RBBP4在HIV转录与潜伏中的作用及机制研究

发布时间：2018-03-02 02:22

本文关键词： RBBP4 HIV LTR HDAC NR2F1　出处：《浙江大学》2016年博士论文　论文类型：学位论文

【摘要】：背景由人类免疫缺陷病毒(human immunodeficiency virus, HIV)所导致的艾滋病是目前最具有威胁性的传染病之一。虽然高效的抗逆转录病毒治疗(highly active anti-retroviral therapy, HAART)能有效的抑制HIV病毒的复制并使病毒载量降低至检测下限,但是由于HIV潜伏库的存在,导致HIV不能被彻底清除,因此当前HIV潜伏库被认为是从体内彻底清除HIV的最后壁垒。HIV潜伏状态本质上是一种可逆的转录沉默状态,这种状态的产生和维持涉及多种宿主转录抑制因子及表观修饰复合体。结合在HIV前病毒基因组长末端重复序列5'LTR (long terminal repeat)上的宿主转录抑制因子及其募集的组蛋白脱乙酰化酶HDACs (histone deacetylase)等表观修饰复合体在HIV的转录沉默和潜伏的建立维持中起着重要作用。视网膜母细胞瘤结合蛋白RBBP4 (retinoblastoma binding protein 4),作为组蛋白分子伴侣,通过与HDACs等表观修饰复合体协同作用,可参与基因的转录沉默。但作为与HDACs密切关联的RBBP4分子,它在HIV的转录以及潜伏中的作用还未见报道。因此,本文将对RBBP4在HIV转录和潜伏中的作用及机制进行研究。目的1.研究RBBP4在正常对照者、临床HIV感染者和治疗者外周血单个核细胞PBMC (peripheral blood mononuclear cells)中的表达变化,并在实验室HIV感染的CEM-ss和TZM-bl细胞模型上对RBBP4的表达变化进行验证；2.研究RBBP4表达的改变对HIV产生的作用,并在HIV的转录环节研究RBBP4对HIV LTR介导的转录调控以及RBBP4与HIV LTR启动子的结合；3.进一步在HIV的感染和潜伏模型中研究RBBP4对HIV转录和潜伏作用的机制,为HIV潜伏库的清除提供实验数据及科学依据。方法1.首先利用荧光定量PCR检测RBBP4分子在临床HIV感染者(30例)、HIV治疗者(30例)和健康对照(30例)的PBMCs中的表达变化。并在实验室HIV感染的CEM-ss和TZM-bl细胞模型上,利用荧光定量PCR、Western blot和免疫荧光技术,对RBBP4的表达变化进行定性和定量检测。2.通过双荧光素酶报告系统检测HIV LTR启动转录的萤火虫荧光素酶活性,来研究RBBP4对HIV LTR启动子的转录调控；利用凝胶迁移实验(electrophoretic mobility shift assay, EMSA研究RBBP4与HIV LTR上NF-κB(-115～-75)和NR2F1(-345--319)结合位点的结合。3.在293T细胞与pNL4-3转染的细胞模型中,利用染色质免疫共沉淀(chrmatin immunoprecipitation analysis, ChIP)研究RBBP4对HDAC1/2和NR2F1在LTR上结合的影响,并在HIV潜伏和激活的CEM-Bru细胞中研究RBBP4、 HDAC1、HDAC2和NR2F1在LTR上的结合变化。结果1.发现RBBP4在临床HIV感染者中的表达明显高于HIV治疗成功者和正常对照(P0.01；P0.01)；在实验室HIV感染的CEM-ss和TZM-bl细胞模型中,RBBP4 mRNA和蛋白的表达均明显高于未感染的对照细胞,其结果与临床发现一致；并进一步发现在CEM-ss和TZM-bl细胞模型中将RBBP4干扰表达后再感染HIV,发现HIV病毒颗粒的产生明显增加；2.过表达RBBP4可减少上清HIV病毒颗粒、病毒载量、单间接转录本、多剪接转录本和未剪接转录本的产生；进一步的研究发现RBBP4可抑制HIV LTR启动的本底转录以及NF-κB和Tat诱导的转录；RBBP4可分别与HIV LTR(-454～+181)上的NF-κB结合位点(-115～-75)和NR2F1结合位点(-345～-319)结合。3. RBBP4在HIV LTR上结合的变化能影响NR2F1、HDAC1/2在LTR上结合,即RBBP4在HIV LTR上的结合增加,NR2F1、HDAC1/2在LTR上的结合也增加,组蛋白乙酰化水平降低,最后导致HIV上清P24和病毒颗粒的产生减少；在HIV潜伏的CEM-Bru细胞中,RBBP4、NR2F1、HDAC1/2在HIV LTR的结合明显高于在激活状态下的结合；在潜伏细胞中将RBBP4干扰表达后能促使HIV起始转录本增加。结论1.首次发现RBBP4在未经治疗的HIV感染者中表达升高,并在HIV感染的TZM-bl和CEM-ss细胞中发现RBBP4的表达升高,表明HIV感染可诱导RBBP4表达增加；2. RBBP4可抑制HIV LTR介导的本底转录；RBBP4可抑制由NF-κB和Tat诱导激活的HIV LTR转录；并能与LTR上的NF-κB结合位点(-115～-75)和NR2F1结合位点(-345～-319)结合；3. RBBP4通过影响转录因子NR2F1和组蛋白脱乙酰酶HDAC1/2在启动子上的结合来抑制HIV的转录和病毒颗粒的产生,并与NR2F1和HDAC1/2共同维持HIV的潜伏状态。
[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.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R512.91

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