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

以作者查詢圖書館館藏

、以作者查詢臺灣博碩士

、以作者查詢全國書目

、勘誤回報

、線上人數：14

、訪客IP：3.138.123.240

姓名

呂冠毅(Kuan-Yi Lu) 查詢紙本館藏

畢業系所

系統生物與生物資訊研究所

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)

相關論文

★ 以生物資訊分析與實驗驗證探討大腸桿菌蛋白質體晶片找出的乳鐵胜肽B胞內目標蛋白	★ 結合奈米脂粒與抗體微陣列晶片的高通量快速檢測系統之發展並應用於婦女子宮頸炎病因之診斷與研究
★ 蛋白質 G 與具硫基反應性的釕複合物之生物接合作為螢光免疫試驗的通用試劑	★ 利用微陣列蛋白質晶片帥選GNRA tetraloop結合蛋白
★ 利用大腸桿菌蛋白質體晶片分析新生兒血液中的免疫球蛋白	★ 利用大腸桿菌蛋白質體晶片找出參與第一型線毛表現之細菌蛋白質
★ 利用大腸桿菌蛋白體微陣列晶片系統性探討抗菌肽的胞內作用目標	★ 利用大腸桿菌蛋白質體晶片找出與2-氧基組胺酸交互作用之蛋白質
★ 發展微珠式96孔過濾盤競爭型免疫分析法偵測硫酸紫菌素	★ 異質性核醣核酸蛋白K (hnRNP K) 抑制成熟miRNA-122轉錄後調控機制之研究
★ 利用酵母菌蛋白質體晶片找出與前信使核糖核酸加工因子19泛素連接?經泛素化作用之受質	★ 腸道共生黴菌與酒精性肝病的相關性
★ 在大腸桿菌與酵母菌蛋白質體晶片中量化其蛋白質的濃度	★ 應用大腸桿菌與酵母菌蛋白質體晶片系統性分析抗菌肽及抗生素作用之目標蛋白質

檔案

[Endnote RIS 格式]

[Bibtex 格式]

[相關文章]

[文章引用]

[完整記錄]

[館藏目錄]

[檢視]

[下載]

本電子論文使用權限為同意立即開放。
已達開放權限電子全文僅授權使用者為學術研究之目的，進行個人非營利性質之檢索、閱讀、列印。
請遵守中華民國著作權法之相關規定，切勿任意重製、散佈、改作、轉貼、播送，以免觸法。

摘要(中)

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。

摘要(英)

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.

關鍵字(中)

★ C 型肝炎病毒
★ 人類蛋白質體微陣列晶片
★ 非轉譯區

關鍵字(英)

