HIV-1基因表达的转录调控

高效抗逆转录病毒治疗（HAART）可以有效地抑制人类免疫缺陷病毒Ⅰ型（HIV-1）的复制及血浆病毒载量,延缓发病进程,改善、提高患者的生活质量和存活时间。但是,一旦停止治疗就会导致血浆病毒血症迅速反弹,HIV-1以原病毒的形式在静息记忆CD4⁺T等细胞中的持续存在是清除HIV-1的一个障碍。HIV-1基因转录的激活与阻抑决定了受感染细胞进入产毒性感染或潜伏感染。本文从原病毒整合位置与转录干扰、细胞转录因子与HIV-1启动子相互作用招募RNA聚合酶起始转录、转录的表观遗传调控和反式激活因子Tat及其相关蛋白促进转录延伸等方面探讨了HIV-1原病毒转录调控机制。     

Glutamate metabolism in HIV-1 infected macrophages: Role of HIV-1 Vpr

HIV-1 infected macrophages play a significant role in the neuropathogenesis of AIDS. HIV-1 viral protein R (Vpr) not only facilitates HIV-1 infection but also contribute to long-lived persistence in macrophages. Our previous studies using SILAC-based proteomic analysis showed that the expression of critical metabolic enzymes in the glycolytic pathway and tricarboxylic acid (TCA) cycle were altered in response to Vpr expression in macrophages. We hypothesized that Vpr-induced modulation of glycolysis and TCA cycle regulates glutamate metabolism and release in HIV-1 infected macrophages.

We assessed the amount of specific metabolites induced by Vpr and HIV-1 in macrophages at the intracellular and extracellular level in a time-dependent manner utilizing multiple reaction monitoring (MRM) targeted metabolomics. In addition, stable isotope-labeled glucose and an MRM targeted metabolomics assay were used to evaluate the de novo synthesis and release of glutamate in Vpr overexpressing macrophages and HIV-1 infected macrophages, throughout the metabolic flux of glycolytic pathway and TCA cycle activation.

The metabolic flux studies demonstrated an increase in glucose uptake, glutamate release and accumulation of α-ketoglutarate (α-KG) and glutamine in the extracellular milieu in Vpr expressing and HIV-1 infected macrophages. Interestingly, glutamate pools and other intracellular intermediates (glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), citrate, malate, α-KG, and glutamine) showed a decreased trend except for fumarate, in contrast to the glutamine accumulation observed in the extracellular space in Vpr overexpressing macrophages.

Our studies demonstrate that dysregulation of mitochondrial glutamate metabolism induced by Vpr in HIV-1 infected macrophages commonly seen, may contribute to neurodegeneration via excitotoxic mechanisms in the context of NeuroAIDS.     

辅调节因子在核受体基因表达调控中的作用

核受体 ( nuclear receptor)在增强或抑制基因转录时 ,需借助于诸多辅调节因子的协同作用 ,使调节更为精细、有效及特异 .辅调节因子 ( coregulator)可区分为辅激活因子 ( coactivator)和辅抑制因子 ( corepressor)两大类 ,均具有多种功能各异的蛋白质因子 ,分别汇聚于核受体上构成不同复合体 .它们的主要作用机理是 :( 1 )促使核小体中的组蛋白乙酰基化 ,导致与 DNA的结合松散 ;或脱乙酰基 ,而使组蛋白与 DNA的结合回复紧密状态 ,从而创造一个有利于转录或封闭转录的局部环境 ;( 2 )作用于通用转录因子及 RNA聚合酶 ,以激活转录或抑制转录 .     

Upregulation of survivin by HIV-1 Vpr

The human survivin gene belongs to the family of inhibitor of apoptosis proteins (IAP) and is involved in apoptosis inhibition and regulation of cell division. The survivin gene is the only member of the IAP family whose expression is known to be regulated through the cell cycle. Survivin expression reaches the highest levels during the G₂/M transition and then is rapidly degraded during the G₁ phase. Here we report that the human immunodeficiency virus type 1 (HIV-1) upregulates Survivin expression via survivin promoter transactivation. Vpr, an HIV-1 accessory protein that induces cell cycle arrest in G₂/M, is necessary and sufficient for this effect. Blocking Vpr-induced G₂/M arrest leads to elimination of the survivin promoter transactivation by Vpr. Our results suggest that Survivin may be actively involved in regulating cell viability during HIV-1 infection.     

选择性剪接在植物逆境相关基因表达调控中的作用

文章介绍了选择性剪接在信号转导分子、转录因子、剪接因子和抗逆功能蛋白等4个层面对与植物逆境胁迫相关基因表达调控的影响,以及植物逆境诱导的选择性剪接机制的研究进展。     

Mitochondrial membrane permeabilization by HIV-1 Vpr

The mitochondrion is a privileged target for apoptosis-modulatory proteins of viral origin. Thus, viral protein R (Vpr) can target mitochondria and induce apoptosis via a specific interaction with the permeability transition pore complex (PTPC). Vpr cooperates with the adenine nucleotide translocator (ANT) to form large conductance channels and to trigger all the hallmarks of mitochondrial membrane permeabilization (MMP). The Vpr/ANT interaction is direct, since it is abolished by the addition of a peptide corresponding to the Vpr binding site of ANT, ADP, ATP, or by Bcl-2. Accordingly, Vpr modulates MMP through direct structural and functional interactions with PTPC proteins.     

