HIV-1潜伏感染相关分子筛查与转录调控

发布时间：2018-05-09 12:05

本文选题：HIV + LTR　；参考：《浙江大学》2013年博士论文

【摘要】：自高效抗逆转录病毒治疗(HAART)广泛应用于临床以来,抗病毒治疗能显著增强HIV-1感染患者的生存期,但是仍未能彻底清除感染。治疗患者体内,HIV-1在静息T细胞等中呈持续性的转录非活跃状态,并在T细胞活化后重新激活。这种潜伏状态使得病毒即使处于HARRT治疗下也长期存在,无法根治。潜伏通常描述成一种病毒可逆的非生产性感染状态,通常无子代毒粒释放。研究发现,HIV-1入侵宿主细胞后急性感染期即开始建立潜伏库。实际的潜伏状态也可以出现基线转录下少量的HIV-1转录、不完整性合成等诸多情况。相对于总体的受染细胞,HIV-1突破宿主防御建立潜伏的频率稀少。但其赋予HIV-1逃逸免疫监视、HAART攻击、可随细胞遗传并重新激活的持续性感染特征,是目前抗病毒清除策略的最大障碍。对于HIV-1潜伏相关细胞分子的系统挖掘,是进一步揭示潜伏机制的前提,也是为今后根治提供科学依据,具有很大的应用价值。国外系列研究已显示微小RNA (miRNA)调节HIV-1的生活周期,但尚无对HIV-1潜伏的miRNA组学报道。本论文以体外HIV-1潜伏感染细胞株为潜伏模型,以HIV-1持续复制细胞及空白细胞为对照,经生物芯片技术进行]miRNA与mRNA的表达谱研究与鉴定。HIV-1潜伏细胞株相比于复制株具不同的miRNA及mRNA转录模式。潜伏感染中差异表达的miRNA不仅有诸如miR-223等已有报道的HIV-1感染相关miRNA,亦发现此前未经报道的miR-363等分子。经基因本体语义(GO)分析,我们发现RNA代谢、核运输相关基因、转录调控是HIV-1潜伏细胞的功能关键,而在HIV-1复制中更多的与细胞周期、凋亡等有关。靶分子通路分析显示潜伏感染中的细胞外基质通路富集,这是潜伏信号源于胞外信息启动的一条重要的线索。miRNA、转录因子等均是基因表达的重要调节子,通过大量的数据分析及汇集,我们进一步建立融合miRNA、mRNA(包括转录因子)的调控网络,通过潜伏与复制状态间的最显著变化挖掘重要的基因调控节点。我们发现miR-223、miR-363等miRNA与调节HIV-1感染相关,转录调节与细胞分化、胞外信号等是潜伏向复制状态变迁的最大特征之一临床长期不进展(LTNP)患者表现出自发性抑制病毒及低残余病毒血症特征。这种表型与HIV-1潜伏类似。同时,受迄今HIV-1潜伏细胞无生物标记可循、无体内模型所限,以LTNP为背景与HIV-1体外模型比较,我们期待发掘更特异性的分子分歧。在高通量芯片数据库内发掘所有的HIV-1潜伏(25例)及LTNP(22例)表达谱,并整合所有表达谱数据经主方差成分分析与启发式聚类评估。结果发现潜伏与LTNP出现456条差异基因。以贝叶斯推断法建立各感染状态下差异基因的调控网络,研究显示不同感染状态下,基因调节网络出现重构。通过中心参数为特征的结构分析,我们亦证明HIV-1感染相关基因的高连接特征。以之为基础鉴定出,HIV-1潜伏中KPNA2与ATP5G3是关键基因,介导RNA代谢与核运输。我们进一步在体外细胞中验证KPNA2、ATP5G3的差异表达及在潜伏细胞中的高表达特性。 HIV-1的潜伏具体发生于细胞核内染色质微环境中,其前提是HIV-1启动子的转录沉默。陆续有潜伏机制的提出,均围绕于转录受抑而展开,诸如不同的转录因子、转录干扰、相关的表观修饰等机制。前述结果也已说明RNA代谢、转录调节是HIV-1潜伏的重要特征。为探查相关分子对HIV-1的转录调控,我们建立起HIV-1启动子的报告系统。以重组PCR技术快速建立HIV-1启动子的各种截短突变体,应用于宿主分子的响应元件定位。RbBP4分子是多个染色质相关蛋白复合体的成员,是一贯穿染色质代谢全过程的协转录分子。过表达与敲低后的荧光素酶报告实验发现,RbBP4分子在在HIV-1Tat依赖方式的转录中起下调作用。综上所述,HIV-1潜伏的机制是多重因素的复合影响。通过高通量的筛选,我们鉴定出一系列的候选分子(包括miRNA、mRNA)及其联系。同时,为贴近体内环境,采用与HIV-1潜伏最为接近的临床LTNP患者作为对照,将HIV-1潜伏的功能关键点逐步缩小于转录调节等范围；这一层次仍将作为今后挖掘潜伏机制的切入点。建立起HIV-1转录调控筛选报告模型,可用于今后一系列宿主分子的筛选。发表相关SCI论文4篇。本研究得出以下结论： 1、首次在HIV-1体外潜伏细胞中绘制miRNA表达谱,合并mRNA表达谱,发现HIV-1潜伏与复制具不同的转录模式。建立自主的miRNA-mRNA联锁分析方法,发现HIV-1潜伏与复制变迁的重要调节点。验证出miR-223、miR-363等是潜伏相关的miRNA。 2、进行细胞水平HIV-1潜伏与体内LTNP样本的比较。通过基因调节网络,发现不同感染状态下的调控重构。KPNA2与ATP5G3等是HIV-1潜伏网络的关键基因。 3、建立快速突变技术,构建HIV-1启动子转录调控筛查与定位系统。RbBP4的筛查显示双相特征,提示细胞染色质微环境的依赖性。 HIV-1的潜伏是当前HARRT治疗的盲点。本研究构建HIV-1感染的基底数据,以系统生物学方式挖掘潜伏为主要导向的关键基因及其调控。通过转录活性的筛选,为进一步的HIV-1潜伏机制研究及抗潜伏设计打下基础。
[Abstract]:Since HAART has been widely used in clinical practice, antiviral therapy can significantly enhance the survival of patients with HIV-1 infection, but it has not been able to completely remove the infection. In the treatment of patients, HIV-1 has a persistent non active state of transcription in resting T cells, and reactivates after the activation of T cells. Even if the virus is under HARRT treatment, it will persist for a long time.