★ hepatitis C virus
★ untranslated region
★ human proteome microarray

論文目次

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

參考文獻

(1) P. Georgel et al., "Virus-host interactions in hepatitis C virus infection: implications
for molecular pathogenesis and antiviral strategies", Trends in molecular medicine, 16,
277, 2010
(2) P. D. Nagy, J. Pogany, "The dependence of viral RNA replication on co-opted host
factors", Nature reviews. Microbiology, 10, 137, 2012
(3) T. L. Tellinghuisen, C. M. Rice, "Interaction between hepatitis C virus proteins and
host cell factors", Current opinion in microbiology, 5, 419, 2002
(4) C. Giannini, C. Brechot, "Hepatitis C virus biology", Cell death and differentiation, 10
Suppl 1, S27, 2003
(5) P. Pileri et al., "Binding of hepatitis C virus to CD81", Science, 282, 938, 1998
(6) M. J. Evans et al., "Claudin-1 is a hepatitis C virus co-receptor required for a late step
in entry", Nature, 446, 801, 2007
(7) S. Hopkins et al., "The cyclophilin inhibitor SCY-635 suppresses viral replication and
induces endogenous interferons in patients with chronic HCV genotype 1 infection",
Journal of hepatology, 2012
(8) A. Albecka et al., "Role of low-density lipoprotein receptor in the hepatitis C virus life
cycle", Hepatology, 55, 998, 2012
(9) A. J. Lyons, J. R. Lytle, J. Gomez, H. D. Robertson, "Hepatitis C virus internal
59
ribosome entry site RNA contains a tertiary structural element in a functional domain
of stem-loop II", Nucleic acids research, 29, 2535, 2001
(10) M. Yanagi, M. St Claire, S. U. Emerson, R. H. Purcell, J. Bukh, "In vivo analysis of
the 3’’ untranslated region of the hepatitis C virus after in vitro mutagenesis of an
infectious cDNA clone", Proceedings of the National Academy of Sciences of the
United States of America, 96, 2291, 1999
(11) C. S. Fraser, J. A. Doudna, "Structural and mechanistic insights into hepatitis C viral
translation initiation", Nature reviews. Microbiology, 5, 29, 2007
(12) J. A. Hiscox, "The interaction of animal cytoplasmic RNA viruses with the nucleus to
facilitate replication", Virus research, 95, 13, 2003
(13) Y. Liu, E. Wimmer, A. V. Paul, "Cis-acting RNA elements in human and animal
plus-strand RNA viruses", Biochimica et biophysica acta, 1789, 495, 2009
(14) E. Martinez-Salas, A. Pacheco, P. Serrano, N. Fernandez, "New insights into internal
ribosome entry site elements relevant for viral gene expression", The Journal of
general virology, 89, 611, 2008
(15) G. A. Otto, J. D. Puglisi, "The pathway of HCV IRES-mediated translation initiation",
Cell, 119, 369, 2004
(16) C. M. Spahn et al., "Hepatitis C virus IRES RNA-induced changes in the
conformation of the 40s ribosomal subunit", Science, 291, 1959, 2001
60
(17) M. Zuker, "Mfold web server for nucleic acid folding and hybridization prediction",
Nucleic acids research, 31, 3406, 2003
(18) D. Moradpour, F. Penin, C. M. Rice, "Replication of hepatitis C virus", Nature reviews.
Microbiology, 5, 453, 2007
(19) R. K. Jangra, M. Yi, S. M. Lemon, "Regulation of hepatitis C virus translation and
infectious virus production by the microRNA miR-122", Journal of virology, 84, 6615,
2010
(20) J. I. Henke et al., "microRNA-122 stimulates translation of hepatitis C virus RNA",
The EMBO journal, 27, 3300, 2008
(21) W. C. Tsai et al., "MicroRNA-122, a tumor suppressor microRNA that regulates
intrahepatic metastasis of hepatocellular carcinoma", Hepatology, 49, 1571, 2009
(22) R. K. Jangra, M. Yi, S. M. Lemon, "Regulation of hepatitis C virus translation and
infectious virus production by the microRNA miR-122", J Virol, 84, 6615,
(23) T. Shimakami et al., "Stabilization of hepatitis C virus RNA by an Ago2-miR-122
complex", Proceedings of the National Academy of Sciences of the United States of
America, 109, 941, 2012
(24) C. S. Chen et al., "A proteome chip approach reveals new DNA damage recognition
activities in Escherichia coli", Nat Methods, 5, 69, 2008
