利用人類蛋白質體微陣列晶片探究病毒核醣核酸與宿主之交互作用;Deciphering the viral RNA-host interactions using human proteome microarrays

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Title:	利用人類蛋白質體微陣列晶片探究病毒核醣核酸與宿主之交互作用;Deciphering the viral RNA-host interactions using human proteome microarrays
Authors:	陳健生
Contributors:	生物資訊與系統生物研究所
Keywords:	蛋白質體晶片;RNA 病毒;病毒-宿主交互作用;Proteome chip;RNA virus;Virus-host interaction;物科學;生物技術(醫)
Date:	2016-08-31
Issue Date:	2016-10-11 17:53:37 (UTC+8)
Publisher:	財團法人國家衛生研究院
Abstract:	過去數十年，RNA 病毒所引起的疾病之發生率與盛行率漸趨頻繁，其嚴重性成為一項全球關注之健康議題。基本上，病毒要成功感染宿主需透過病毒核醣核酸與宿主之蛋白質交互作用。而位於病毒核醣核酸基因體上的非編碼區在病毒基因體複製與病毒顆粒之增生扮演著相當關鍵的角色。因此，深入且通盤了解蛋白質與非編碼區核醣核酸之交互作用能促進對其致病機轉的認識，並提供潛在之治療標的以發展抗病毒劑。然而，研究蛋白質-核醣核酸交互作用最常面臨的挑戰在於缺乏合適之分析平台，逐次測試單一交互作用過於繁複且不切實際。為解決上述難題，本實驗室之合作團隊於近期成功建構出約含一萬七千種非重複人類蛋白質之微陣列晶片。於此，本團隊將利用此人類蛋白質體晶片分析鑑定出能辨認 C 型肝炎病毒之非轉譯區，並與之作用的蛋白質。此外，研究指出微核醣核酸-122 (miR-122) 透過與 C 型肝炎病毒非轉譯區之互補序列結合，進而提高病毒轉譯及複製效率。本團隊亦將剖析能與微核醣核酸-122 以及 C 型肝炎病毒非轉譯區兩者進行專一作用的蛋白質，以探求微核醣核酸-122 在 C 型肝炎中所扮演之功能角色。此角色包含能幫助微核醣核酸-122 與非轉譯區結合之伴護蛋白 haperon)，抑或阻止其結合之抑制蛋白。本計畫亦嘗試將鑑定所得之蛋白進行純化並製成晶片，此平台未來可供迅速篩檢此交互作用之抑制劑，並能大幅降低成本。據此，一系列具抗病毒性之抑制劑可望於本計畫被鑑定。前述所得之交互作用與抑制劑之功效亦將進一步驗證其生物意義。日前，本團隊已成功利用此人類蛋白質體晶片鑑定出能與 C 型肝炎病毒之 5’端莖環構造 (stem loop 1) 結合之候選蛋白。其中，本團隊成功發現並驗證出 hnRNP K 能與此莖環構造之環狀部位以及微核醣核酸-122 之互補序列進行專一性結合。在 hnRNP K 基因減弱 (knockdown) 之實驗中，我們也觀察到 C 型肝炎病毒之核醣核酸下降約四倍，代表 hnRNP K 極有可能透過與 C 型肝炎病毒之莖環構造結合並促進病毒基因體複製增生。上述重大突破一再顯示本計畫之可行性與重要性。 ;Over the past few decades, the incidence and prevalence of diseases caused from RNA viruses is becoming more frequent and serious, bringing a critical health concern all over the world. Basically, a successful infection by the virus requires proper interactions between viral RNA molecules and cellular proteins in the host. Among the interactions, the untranslated regions (UTRs) of virus RNA genome play a pivotal role in genome accumulation and virion production. Thus, deciphering of the protein-viral UTR interactions allows a better understanding of those pathogenic mechanisms and provides potential therapeutic targets for developing antivirals. However, the most considerable challenge in the study of protein-RNA interactions is that testing a singular interaction at a time is cumbersome and not practical. To study the interactions in a high-throughput manner, an array of ~17,000 non-redundant human proteins has been recently established in our collaborative team. Herein, we intend to implement the comprehensive proteome chip analysis to identify proteins that recognize the conserved UTRs of hepatitis C virus (HCV). In addition, it has been implicated that microRNA-122 (miR-122) participates in virion production and stimulation of virus ranslation through binding to its target sequence on HCV 5’ UTR. To globally identify the functional roles of miR-122 in the context of HCV infection, profiling of the miR-122 interacting proteins will be carried out using the human proteome chip. Proteins with binding capacities to both SL1 and miR-122 might be directly involved in HCV biology. After identifying HCV UTR-binding proteins, those proteins will be high-throughput purified and spotted on aldehyde slides to construct the “boutique” chips. Compared to the human proteome chip, this boutique chip will be easier to fabricate at lower cost, and will allow us to efficiently identify the interaction inhibitors in the future. To demonstrate the utility of the novel screening platform, a cohort of FDA approved NA-binding antibiotics will be used to probe the boutique chips. The antibiotic that sequesters the protein-viral UTR interactions could be a potential antiviral drug against HCV infection. All the biological significance of identified interactions and the efficacy of inhibitors will be further studied to complete our aims. Remarkably, our team has recently identified a protein called hnRNP K which specifically binds to the tetraloop and the miR-122 seed sequence in the stem loop 1 (SL1) of HCV 5’ UTR. We showed that hnRNP K knockdown contributes to HCV RNA decreased by approximately 4-fold. These current evidences indicate that hnRNP K served as a chaperon that directs miR-122 to bind HCV SL1 and enhance HCV genome replication. Obviously, our preliminary data strongly supports the feasibility and high impact of the proposed project. ;研究期間：10501 ~ 10512
Relation:	財團法人國家實驗研究院科技政策研究與資訊中心
Appears in Collections:	[Institute of Systems Biology and Bioinformatics] Research Project

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