The latent state is usually described as a non productive infection state of a virus reversible, usually without the release of the progeny. The study found that HIV-1 invaded the host cells after the acute infection period began to establish the latent library. The actual latent state could also occur in a small amount of HIV-1 transcriptional, incomplete synthesis, and so on. In infected cells, HIV-1 breaks through the host defense to establish a rare latent frequency. But it gives HIV-1 escape immune surveillance, HAART attack, and can be inherited and reactivated by cell inheritance and persistent infection characteristics, which is the biggest obstacle to the current antiviral clearance strategy. System mining for HIV-1 latent cell molecules is to further reveal the latent mechanism. The premise also provides a scientific basis for radical treatment in the future. It has great application value.
A series of foreign studies have shown that small RNA (miRNA) regulates the life cycle of HIV-1, but there is no miRNA group report on HIV-1 latent. This paper takes the latent model of HIV-1 latent infection cells in vitro as the latent model, and takes the HIV-1 continuous replicating and blank cells as the control, and studies the expression of]miRNA and mRNA by biochip technique and identifies.HIV-1. The latent cell lines have different miRNA and mRNA transcriptional patterns compared to the replicas. The differentially expressed miRNA in latent infection not only contains miRNA related to HIV-1 infection, such as miR-223, but also previously unreported miR-363 and other molecules. Through genetic semantic (GO) analysis, we found RNA metabolism, nuclear transport related genes, and transcription. Regulation is the key to the function of HIV-1 latent cells, and more in HIV-1 replication is related to cell cycle and apoptosis. Target molecular pathway analysis shows that the extracellular matrix pathway in latent infection is enriched, which is an important clue that the latent signal is derived from the initiation of extracellular information, and the transcription factor is an important regulation of gene expression. Through a large number of data analysis and aggregation, we further establish a regulatory network that integrates miRNA, mRNA (including transcription factors), and mining important gene regulation nodes through the most significant changes between latent and replicative states. We find that miR-223, miR-363, and other miRNA are associated with HIV-1 infection, transcription regulation and cell differentiation, and extracellular signal And so on is one of the biggest characteristics of latent change to copying state.
