本文關鍵詞： RBBP4 HIV LTR HDAC NR2F1　出處：《浙江大學》2016年博士論文　論文類型：學位論文

【摘要】：背景由人類免疫缺陷病毒(human immunodeficiency virus, HIV)所導致的艾滋病是目前最具有威脅性的傳染病之一。雖然高效的抗逆轉錄病毒治療(highly active anti-retroviral therapy, HAART)能有效的抑制HIV病毒的復制并使病毒載量降低至檢測下限,但是由于HIV潛伏庫的存在,導致HIV不能被徹底清除,因此當前HIV潛伏庫被認為是從體內徹底清除HIV的最后壁壘。HIV潛伏狀態(tài)本質上是一種可逆的轉錄沉默狀態(tài),這種狀態(tài)的產生和維持涉及多種宿主轉錄抑制因子及表觀修飾復合體。結合在HIV前病毒基因組長末端重復序列5'LTR (long terminal repeat)上的宿主轉錄抑制因子及其募集的組蛋白脫乙�；窰DACs (histone deacetylase)等表觀修飾復合體在HIV的轉錄沉默和潛伏的建立維持中起著重要作用。視網膜母細胞瘤結合蛋白RBBP4 (retinoblastoma binding protein 4),作為組蛋白分子伴侶,通過與HDACs等表觀修飾復合體協同作用,可參與基因的轉錄沉默。但作為與HDACs密切關聯的RBBP4分子,它在HIV的轉錄以及潛伏中的作用還未見報道。因此,本文將對RBBP4在HIV轉錄和潛伏中的作用及機制進行研究。目的1.研究RBBP4在正常對照者、臨床HIV感染者和治療者外周血單個核細胞PBMC (peripheral blood mononuclear cells)中的表達變化,并在實驗室HIV感染的CEM-ss和TZM-bl細胞模型上對RBBP4的表達變化進行驗證；2.研究RBBP4表達的改變對HIV產生的作用,并在HIV的轉錄環(huán)節(jié)研究RBBP4對HIV LTR介導的轉錄調控以及RBBP4與HIV LTR啟動子的結合；3.進一步在HIV的感染和潛伏模型中研究RBBP4對HIV轉錄和潛伏作用的機制,為HIV潛伏庫的清除提供實驗數據及科學依據。方法1.首先利用熒光定量PCR檢測RBBP4分子在臨床HIV感染者(30例)、HIV治療者(30例)和健康對照(30例)的PBMCs中的表達變化。并在實驗室HIV感染的CEM-ss和TZM-bl細胞模型上,利用熒光定量PCR、Western blot和免疫熒光技術,對RBBP4的表達變化進行定性和定量檢測。2.通過雙熒光素酶報告系統(tǒng)檢測HIV LTR啟動轉錄的螢火蟲熒光素酶活性,來研究RBBP4對HIV LTR啟動子的轉錄調控；利用凝膠遷移實驗(electrophoretic mobility shift assay, EMSA研究RBBP4與HIV LTR上NF-κB(-115～-75)和NR2F1(-345--319)結合位點的結合。3.在293T細胞與pNL4-3轉染的細胞模型中,利用染色質免疫共沉淀(chrmatin immunoprecipitation analysis, ChIP)研究RBBP4對HDAC1/2和NR2F1在LTR上結合的影響,并在HIV潛伏和激活的CEM-Bru細胞中研究RBBP4、 HDAC1、HDAC2和NR2F1在LTR上的結合變化。結果1.發(fā)現RBBP4在臨床HIV感染者中的表達明顯高于HIV治療成功者和正常對照(P0.01；P0.01)；在實驗室HIV感染的CEM-ss和TZM-bl細胞模型中,RBBP4 mRNA和蛋白的表達均明顯高于未感染的對照細胞,其結果與臨床發(fā)現一致；并進一步發(fā)現在CEM-ss和TZM-bl細胞模型中將RBBP4干擾表達后再感染HIV,發(fā)現HIV病毒顆粒的產生明顯增加；2.過表達RBBP4可減少上清HIV病毒顆粒、病毒載量、單間接轉錄本、多剪接轉錄本和未剪接轉錄本的產生；進一步的研究發(fā)現RBBP4可抑制HIV LTR啟動的本底轉錄以及NF-κB和Tat誘導的轉錄；RBBP4可分別與HIV LTR(-454～+181)上的NF-κB結合位點(-115～-75)和NR2F1結合位點(-345～-319)結合。3. RBBP4在HIV LTR上結合的變化能影響NR2F1、HDAC1/2在LTR上結合,即RBBP4在HIV LTR上的結合增加,NR2F1、HDAC1/2在LTR上的結合也增加,組蛋白乙酰化水平降低,最后導致HIV上清P24和病毒顆粒的產生減少；在HIV潛伏的CEM-Bru細胞中,RBBP4、NR2F1、HDAC1/2在HIV LTR的結合明顯高于在激活狀態(tài)下的結合；在潛伏細胞中將RBBP4干擾表達后能促使HIV起始轉錄本增加。結論1.首次發(fā)現RBBP4在未經治療的HIV感染者中表達升高,并在HIV感染的TZM-bl和CEM-ss細胞中發(fā)現RBBP4的表達升高,表明HIV感染可誘導RBBP4表達增加；2. RBBP4可抑制HIV LTR介導的本底轉錄；RBBP4可抑制由NF-κB和Tat誘導激活的HIV LTR轉錄；并能與LTR上的NF-κB結合位點(-115～-75)和NR2F1結合位點(-345～-319)結合；3. RBBP4通過影響轉錄因子NR2F1和組蛋白脫乙酰酶HDAC1/2在啟動子上的結合來抑制HIV的轉錄和病毒顆粒的產生,并與NR2F1和HDAC1/2共同維持HIV的潛伏狀態(tài)。
[Abstract]:The background by the human immunodeficiency virus (human immunodeficiency, virus, HIV) caused by AIDS is one of the most threatening infectious disease. Although antiretroviral treatment (highly active anti-retroviral therapy, HAART) can effectively inhibit the replication of the HIV virus and the virus load to reduce the detection limit, but because of latent HIV the library exists, resulting in HIV cannot be completely eliminated, so the latent HIV library is considered to be.HIV the last barrier completely cleared from the body of HIV latency is essentially a reversible transcription silence state, this state and maintenance involves a variety of host transcriptional inhibitor and epigenetic modification combined with in front of the HIV complex. Virus long terminal repeat 5'LTR (long terminal repeat) on the inhibition of host transcription factor and recruitment of histone deacetylase HDACs (histo NE deacetylase) and other epigenetic transcriptional silencing complex in HIV and latent building plays an important role in maintaining. Retinoblastoma binding protein RBBP4 (retinoblastoma binding protein 4), as a histone chaperone, through epigenetic modification and HDACs complex synergistic effect, can participate in the transcriptional silencing of genes. But as RBBP4 molecules are closely associated with the HDACs, it has not been reported in HIV transcription and the latent role. Therefore, this paper studies RBBP4 in HIV transcription and latent in effect and mechanism. To study on 1. RBBP4 in normal controls, PBMC peripheral blood mononuclear cells and the clinical treatment of HIV infection