利用人類蛋白質體微陣列晶片探究C型肝炎病毒非轉譯區與宿主之交互作用

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研究生：	呂冠毅 Kuan-Yi Lu
論文名稱：	利用人類蛋白質體微陣列晶片探究C型肝炎病毒非轉譯區與宿主之交互作用 Deciphering the hepatitis C virus untranslated region-host interactions using human proteome microarrays
指導教授：	陳健生 Chien-Sheng Chen
口試委員:
學位類別：	碩士 Master
系所名稱：	生醫理工學院 - 系統生物與生物資訊研究所 Graduate Institute of Systems Biology and Bioinformatics
畢業學年度：	100
語文別：	英文
論文頁數：	100
中文關鍵詞：	C 型肝炎病毒、人類蛋白質體微陣列晶片、非轉譯區
外文關鍵詞：	hepatitis C virus, untranslated region, human proteome microarray
相關次數：	點閱：17 下載：0
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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 (untranslated region) 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，本團隊已成功利用此人類蛋白質體晶片分析鑑定出能辨認 C 型肝炎病毒非轉譯區上之 5’ 端莖環構造 (stem-loop I)，並與之結合之候選蛋白。其中，本團隊成功發現並驗證出 hnRNP K 蛋白質能與此莖環構造之環狀部位 (tetra-loop) 以及其微核醣核酸-122 (microRNA-122) 之互補序列進行專一性結合。在 hnRNP K 基因減弱 (knock-down) 之實驗中，我們也觀察到 C 型肝炎病毒之核醣核酸量隨著 hnRNP K 表現量下降而劇減。透過免疫螢光染色，我們發現在部分內含 C 型肝炎病毒 RNA 複製子 (HCV RNA replicon) 之人類肝細胞株中，hnRNP K 蛋白質的表現由細胞核部份轉移至細胞質中。以上結果顯示 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。此外，研究指出微核醣核酸-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。因此，本團隊亦剖析能與微核醣核酸-122 進行專一作用之蛋白質，以探求微核醣核酸-122 在 C 型肝炎中所扮演之功能角色。篩選所得之蛋白質扮演的角色包含能幫助微核醣核酸-122 與 C 型肝炎病毒非轉譯區結合之伴護蛋白 (chaperon)，抑或阻止其結合之抑制蛋白。

Over the past few decades, the incidence and prevalence of diseases caused from RNA viruses are frequent and serious, bringing a critical health concern all over the world. A
successful infection by RNA viruses requires proper interactions between viral RNA molecules and cellular proteins in the host. The untranslated regions (UTRs) of virus RNA genome are known to play pivotal roles in genome accumulation and virion production. Thus, deciphering the protein-viral UTR interactions allows a better understanding of the viral multiplication and provides potential therapeutic targets for developing antivirals. However,
the most considerable challenge in the study of protein-RNA interactions is that testing an individual interaction at a time is cumbersome. To study the interactions in a high-throughput manner, an array of ~17,000 non-redundant human proteins has been recently established by our collaborative team. Herein, we implemented the comprehensive proteome chip analysis to identify proteins that recognized the conserved stem-loop I (SL1) in hepatitis C virus (HCV) 5’ UTR. Remarkably, we identified a protein termed hnRNP K that specifically bound to the tetra-loop structure and the microRNA-122 (miR-122) seed sequence of the HCV SL1. We showed that the hnRNP K knock-down contributed to the decrease in HCV RNA levels in a dose response manner. We also found that hnRNP K in 5% of cells translocated from nucleus to the cytoplasm in the presence of HCV RNA replicons. These evidences indicated that hnRNP K might enhance the virus replication through the binding to SL1. In addition, we screened the proteins that interacted with the miR-122 using the same human chips. The miR-122 has been reported to stabilize the HCV RNA genome through binding to its seed sequence in SL1 and stimulates the HCV infection. Therefore, identified miR-122-binding proteins could be involved in the context of HCV infection or in the microRNA processing itself. Herein, our result supported that hnRNP K was one of the identified proteins with binding capacities to both SL1 and miR-122, which might have critical roles for HCV replication or persistence in the biology of HCV.

中文摘要 ....................................................................................................................................i
ABSTRACT ..............................................................................................................................iii
ABBREVIATION......................................................................................................................v
致謝..........................................................................................................................................vi
CONTENTS .............................................................................................................................vii
LIST OF FIGURES....................................................................................................................x
LIST OF TABLES....................................................................................................................xii
I. INTRODUCTION..............................................................................................................1
I. 1. RNA virus...........................................................................................................1
I. 2. Hepatitis C virus .................................................................................................1
I. 3. The HCV untranslated regions ...........................................................................2
I. 4. The study of HCV RNA-host protein interactions .............................................4
I. 5. microRNA-122 ...................................................................................................5
I. 6. Proteome microarrays.........................................................................................6
I. 6. 1. Applications of proteome microarrays in virology.....................................6
I. 6. 2. Construction of human proteome microarrays ...........................................7
I. 7. Study outline.......................................................................................................9
II. MATERIALS AND METHODS...................................................................................... 11
viii
II. 1. Proteome chip assays........................................................................................ 11
II. 2. High-throughput yeast protein purification ......................................................12
II. 3. Bead-based affinity assays................................................................................13
II. 4. Protein-RNA UV-crosslinking assays...............................................................14
II. 5. siRNA knock-down on HCV SL1-binding proteins in HCV replicon cells.....14
II. 6. Characterization of hnRNP K-binding sites in the HCV SL1 ..........................17
II. 7. hnRNP K knock-down assay ............................................................................17
II. 8. Immunofluorescence assay of hnRNP K in the presence of HCV replicon .....18
II. 9. Databases..........................................................................................................19
III. RESULTS .........................................................................................................................20
III. 1. Identification of proteins that bound to the stem-loop I (SL1) of HCV 5’ UTR
using human proteome chips ............................................................................................20
III. 2. Identification of proteins that bound to the miR-122 using human proteome
chips…….........................................................................................................................24
III. 3. Global analyses of identified HCV SL1- and miR-122-binding proteins ........27
III. 4. Establishment of the high-throughput yeast protein purification system.........32
III. 5. Establishment of the bacterial stocks that harbored candidate genes...............35
III. 6. Validation of HCV SL1-binding proteins using the bead-based affinity assay 39
III. 7. Validation of HCV SL1-binding proteins using UV-crosslinking assays.........41
ix
III. 8. Knock-down effects on SL1-binding proteins in HCV replicon cells..............43
III. 9. Characterization of hnRNP K-binding sites in the HCV SL1 RNA.................45
III. 10. Knock-down effect on hnRNP K in HCV replicon cells..................................49
III. 11. Subcellular localization of hnRNP K in the presence of HCV RNA ...............51
IV. CONCLUSION AND DISCUSSION ..............................................................................53
V. REFERENCES.................................................................................................................58
VI. APPENDIXES..................................................................................................................67
Appendix 1. The 313 identified HCV SL1-binding proteins. ..........................................67
Appendix 2. The 273 identified miR-122-binding proteins. ............................................82

                                

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