(25) C. S. Chen et al., "Identification of novel serological biomarkers for inflammatory
61
bowel disease using Escherichia coli proteome chip", Mol Cell Proteomics, 8, 1765,
2009
(26) H. Zhu et al., "Global analysis of protein activities using proteome chips", Science,
293, 2101, 2001
(27) H. Zhu, M. Bilgin, M. Snyder, "Proteomics", Annu Rev Biochem, 72, 783, 2003
(28) J. Zhu et al., "RNA-binding proteins that inhibit RNA virus infection", Proceedings of
the National Academy of Sciences of the United States of America, 104, 3129, 2007
(29) Y. Y. Lin et al., "Protein acetylation microarray reveals that NuA4 controls key
metabolic target regulating gluconeogenesis", Cell, 136, 1073, 2009
(30) S. C. Tao, C. S. Chen, H. Zhu, "Applications of protein microarray technology", Comb
Chem High Throughput Screen, 10, 706, 2007
(31) J. Zhu et al., "Protein array identification of substrates of the Epstein-Barr virus
protein kinase BGLF4", J Virol, 83, 5219, 2009
(32) L. A. Kung et al., "Global analysis of the glycoproteome in Saccharomyces cerevisiae
reveals new roles for protein glycosylation in eukaryotes", Mol Syst Biol, 5, 308, 2009
(33) S. Hu et al., "Profiling the human protein-DNA interactome reveals ERK2 as a
transcriptional repressor of interferon signaling", Cell, 139, 610, 2009
(34) R. Li et al., "Conserved herpesvirus kinases target the DNA damage response pathway
and TIP60 histone acetyltransferase to promote virus replication", Cell host & microbe,
62
10, 390, 2011
(35) M. R. Chapman, C. C. Kao, "A minimal RNA promoter for minus-strand RNA
synthesis by the brome mosaic virus polymerase complex", J Mol Biol, 286, 709, 1999
(36) K. Sivakumaran, M. Hema, C. C. Kao, "Brome mosaic virus RNA syntheses in vitro
and in barley protoplasts", J Virol, 77, 5703, 2003
(37) K. J. Livak, T. D. Schmittgen, "Analysis of relative gene expression data using
real-time quantitative PCR and the 2(-Delta Delta C(T)) Method", Methods, 25, 402,
2001
(38) A. Kozomara, S. Griffiths-Jones, "miRBase: integrating microRNA annotation and
deep-sequencing data", Nucleic acids research, 39, D152, 2011
(39) C. UniProt, "Reorganizing the protein space at the Universal Protein Resource
(UniProt)", Nucleic acids research, 40, D71, 2012
(40) T. Y. Hsieh et al., "Hepatitis C virus core protein interacts with heterogeneous nuclear
ribonucleoprotein K", The Journal of biological chemistry, 273, 17651, 1998
(41) M. Costa, J. L. Rodriguez-Sanchez, A. J. Czaja, C. Gelpi, "Isolation and
characterization of cDNA encoding the antigenic protein of the human tRNP(Ser)Sec
complex recognized by autoantibodies from patients withtype-1 autoimmune
hepatitis", Clinical and experimental immunology, 121, 364, 2000
(42) M. Volkmann et al., "Soluble liver antigen: isolation of a 35-kd recombinant protein
63
(SLA-p35) specifically recognizing sera from patients with autoimmune hepatitis",
Hepatology, 33, 591, 2001
(43) I. Wies et al., "Identification of target antigen for SLA/LP autoantibodies in
autoimmune hepatitis", Lancet, 355, 1510, 2000
(44) J. Wang et al., "Hepatitis C virus non-structural protein NS5A interacts with FKBP38
and inhibits apoptosis in Huh7 hepatoma cells", FEBS letters, 580, 4392, 2006
(45) H. L. Bonkovsky et al., "Porphyria cutanea tarda, hepatitis C, and HFE gene mutations
in North America", Hepatology, 27, 1661, 1998
(46) T. Tsutsumi et al., "Interaction of hepatitis C virus core protein with retinoid X
receptor alpha modulates its transcriptional activity", Hepatology, 35, 937, 2002
(47) W. H. Huang, B. Y. Yung, W. J. Syu, Y. H. Lee, "The nucleolar phosphoprotein B23
interacts with hepatitis delta antigens and modulates the hepatitis delta virus RNA
replication", The Journal of biological chemistry, 276, 25166, 2001
(48) K. Yuksek, W. L. Chen, D. Chien, J. H. Ou, "Ubiquitin-independent degradation of
hepatitis C virus F protein", Journal of virology, 83, 612, 2009