outside (peripheral blood mononuclear cells) expression, and in the Laboratory of CEM-ss HIV infection and TZM-bl cell model on the expression of RBBP4 to test the expression of 2. RBBP4; the change of research The effects of HIV, HIV and LTR in RBBP4 to the study of transcription of HIV transcription regulation and RBBP4 with HIV LTR promoter binding; a further 3. in HIV infection and a potential mechanism model of RBBP4 on HIV transcription and latent function, provide experimental data and scientific basis for the removal of the latent reservoir HIV methods 1.. Using fluorescence quantitative PCR detection of RBBP4 molecules in clinical HIV infection (30 cases), HIV treatment (30 cases) and healthy controls (n = 30). The expression of PBMCs and CEM-ss in the laboratory of HIV infection and TZM-bl cell model, using fluorescence quantitative PCR, Western and blot immunofluorescence technique. The expression changes of RBBP4 were qualitative and quantitative detection of.2. by dual luciferase reporter assay HIV LTR promoter firefly luciferase activity to transcription, transcription of RBBP4 promoter of HIV LTR; the use of gel migration Electrophoretic mobility shift assay (shift experiment, EMSA RBBP4 and HIV LTR NF- of kappa B (-115 ~ -75) and NR2F1 (-345--319) combined with.3. in the cell model of 293T cells transfected with pNL4-3 loci, using chromatin immunoprecipitation (chrmatin immunoprecipitation analysis, ChIP) the influence of RBBP4 on HDAC1/2 and NR2F1 in the LTR with the HDAC1 and RBBP4 in the HIV study, latent and activated CEM-Bru cells, HDAC2 and NR2F1 in combination with the change of LTR. The results showed the expression of RBBP4 in 1. clinical HIV infection were significantly higher than in HIV treatment and normal control (P0.01; P0.01); in the laboratory of CEM-ss HIV infection the TZM-bl cell model, the expression of RBBP4 protein and mRNA were significantly higher than that of uninfected control cells, the result is consistent with clinical findings; and further found in CEM-ss and TZM-bl cell model, RBBP4 interference HIV expression after infection, HIV virus particles produced significantly increased 2.; overexpression of RBBP4 can reduce the supernatant of HIV virus particles, viral load, single connected transcripts, multiple splicing transcripts and spliced transcripts did not produce; further study found that RBBP4 can inhibit the HIV LTR promoter of the basal transcription and NF- K B and Tat induced transcription; RBBP4 and HIV LTR respectively (-454 ~ +181) of the NF- B binding site (-115 ~ -75) and NR2F1 binding sites (-345 ~ -319) combined with.3. RBBP4 based on HIV LTR can affect NR2F1, HDAC1/2 in LTR with RBBP4 in HIV LTR combined with NR2F1, HDAC1/2 increased in LTR with increased histone acetylation levels decreased, finally leads to the generation of HIV supernatant of P24 and virus particles decreased; in HIV latent CEM-Bru cells, RBBP4, NR2F1, HDAC1/2 in HIV combined with LTR was significantly higher than that in the activated state The combination of HIV can promote the initiation of transcription; increase in latency in cell RBBP4 interference expression. Conclusion 1. we found that the expression of RBBP4 in untreated HIV infection increased, and in HIV infected TZM-bl and CEM-ss cells showed increased expression of RBBP4, suggesting that HIV infection can induce increased expression of RBBP4; 2. RBBP4 can inhibit HIV mediated LTR RBBP4 can inhibit the basal transcription; HIV LTR transcription induced by NF- kappa B and Tat activation; and binding sites and LTR NF- kappa B (-115 ~ -75) and NR2F1 binding sites (-345 ~ -319) combined with 3. RBBP4; through the influence of transcription factor NR2F1 and histone deacetylase HDAC1/2 promoter to inhibit transcription and HIV virus production, and NR2F1 and HDAC1/2 to maintain HIV latency.

【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R512.91

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