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研究生:江卓穎
研究生(外文):Cho-ying Chiang
論文名稱:人類免疫缺失病毒核鞘蛋白網絡及其功能性聯結IMP1蛋白微聚體以串接病毒Gag-RNA的交互作用至含有CD81的微膜區域
論文名稱(外文):The proteome of HIV nucleocapsid and its functional link to IMP1 granules in bridging Gag-RNA interactions and association to CD81-containing micromembrane domain
指導教授:王憲威
指導教授(外文):Shainn-Wei Wang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:分子醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:70
中文關鍵詞:類胰島素生長因子訊息核醣核酸結合蛋白多液泡囊輸出體人類免疫缺失病毒核酸鞘蛋白
外文關鍵詞:GaghStau1nucleocapsidMVBIMP1Human Immunodeficient Virus
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人類免疫缺失病毒核酸鞘蛋白(NC)為Gag前驅蛋白內之ㄧ結構單位,在病毒Gag的組合與基因(ΨRNA)包裝時扮演中樞角色。儘管NC主導Gag之同聚作用及RNA的親和性,但最近證實寄主細胞負責mRNA運送和代謝的蛋白,例如IMP1和hSTAU1,會透過NC的鋅指區(Zinc-finger motifs)參與Gag-ΨRNA的交互作用。然而此兩種蛋白如何在不同時期參與GagNC的交互作用或是否有其他蛋白參與其間尚不清楚。我們利用串接親合純化技術找到27個可能和NC有交互作用的蛋白,並結合質譜、網路資料庫(UniHi)分析,推測他們彼此有極親密的交互作用網絡。透過免疫沉澱實驗,證實我們所預測的IMP1/DHX9/HNRPU網絡與文獻報導的IMP1微聚體之組成相似外,FLAG-IMP1可和NC、Gag、甚或喪失對RNA親和力的變異種Gag產生交互作用。此結果加強確認了最近文獻中報導IMP1可透過NC鋅手指在不需要病毒RNA參與下就可和Gag產生交互作用;同時也提供了一個可能性觀點,即ΨRNA在運送的過程中是以IMP1-ΨRNP聚合型式為主,隨後再和Gag產生交互作用。然而相較於IMP1,hSTAU1並沒有被NC串接親和實驗所純化出來,也沒出現在文獻中利用修飾過的Gag以相似的串接親和實驗所純化出來。利用FLAG-hSTAU1在病毒轉染的細胞進行免疫沉澱也發現Gag幾乎不被偵測到,極可能hSTAU1相較於IMP1對可溶性NC或Gag的親和力較差。我們也發現當細胞過度表現IMP1會造成Gag提早進行病毒蛋白酶切割而成熟;但當細胞表達蛋白質分解酶缺失的病毒時,Gag的產量會隨著IMP1的劑量而增多。我們推測IMP1在Gag的轉譯或後轉譯調控伴隨的成熟過程中扮演被動的角色。更重要的是,不論Gag存在與否,hSTAU1及IMP1彼此並不互相被免疫沉澱,推測此兩種蛋白屬於不同微聚體。為了進一步描繪兩者和Gag在細胞內的交互作用,我們利用了密度梯度離心(density fractionation gradient)及細胞膜漂浮(membrane floatation experiment)技術分離293T細胞的細胞質液和細胞膜以觀察Gag產成後之區間物(intermediates)與兩者在組合過程中的關聯。我們發現IMP1主要出現在細胞質液之清潔劑不溶性DRC Gag (detergent resistant complexes)的梯度位址並與細胞膜含CD81之微膜脂質蛋白共層,而後者亦與hSTAU1座落同層。由於CD-81與外送體(exsosome)或多液泡囊輸出體(MVB; Multi-vescularbodies)上之raft tetraspaning complex的形成高度相關,因此綜合上述證據,我們推測IMP1-ΨRNP結合Gag運送到具有hSTAU1的MVB區域以利用囊泡(vesicles)運送到細胞漿膜(plasma membrane)或是直接在MVB出芽(budding)。此外,DHX9與hnRPU也證實只與細胞質液內的Gag區間物結合而似乎有動態的交互作用,與其具有RNA解旋酶活性的特質相當,可能與短暫的調控RNA結構以提供Gag-ΨRNA彼此的交互作用。此研究不只提供HIV NC蛋白和細胞蛋白可能的交互作用網路,據此也可利用這些特定細胞蛋白來作為Gag-ΨRNP運送過程上的追蹤標的及其可能參與的作用機制以提供抗HIV藥物的開發。
Human immunodeficiency virus-1 (HIV-1) nucleocapsid (NC) is encoded as a structural domain within the Gag precursors and plays a central role assisting Gag assembly and genomic RNA (ΨRNA) packaging processes. Despite that NC domain has unique characters for homotypic Gag interaction and RNA affinity, cellular proteins functioned in mRNA translocation and metabolism, such as IMP1 and hSTAU1, are demonstrated to participate in the Gag-ΨRNA interaction through NC Zinc-finger motifs. However, how these two proteins interact spatiotemporally with the NC domain and whether other proteins are involved in the interplay remain unclear. By employing a Tandem Affinity Tag Purification (TAP) based proteomic approach and bioinformatic research through Unified Human Interactome (UniHi), we consistently identified 27 NC-associated proteins in a network of interplays. A hypothetical interactome consisted of IMP1/DHX9/hnRPU, consistent to the relative composition of reported IMP1 granules, was found to interact with NC, Gag, and a GagNC mutant lacking general RNA affinity through confirmatory co-immunoprecipitation experiments. This result not only corroborates with a recent finding that IMP1 interacts with Gag through NC zinc fingers independent of viral RNA interaction, but also supports in part that ΨRNA traffics as an IMP1-ΨRNP complex for subsequent interaction with Gag. However, hSTAU1 was not detected in our NC