus ORF6 protein
Authors:	李姿穎;Tzu-Ying LEE
Contributors:	中國醫藥大學：醫學檢驗生物技術學系
Keywords:	嚴重急性呼吸道症候群;第一型干擾素;ORF6 protein
Date:	2008-07-10
Issue Date:	2009-08-12 17:09:53 (UTC+8)
Abstract:	嚴重急性呼吸道症候群(SARS)是一種新興的傳染性疾病，為一種新型的冠狀病毒(SARS-CoV)所引起。嚴重急性呼吸道症候群冠狀病毒已經被發現可以抑制干擾素反應的訊息傳遞路徑。SARS-CoV ORF6 蛋白被報導與第一型干擾素產生拮抗作用，以及細胞內抑制STAT-1 進入細胞核內之作用。本篇論文的主要目的為利用噬菌體呈現技術鑑定與SARS-CoV ORF6 蛋白結合之細胞宿主蛋白及其交互作用與拮抗第一型干擾素訊息傳遞之相關性。本論文首先構築SARS-CoV ORF6 重組載體，藉細菌表現系統，製備ORF6 重組蛋白。噬菌體表現人類肺cDNA 基因庫，經過五次bio-panning 後，找出與ORF6 蛋白結合的肺細胞蛋白。PI-3 kinase-related kinase SMG-1 為其中之一。我們以共軛焦顯微鏡觀察確認病毒蛋白ORF6 與宿主細胞蛋白SMG-1 在細胞中有co-locolization 的現象。之後以ISRE-reporter gene assay 以及Real-time RT-PCR 對表現SARS CoV ORF6 蛋白的HL-CZ 細胞及對照組細胞加入第一型干擾素作用4 小時，發現與對照組相較下， SARS CoV ORF6 蛋白表現細胞的ISRE 啟動子的活性表現量以及ISRE 下游PKR mRNA 有明顯被抑制的現象。而後同樣以ISRE-reporter gene assay 對SARS CoV ORF6 蛋白與SMG-1 蛋白共同表現的細胞同樣加入第一型干擾素作用，相較下發現ISRE 啟動子的活性表現量因有SMG-1 蛋白的存在而明顯有回復升高的現象。接著，我們使用共軛焦顯微鏡觀察發現單獨表現ORF6 蛋白的細胞株加入第一型干擾素作用會抑制STAT-1 進入細胞核內，但是SARS CoV ORF6 蛋白與 SMG-1 蛋白共同表現的細胞株加入第一型干擾素作用後，會有使STAT-1進入細胞核內的現象。同樣地，以西方點墨法來觀察STAT-1 磷酸化情形，發現單獨表現ORF6 蛋白的細胞株加入第一型干擾素作用無STAT-1磷酸化，但是SARS CoV ORF6 蛋白與SMG-1 蛋白共同表現的細胞株加入第一型干擾素作用後，會有使STAT-1 產生磷酸化作用。由上述推測SARS CoV ORF6 蛋白抑制第一型干擾素訊息傳遞之現象會因共同表現宿主細胞SMG-1蛋白而對干擾素反應，如ISRE 啟動子活性以及下游PKR基因表現與單獨表現病毒蛋白ORF6 的細胞株相較之下有回復提升活性的現象。由此我們推測會與病毒蛋白ORF6 作用的宿主細胞蛋白SMG-1在對於嚴重急性呼吸道症候群冠狀病毒拮抗第一型干擾素免疫反應中可能扮演重要腳色。 Abstract Severe acute respiratory syndrome (SARS) is a newly emerged infectious disease. The causative agent of SARS has been identified to be a new type of coronavirus, namely SARS coronavirus (SARS-CoV).SARS-CoV proteins including the ORF6 protein have been reported to inhibit interferon signaling response. The goal of this study is to identify human SARS-CoV ORF6-interacting proteins using the phage displayed human lung cDNA libraries and to investigate their interaction on the type I interferon antagonist function. At first, the recombinant ORF6 protein was generated in BL21(DE3) and purified by using IMAC (immobilized mental-affinity chromatography). After five rounds of biopanning, PI-3 kinase-related kinase SMG-1 protein was identified as SARS-CoV ORF6 interacting protein from phage displayed lung cDNA library. Confocal imaging revealed co-localization of SARS-CoV ORF6 protein with SMG-1 protein in HL-CZ cells. In vivo signaling pathway assay and real time RT-PCR showed that single gene expression of SARS-CoV ORF6-expressing cells blocked the INFα/β-induced responses, such as ISRE-responsive firefly luciferase activity and the expression of PKR. However, both gene expression of SARS-CoV ORF6 and SMG-1 had a significantly higher relative activities of INFα/β-induced ISRE-responsive firefly luciferase than single gene expression of SARS-CoV ORF6 in HL-CZ cells. In addition, confocal imaging and Western blotting assays revealed that the translocation of STAT-1 into nucleus and the STAT-1 phophorylation were found in the transfected cells expressing both genes of ORF6 and SMG-1, but not in the ORF 6-expressing cells in response to INFα/β. Therefore, cell expression of PI-3 kinase-related kinase SMG-1 could restore the INFα /β responses in SARS-CoV ORF6 expressing cells. The interaction of SARS CoV ORF6 and SMG-1 may be responsible for the inhibition of type I IFN response by SARS-CoV.
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