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为了抑制Tat蛋白的反式激活作用,在细胞内大量表达外源TARRNA使其与Tat蛋白结合,从而竞争性抑制其与HIV-1LTR的TARRNA元件结合.构建了以HIV-1LTRYL158(-158~+180)为启动子,分别含有4,8和15个拷贝的TAR-CoreRNA诱饵(decoy)表达质粒;以荧光酶基因为报告基因,检测了瞬时共转染体系中含不同拷贝数的TAR-CoreDNA转录产物对Tat蛋白反式激活作用的影响.结果证明,TAR-CoreRNA诱饵对Tat蛋白活性具有很强的抑制作用,其抑制程度与TAR-CoreDNA串联体的拷贝数有关. 相似文献
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Aarthi Narayanan Sergey Iordanskiy Ravi Das Rachel Van Duyne Steven Santos Elizabeth Jaworski Irene Guendel Gavin Sampey Elizabeth Dalby Maria Iglesias-Ussel Anastas Popratiloff Ramin Hakami Kylene Kehn-Hall Mary Young Caroline Subra Caroline Gilbert Charles Bailey Fabio Romerio Fatah Kashanchi 《The Journal of biological chemistry》2013,288(27):20014-20033
Exosomes are nano-sized vesicles produced by healthy and virus-infected cells. Exosomes derived from infected cells have been shown to contain viral microRNAs (miRNAs). HIV-1 encodes its own miRNAs that regulate viral and host gene expression. The most abundant HIV-1-derived miRNA, first reported by us and later by others using deep sequencing, is the trans-activation response element (TAR) miRNA. In this study, we demonstrate the presence of TAR RNA in exosomes from cell culture supernatants of HIV-1-infected cells and patient sera. TAR miRNA was not in Ago2 complexes outside the exosomes but enclosed within the exosomes. We detected the host miRNA machinery proteins Dicer and Drosha in exosomes from infected cells. We report that transport of TAR RNA from the nucleus into exosomes is a CRM1 (chromosome region maintenance 1)-dependent active process. Prior exposure of naive cells to exosomes from infected cells increased susceptibility of the recipient cells to HIV-1 infection. Exosomal TAR RNA down-regulated apoptosis by lowering Bim and Cdk9 proteins in recipient cells. We found 104–106 copies/ml TAR RNA in exosomes derived from infected culture supernatants and 103 copies/ml TAR RNA in the serum exosomes of highly active antiretroviral therapy-treated patients or long term nonprogressors. Taken together, our experiments demonstrated that HIV-1-infected cells produced exosomes that are uniquely characterized by their proteomic and RNA profiles that may contribute to disease pathology in AIDS. 相似文献
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研究了具有抗HIV-1活性的大环多胺类化合物与RNA的识别作用,以及其对cos-7细胞凋亡的影响,以进一步探讨其抗HIV-1的作用机理.实验采用琼脂糖凝胶电泳方法, 观察化合物与RNA的识别作用;通过流式细胞计数法探讨其对cos-7细胞凋亡的影响;运用计算机分子模型, 从理论上Docking计算化合物与TAR RNA结合的可能性.结果表明, 大环多胺类化合物MP-1、MP-2和MP-3不仅具有断裂RNA的作用,并可抑制Tat-RNA的相互作用, 还可影响cos-7细胞亚二倍体的含量; 理论化学计算数据与实验结果基本一致.这一结果提示化合物的抗HIV-1活性可能通过作用于病毒基因组RNA而发挥作用,是多靶作用的结果. 相似文献
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研究了具有抗HIV-1活性的大环多胺类化合物与RNA的识别作用,以及其对cos-7细胞凋亡的影响,以进一步探讨其抗HIV-1的作用机理。实验采用琼脂糖凝胶电泳方法。观察化合物与RNA的识别作用。通过流式细胞计数法探讨其对cos-7细胞凋亡的影响。运用计算机分子模型,从理论上Docking计算化合物与TAR RNA结合的可能性。结果表明,大环多胺类化合物MP-1、MP-2和MP-3不仅具有断裂RNA的作用,并可抑制Tat-RNA的相互作用,还可影响cos-7细胞亚二倍体的含量;理论化学计算数据与实验结果基本一致,这一结果提示合物的抗HIV-1活性可能通过作用于病毒基因组RNA而发挥作用,是多靶作用的结果。 相似文献
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Analysis of trans-acting response decoy RNA-mediated inhibition of human immunodeficiency virus type 1 transactivation. 总被引:18,自引:12,他引:6
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Overexpression of trans-acting response element (TAR)-containing sequences (TAR decoys) in CEM SS cells renders cells resistant to human immunodeficiency type 1 (HIV-1) replication. Mutagenesis of TAR was used to investigate the molecular mechanism underlying the observed inhibition. A nucleotide change which disrupts the stem structure of TAR or sequence alterations in the loop abolish the ability of the corresponding TAR decoy RNAs to inhibit HIV replication. A compensatory mutation which restores the stem structure also restores TAR decoy RNA function. Synthesis of viral RNA is drastically reduced in cells expressing a functional TAR decoy RNA, but it is unaffected in cells expressing a mutant form of TAR decoy RNA. It is therefore concluded that overexpression of TAR-containing sequences in CEM SS cells interferes with the process of Tat-mediated transactivation of viral gene expression. However, the phenotype of several mutations suggests that TAR decoy RNA does not inhibit HIV-1 gene expression by simply sequestering Tat but rather does so by sequestering a transactivation protein complex, implying that transactivation requires the cooperative binding of both Tat and a loop-binding cellular factor(s) to TAR. Expression of wild-type or mutant forms of TAR had no discernible effects on cell viability, thus reducing concerns about using TAR decoy RNAs as part of an intracellular immunization protocol for the treatment of AIDS. 相似文献
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Inhibition of HIV-LTR gene expression by oligonucleotides targeted to the TAR element. 总被引:15,自引:4,他引:11
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T Vickers B F Baker P D Cook M Zounes R W Buckheit Jr J Germany D J Ecker 《Nucleic acids research》1991,19(12):3359-3368