The clinical long-term non progression (LTNP) patients showed the characteristics of self induced virus and low residual viremia. This phenotype was similar to that of HIV-1 latent. At the same time, the HIV-1 latent cells had no biomarkers to follow, and no body model was limited. Compared with the HIV-1 extracorporeal model, we expect to explore more specific molecular differences with the LTNP as the background. The expression profiles of all HIV-1 latent (25 cases) and LTNP (22 cases) were discovered in the flux chip database, and all the expression profiles were integrated by the principal variance component analysis and the heuristic clustering evaluation. The results showed that 456 differentially genes were found in the latent and LTNP. In the same infection state, the gene regulatory network was reconstructed. Through the structural analysis characterized by central parameters, we also demonstrated the high connection characteristics of HIV-1 related genes. Based on it, it was identified that KPNA2 and ATP5G3 were the key genes in HIV-1 incubation, mediating RNA metabolism and nuclear transport. We further verified KPNA2, ATP5G3 in vitro cells. Differential expression and high expression in latent cells.
The latent period of HIV-1 occurs specifically in the chromatin microenvironment of the nucleus. The premise is the transcriptional silence of the HIV-1 promoter. The latent mechanism is put forward in succession around the inhibition of transcription, such as different transcription factors, transcriptional interference, and related apparent modification. The previous results also indicate that the metabolism of RNA, and the transcription regulation is HIV-1 latent An important feature of volt. In order to detect the transcriptional regulation of HIV-1 by related molecules, we have established a reporting system for the HIV-1 promoter. A variety of truncated mutants of the HIV-1 promoter are quickly established by recombinant PCR technology. The.RbBP4 molecule of the host molecule's response element is a member of multiple chromatin related protein complexes and is consistently dyed. The co transcriptional molecules in the whole process of mass metabolism. Over expression and after knockout luciferase report experiments found that RbBP4 molecules play a downregulation role in HIV-1Tat dependent transcription.
To sum up, HIV-1's latent mechanism is a complex effect of multiple factors. Through high throughput screening, we identified a series of candidate molecules (including miRNA, mRNA) and their connections. At the same time, the key points of the latent function of HIV-1 were gradually reduced to a comparison of the clinical LTNP patients closest to the HIV-1. Transcriptional regulation and so on; this level will still be the breakthrough point for the potential mechanism in the future. The HIV-1 transcriptional regulation screening report model can be established for the screening of a series of host molecules in the future. 4 articles related to SCI are published.
This study draws the following conclusions:
1, for the first time, miRNA expression profiles were plotted in HIV-1 extracorporeal cells, and mRNA expression profiles were combined. The latent and replication modes of HIV-1 were found to be different. The independent miRNA-mRNA interlocking analysis method was established to find the important adjustment point of HIV-1 latent and replication change. It was proved that miR-223, miR-363 and so on were latent related miRNA..
2, compare the latent HIV-1 latency with the LTNP samples in the body. Through the gene regulation network, it is found that the key genes of the HIV-1 latent network are the regulation of the reconfiguration of.KPNA2 and ATP5G3 under different infection states.
3, rapid mutation technology was established, and the screening and positioning system of HIV-1 promoter transcriptional screening and positioning system.RbBP4 screening showed biphasic characteristics, suggesting the dependence of cell chromatin microenvironment.
The latent of HIV-1 is the blind spot of current HARRT therapy. This study constructs the base data of HIV-1 infection, excavates the key genes and their regulation in a systematic biological way. Through the screening of transcriptional activity, it lays the foundation for further study of the latent mechanism of HIV-1 and the anti latent design.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R512.91

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