The Role of Vpr in HIV-1 Disease Progression is Independent of Its G2 Arrest Induction Function

Vpr (viral protein R) is a vital HIV-1 accessory protein with multiple functions in the viral life cycle, including nuclear import of preintegration complex, induction of apoptosis and G2 cell cycle arrest. The cell cycle perturbation activity of Vpr requires activation of the ATR (Ataxia-Telangiectasia and Rad3-related) pathway and the integrity of Vpr C-terminal motif that is crucial for chromatin binding. Recent studies also demonstrated Vpr as one of the viral factors that influence HIV disease progression, as mutations in Vpr were overrepresented in some cohorts of long-term nonprogressors (LTNP). The LTNP-associated mutations of Vpr are frequently observed in the C-terminal domain. This raises the question whether the LTNP phenotype of Vpr is the result of the loss its ability to induce G2 arrest. Here we report that the LTNP-associated mutants of Vpr function normally in the induction of G2 arrest. No defects in ATR activation and direct binding to chromatin are observed. These mutants also show similar levels of apoptosis induction as wild-type Vpr. These data differentiate the LTNP-associated mutations of Vpr with those defective in inducing G2 arrest. We propose that the G2 arrest function of Vpr is separated from the LTNP phenotype, and the role of Vpr in HIV disease progression may involve other functions of Vpr.     

Effects of HIV-1 Vpr on neuroinvasion and neuropathogenesis

Human immunodeficiency virus type I (HIV-1) infection leads to penetration of the central nervous system (CNS) in virtually all infected individuals and HIV-1-induced encephalopathy in a significant number of untreated patients. The molecular mechanisms by which HIV-1 enters the CNS and yields CNS dysfunction are still unclear. Our laboratories and others have begun to explore the direct effects of prioritized HIV-1-specific proteins on diverse human CNS cell types. One of these proteins, the accessory HIV-1 protein Vpr, is a critical moiety in these studies, and will be discussed in this article.     

Effect of HIV-1 Vpr on cell cycle regulators

Cell cycle is one of the most complex processes in the life of a dividing cell. It involves numerous regulatory proteins, which direct the cell through a specific sequence of events for the production of two daughter cells. Cyclin-dependent kinases (cdks), which complex with the cyclin proteins, are the main players in the cell cycle. They can regulate the progression of the cells through different stages regulated by several proteins including p53, p21(WAF1), p19, p16, and cdc25. Downstream targets of cyclin-cdk complexes include pRB and E2F. A cell cycle can be altered to the advantage of many viral agents, most notably polyomaviruses, papillomaviruses, adenoviruses, and retroviruses. In addition, viral protein R (Vpr) is a protein encoded by the human immunodeficiency virus type 1 (HIV-1). HIV-1, the causative agent of acquired immunodeficiency syndrome (AIDS), is a member of the lentivirus class of retroviruses. This accessory protein plays an important role in the regulation of the cell cycle by causing G(2) arrest and affecting cell cycle regulators. Vpr prevents infected cells from proliferating, and collaborates with the matrix protein (MA) to enable HIV-1 to enter the nucleus of nondividing cells. Studies from different labs including ours showed that Vpr affects the functions of cell cycle proteins, including p53 and p21(WAF1). Thus, the replication of HIV-1, and ultimately its pathogenesis, are intrinsically tied to cell-cycle control.     

siRNA抑制HIV-1基因表达的研究

RNA干扰(RNAinterfering,RNAi)现象是动植物体内的一种序列特异性的、转录后基因沉默的过程.在哺乳动物细胞中,19～25个核苷酸长短的双链siRNA(smallinterferingRNA)可有效地抑制基因表达.利用pBS/H1SP载体,它表达的双链RNA的转录产物可以用来抑制特异性HIV-1基因的表达.在siRNA实验中,为了比较由H1启动子转录的siRNA在细胞内的作用,使用以绿色荧光蛋白(EGFP)为报告基因的载体——pEGFP-C1质粒,在荧光显微镜下,很容易地看到EGFP在细胞中表达.将HIV-1siRNA表达载体与表达相应EGFP-HIV融合基因的质粒,共转染人胚肾293细胞,结果表明,和对照质控载体相比,转染了pHIV-siRNA质粒的细胞中EGFP-HIV的表达得到显著抑制.通过该途径,筛选出能抑制HIV-1基因表达的有效siRNA.此外,还在同一载体上表达两种或三种siRNA,分别针对不同的HIV基因,获得了良好的抑制效果.     

植物叶绿体基因组基因表达调控的研究

叶绿体基因的表达在许多方面与原核基因表达相似,所以最早的理论认为叶绿体基因的表达与原核相似,是在转录起始水平上的调控,进一步的研究认为叶绿体基因表达调控是在不同水平上进行的如：转录水平的调节、转录后调节与修饰、翻译和翻译后修饰等。