All human immunodeficiency virus mRNAs contain a sequence known as TAR (trans-activating responsive sequence). The TAR element forms a stable RNA stem-loop structure which binds the HIV tat (trans-activator) protein and mediates increased viral gene expression. In principle, molecules which bind to the TAR RNA structure would inhibit trans-activation by perturbing the native RNA secondary structure. We have constructed a series of phosphodiester and phosphorothioate antisense oligonucleotides which specifically bind to the HIV TAR element. Specific binding to the TAR element was demonstrated in vitro with enzymatically synthesized TAR RNA. The TAR-directed phosphorothioates inhibited trans-activation in a sequence-dependent fashion in a cell culture model using an HIV LTR/human placental alkaline phosphatase gene fusion and tat protein supplied in trans. The molecules also inhibited HIV replication in both acute and chronically infected viral assays, but without sequence specificity. We have constructed a series of vectors consisting of the MMTV promoter and 5'-untranslated region of four different mRNAs, including the TAR region, to study the effect of TAR on gene expression in heterologous systems. The results suggest that, in the absence of the HIV LTR, the TAR element has a repressive effect on gene expression, which is relieved by tat. 相似文献
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A Daher M Longuet D Dorin F Bois E Segeral S Bannwarth P L Battisti D F Purcell R Benarous C Vaquero E F Meurs A Gatignol 《The Journal of biological chemistry》2001,276(36):33899-33905
Trans-activation response (TAR) RNA-binding protein (TRBP) is a cellular protein that binds to the human immunodeficiency virus-1 (HIV-1) TAR element RNA. It has two double-stranded RNA binding domains (dsRBDs), but only one is functional for TAR binding. TRBP interacts with the interferon-induced protein kinase R (PKR) and inhibits its activity. We used the yeast two-hybrid assay to map the interaction sites between the two proteins. We show that TRBP and PKR-N (178 first amino acids of PKR) interact with PKR wild type and inhibit the PKR-induced yeast growth defect in this assay. We characterized two independent PKR-binding sites in TRBP. These sites are located in each dsRBD in TRBP, indicating that PKR-TRBP interaction does not require the RNA binding activity present only in dsRBD2. TRBP and its fragments that interact with PKR reverse the PKR-induced suppression of HIV-1 long terminal repeat expression. In addition, TRBP activates the HIV-1 long terminal repeat expression to a larger extent than the addition of each domain. These data suggest that TRBP activates gene expression in PKR-dependent and PKR-independent manners. 相似文献
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A mammalian gene expression vector based on cytomegalovirus (CMV)enhancer/promoter (CMVe/p) for the regulation of gene expression
was further optimized by adding oriP elements derived from Epstein-Barr virus (EBV) and the Tat/TAR transactivation axisfrom human immunodeficiency virus type
1 (HIV-1). Using the Tat/TAR-oriP expression vector, a transient transfection system was optimized for an extended culture period to produce large amounts
of secreted IL-2SA (an IL-2 mutein) in HKB11 cells. We observed a 4-fold increase in IL-2SA expression in cells transfected
with vectors containing the HIV-1 transactivation axis (Tat/TAR) or oriP elements alone when compared to cells transfected with the control vector having a CMVe/p. Cells transfected with expression
vectors equipped with both oriP and Tat/TAR showed an 18-fold increase in IL-2SA expression. This transient transfection system maintained high secretion
of IL-2SA for a period of 10-day with no appreciable loss in expression. We demonstrate that during this 10-day culture period,
it was possible to produce 1–100 mg of proteins using 500 μg of plasmid DNA.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
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Activation of double-stranded RNA-dependent kinase (dsl) by the TAR region of HIV-1 mRNA: a novel translational control mechanism 总被引:30,自引:0,他引:30
All mRNAs of human immunodeficiency virus 1 (HIV-1) contain in their 5' untranslated region a sequence termed TAR that responds to trans-activation by the tat (trans-activating) protein. This RNA sequence assumes a stable secondary structure, and its cap structure is relatively inaccessible. Here we report that these structural properties of the TAR sequence underlie the ability of TAR to inhibit in trans the translation of other mRNAs. This mechanism of translation inhibition involves the activation of the double-stranded RNA-dependent kinase (dsl), which in turn phosphorylates the protein synthesis initiation factor 2 (eIF-2). Mutations in the TAR region that diminish the stability of the secondary structure cause a significant reduction in the trans-inhibition. A similar reduction in the dsl activation occurs when TAR is placed further downstream of the cap structure. This is a clear demonstration of a specific naturally occurring mRNA sequence that can activate dsl. We suggest a novel translational regulatory mechanism that interdigitates the activities of eIF-2 and eIF-4F. 相似文献
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Shuguang Chen Ran Chen Meizi He Ruifang Pang Zhiwu Tan Ming Yang 《Bioorganic & medicinal chemistry》2009,17(5):1948-1956
Thirty-two quinoline derivatives were designed and synthesized as HIV-1 Tat–TAR interaction inhibitors. All the compounds showed high antiviral activities in inhibiting the formation of SIV-induced syncytium in CEM174 cells. Nine of them with low cytotoxicities were evaluated by Tat dependent HIV-1 LTR-driven CAT gene expression colorimetric enzyme assay in human 293T cells, indicating effective inhibitory activities of blocking the Tat–TAR interaction. Molecular modeling experiments indicated that these compounds may inhibit Tat–TAR interaction by binding to Tat protein instead of TAR RNA. 相似文献