(49) H. Aoki, J. Hayashi, M. Moriyama, Y. Arakawa, O. Hino, "Hepatitis C virus core
protein interacts with 14-3-3 protein and activates the kinase Raf-1", Journal of
virology, 74, 1736, 2000
(50) T. H. Lee, S. J. Elledge, J. S. Butel, "Hepatitis B virus X protein interacts with a
64
probable cellular DNA repair protein", Journal of virology, 69, 1107, 1995
(51) O. Leupin, S. Bontron, M. Strubin, "Hepatitis B virus X protein and simian virus 5 V
protein exhibit similar UV-DDB1 binding properties to mediate distinct activities",
Journal of virology, 77, 6274, 2003
(52) N. Lin-Marq, S. Bontron, O. Leupin, M. Strubin, "Hepatitis B virus X protein
interferes with cell viability through interaction with the p127-kDa UV-damaged
DNA-binding protein", Virology, 287, 266, 2001
(53) N. O. Ku, S. Michie, R. G. Oshima, M. B. Omary, "Chronic hepatitis, hepatocyte
fragility, and increased soluble phosphoglycokeratins in transgenic mice expressing a
keratin 18 conserved arginine mutant", The Journal of cell biology, 131, 1303, 1995
(54) J. Y. Lin et al., "Heterogeneous nuclear ribonuclear protein K interacts with the
enterovirus 71 5’’ untranslated region and participates in virus replication", The Journal
of general virology, 89, 2540, 2008
(55) M. J. Matunis, W. M. Michael, G. Dreyfuss, "Characterization and primary structure
of the poly(C)-binding heterogeneous nuclear ribonucleoprotein complex K protein",
Molecular and cellular biology, 12, 164, 1992
(56) D. Dhar et al., "Human ribosomal protein L18a interacts with hepatitis C virus internal
ribosome entry site", Archives of virology, 151, 509, 2006
(57) Y. Inoue et al., "Ubiquitous presence of cellular proteins that specifically bind to the 3’’
65
terminal region of hepatitis C virus", Biochemical and biophysical research
communications, 245, 198, 1998
(58) G. Luo, "Cellular proteins bind to the poly(U) tract of the 3’’ untranslated region of
hepatitis C virus RNA genome", Virology, 256, 105, 1999
(59) J. Wood, R. M. Frederickson, S. Fields, A. H. Patel, "Hepatitis C virus 3’’X region
interacts with human ribosomal proteins", Journal of virology, 75, 1348, 2001
(60) S. Hu, Z. Xie, J. Qian, S. Blackshaw, H. Zhu, "Functional protein microarray
technology", Wiley interdisciplinary reviews. Systems biology and medicine, 3, 255,
2011
(61) L. Peyrin-Biroulet et al., "Interaction of ribavirin with azathioprine metabolism
potentially induces myelosuppression", Alimentary pharmacology & therapeutics, 28,
984, 2008
(62) T. Reiberger et al., "Mitochondrial toxicity is associated with virological response in
patients with HIV and hepatitis C virus coinfection treated with ribavirin and highly
active antiretroviral therapy", The Journal of infectious diseases, 202, 156, 2010
(63) B. Horoldt et al., "Results of combination treatment with pegylated interferon and
ribavirin in cirrhotic patients with hepatitis C infection", Liver international : official
journal of the International Association for the Study of the Liver, 26, 650, 2006
(64) C. F. Huang et al., "Efficacy and safety of pegylated interferon combined with
66
ribavirin for the treatment of older patients with chronic hepatitis C", The Journal of
infectious diseases, 201, 751, 2010
(65) M. L. Vachon, D. T. Dieterich, "The era of direct-acting antivirals has begun: the
beginning of the end for HCV?", Seminars in liver disease, 31, 399, 2011
(66) C. Welsch, A. Jesudian, S. Zeuzem, I. Jacobson, "New direct-acting antiviral agents
for the treatment of hepatitis C virus infection and perspectives", Gut, 61 Suppl 1, i36,
2012
(67) G. T. Strickland, S. S. El-Kamary, P. Klenerman, A. Nicosia, "Hepatitis C vaccine:
supply and demand", The Lancet infectious diseases, 8, 379, 2008
(68) K. J. Blight, J. A. McKeating, C. M. Rice, "Highly permissive cell lines for
subgenomic and genomic hepatitis C virus RNA replication", Journal of virology, 76,
13001, 2002

指導教授

陳健生(Chien-Sheng Chen)

審核日期

2012-7-24

推文