purification profile nor was it evident in a reported profile using a modified Gag in a similar TAP approach. Given that flag-hSTAU1 barely co-immunoprecipitated HIV-1 Gag, it is possible that hSTAU1 has a relative weaker affinity than IMP1 to interact with soluble NC or Gag. We also found that IMP1 overexpression resulted in increased intracellular Gag processing; But when HIV protease is inactivated, IMP1 enhanced intracellular Gag productions in a dose dependent manner. These results indicate that IMP1 has a passive role in translational or post-translational control in couples with Gag maturation process. Moreover, hSTAU1 and IMP1 were not pull-downed by each others regardless of the presence of Gag or not, suggesting that they are distinct granule proteins in action. To understand further the interplays of these granule proteins to Gag, we investigated their association in cytosolic and membrane compartments of 293T cell by density fractionation gradient and membrane floatation experiments. IMP1 preferentially associated with cytosolic detergent resistant Gag complexes (DRC) and Gag-laden CD81 associated membrane fractions, while the later is also associated with hSTAU1. Since CD81 belongs to a raft tetraspanin complex exported through exosomes or multivescular bodies (MVB), our results together support a possible interplay of IMP1-ΨRNP complex to Gag and their transition to hSTAU1 in a tetraspanin microdomain for subsequent vesicular trafficking to plasma membrane or direct budding into the lumen of MVB. In addition, DHX9/hnRPU interacted only with cytosolic Gag intermediates, consistent with their transient role as RNA helicase for RNA remodelings during Gag-ΨRNA interaction. This study not only provides significant insights into the possible NC-cellular protein interacting networks, but also demonstrates critical prospects using certain of these proteins as markers for Gag-ΨRNP interaction or trafficking and as potential targets for anti-HIV drug development.
中文摘要 3
英文摘要 5
致謝 7
目錄 8
表目錄 11
圖目錄 12
附錄 13
符號及縮寫 14
緒論 15
一、 人類免疫缺失病毒 15
1. 人類免疫缺失病毒結構介紹 15
2. 人類免疫缺失病毒顆粒組裝機制 16
3. 人類免疫缺失病毒的NC蛋白區域功能介紹 17
二、 與人類免疫缺失病毒NC蛋白作用之寄主蛋白 17
1. IMP1 (Insulin-like growth factor II mRNA binding protein1)蛋白 17
2. hStaufen1蛋白 18
三、 研究目標 18
實驗材料與方法 20
一、 磷酸鈣細胞轉染法 (Calcium phosphate coprecipitation method) 20
二、 電擊轉染法 (Electroporation method) 20
三、 細胞培養及過度性轉染 (Cell Cultures and Transient Transfection) 20
四、 建立持續表現HR’-Green的細胞株 (Stable CEM-Green reportor cell lines) 21
五、 製備有感染力的病毒原液 (Preparation of infectious virus stock) 21
六、 定量病毒感染力 (Quantitative assays for virus infectivity) 22
七、 人類免疫缺失病毒感染細胞的方法 (HIV-1 infection) 22
八、 質體構築 (Plasmid constructs) 22
九、 反轉錄PCR (RT (reverse transcriptase)-PCR) 23
十、 西方點墨法 (Western blot analysis) 23
十一、 TCA蛋白質沉澱法 (TCA protein precipitation) 24
十二、 免疫沉澱 (Immunoprecipitation) 24
十三、 次細胞分層分析與蔗醣密度梯度離心 (Subcellular fractionation and sucrose density gradient preparation) 25
十四、 Optiprep密度梯度離心 (Optiprep gradient centrifugation) 25
十五、 細胞膜漂浮實驗 (Membrane flotation assay) 26
十六、 RNA點墨實驗 (RNA dot-blot) 26
結果 28
一、 網路資料庫(UniHi)分析與NC有交互作用的蛋白 28
二、 IMP1蛋白以微聚體的型式和Gag蛋白交互作用 28
三、 過度表現IMP1蛋白時會造成Gag蛋白不正常的成熟 29
四、 IMP1蛋白和Gag蛋白的交互作用不需要ΨRNA參與 30
五、 pr55 Gag 蛋白和 hSTAU1 微聚體交互作用並不強烈 30
六、 IMP1 與細胞質液和細胞膜Gag區間產物的聚合關係 31
七、 IMP1蛋白和Gag蛋白的轉譯或後轉譯調控有關 33
八、 IMP1 可能藉由和vRNA的交互作用影響Gag的組裝及運送 34
九、 IMP1蛋白可在試管外搶奪ΨRNA不需要Gag蛋白參與 36
十、 過度表現IMP1蛋白會增加pr55 Gag 蛋白的產量 36
討論 38
參考文獻 42
圖 47
表 59
附錄 64
自述 70
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