共查询到20条相似文献,搜索用时 31 毫秒
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Jan Snášel Ivan Rosenberg Ondřej Pačes 《Journal of enzyme inhibition and medicinal chemistry》2013,28(1):241-246
Retroviral integrase participates in two catalytic reactions, which require interactions with the two ends of the viral DNA in the 3′processing reaction, and with a targeted host DNA in the strand transfer reaction. The 3′-hydroxyl group of 2′-deoxyadenosine resulting from the specific removing of GT dinucleotide from the viral DNA in the processing reaction provides the attachment site for the host DNA in a transesterification reaction. We synthesized oligonucleotides (ONs) of various lengths that mimic the processed HIV-1 U5 terminus of the proviral long terminal repeat (LTR) and are ended by 2′-deoxyadenosine containing a 3′-O-phosphonomethyl group. The duplex stability of phosphonomethyl ONs was increased by covalent linkage of the modified strand with its complementary strand by a triethylene glycol loop (TEG). Modified ONs containing up to 10 bases inhibited in vitro the strand transfer reaction catalyzed by HIV-1 integrase at nanomolar concentrations. 相似文献
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Brown D Arzumanov AA Turner JJ Stetsenko DA Lever AM Gait MJ 《Nucleosides, nucleotides & nucleic acids》2005,24(5-7):393-396
Mixmer oligonucleotides consisting of residues of both 2'-O-methylnucleosides (OMe) and locked nucleic acids (LNA) were designed targeting two stem-loops in the 5'-UTR of HIV-1 RNA, the transactivation response region (TAR), which is the site of binding of the Tat protein, and the SL3 loop, which is the primary packaging element that binds the Gag polyprotein. These oligonucleotides were found to inhibit syncitia formation dose- and sequence-dependently when delivered to HeLa T4 LTR beta-Gal cells and subsequently infected with HIV-1. 相似文献
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Jing N Marchand C Guan Y Liu J Pallansch L Lackman-Smith C De Clercq E Pommier Y 《DNA and cell biology》2001,20(8):499-508
As novel anti-HIV agents, the G-tetrad-forming oligonucleotides have been explored for their structure-activity relations with regard to inhibition of integrase (IN) (N. Jing, Expert Opin. Investig. Drugs (2000) 9, 1777-1785). We have now developed two families of G-quartet oligonucleotides: T40217-T40222, with potential formation of a tail-to-tail G-quartet dimer, and T40224-T40227, with phosphorothioate (PT) linkages in the guanine loops. The results obtained from biophysical measurements and the assays of the inhibition of HIV-1 IN and virus replication demonstrated that an increase in the length of the G-quartet structure from a monomer (15A) to a tail-to-tail dimer (47A) does not distinctly disrupt the inhibition of HIV-1 IN activity or the inhibition of HIV-1 replication in cell cultures. G-quartet oligonucleotides were observed to induce molecular aggregation of HIV-1 IN and interrupt the binding of viral DNA to HIV-1 IN. Also, PT substitutions did not confer any advantages compared with the regular phosphodiesters for the inhibition of HIV-1 replication by intramolecular G-quartets. The G-quartet motif is the primary requirement for the remarkable nuclease resistance and pronounced biological efficacy of these oligonucleotides. 相似文献
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Potassium-dependent folding: a key to intracellular delivery of G-quartet oligonucleotides as HIV inhibitors 总被引:6,自引:0,他引:6
Several groups have demonstrated that G-rich oligonucleotides forming G-quartet structures display activity as potential drugs, such as potent HIV inhibitors. The delivery of G-quartet oligonucleotides to their intracellular targets is a key obstacle to overcome for their clinical success. Here we have developed a novel system to deliver G-rich oligonucleotides into the cell nucleus, e.g., the site of HIV integration. On the basis of the property of potassium-induced formation of G-quartet structure, we explored the difference of K(+) concentrations inside (140 mM) and outside (4 mM) cells to induce the G-rich oligonucleotides to form different structures inside and outside cells. The key steps of this delivery system include the following: (i) First, the G-quartet structure is denatured to form a lipid-DNA complex, so that the molecules can be well delivered into cells. (ii) Then the delivered molecules are induced to form G-quartet structures by potassium inside cells since the G-quartet structure is the primary requirement for inhibition of HIV-1 HIV integrase (IN) activity. The molecules of a novel G-quartet HIV inhibitor, T40214, with the sequence of (GGGC)(4) were successfully delivered into the nuclei of target cells, which significantly decreased HIV-1 replication and increased the probability to target HIV-1 IN in infected cells. 相似文献
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Both substrate and target oligonucleotide sequences affect in vitro integration mediated by human immunodeficiency virus type 1 integrase protein produced in Saccharomyces cerevisiae. 总被引:29,自引:15,他引:14
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Integration of retroviral DNA into the host cell genome requires the interaction of retroviral integrase (IN) protein with the outer ends of both viral long terminal repeats (LTRs) to remove two nucleotides from the 3' ends (3' processing) and to join the 3' ends to newly created 5' ends in target DNA (strand transfer). We have purified the IN protein of human immunodeficiency virus type 1 (HIV-1) after production in Saccharomyces cerevisiae and found it to have many of the properties described for retroviral IN proteins. The protein performs both 3' processing and strand transfer reactions by using HIV-1 or HIV-2 attachment (att) site oligonucleotides. A highly conserved CA dinucleotide adjacent to the 3' processing site of HIV-1 is important for both the 3' processing and strand transfer reactions; however, it is not sufficient for full IN activity, since alteration of nucleotide sequences internal to the HIV-1 U5 CA also impairs IN function, and Moloney murine leukemia virus att site oligonucleotides are poor substrates for HIV-1 IN. When HIV-1 att sequences are positioned internally in an LTR-LTR circle junction substrate, HIV-1 IN fails to cleave the substrate preferentially at positions coinciding with correct 3' processing, implying a requirement for positioning att sites near DNA ends. The 2 bp normally located beyond the 3' CA in linear DNA are not essential for in vitro integration, since mutant oligonucleotides with single-stranded 3' or 5' extensions or with no residues beyond the CA dinucleotide are efficiently used. Selection of target sites is nonrandom when att site oligonucleotides are joined to each other in vitro. We modified an in vitro assay to distinguish oligonucleotides serving as the substrate for 3' processing and as the target for strand transfer. The modified assay demonstrates that nonrandom usage of target sites is dependent on the target oligonucleotide sequence and independent of the oligonucleotide used as the substrate for 3' processing. 相似文献
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Andreola ML Pileur F Calmels C Ventura M Tarrago-Litvak L Toulmé JJ Litvak S 《Biochemistry》2001,40(34):10087-10094
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《Biophysical journal》2021,120(21):4874-4890
During HIV-1 assembly, the viral Gag polyprotein specifically selects the dimeric RNA genome for packaging into new virions. The 5′ untranslated region (5′UTR) of the dimeric genome may adopt a conformation that is optimal for recognition by Gag. Further conformational rearrangement of the 5′UTR, promoted by the nucleocapsid (NC) domain of Gag, is predicted during virus maturation. Two 5′UTR dimer conformations, the kissing dimer (KD) and the extended dimer (ED), have been identified in vitro, which differ in the extent of intermolecular basepairing. Whether 5′UTRs from different HIV-1 strains with distinct sequences have access to the same dimer conformations has not been determined. Here, we applied fluorescence cross-correlation spectroscopy and single-molecule Förster resonance energy transfer imaging to demonstrate that 5′UTRs from two different HIV-1 subtypes form (KDs) with divergent stabilities. We further show that both 5′UTRs convert to a stable dimer in the presence of the viral NC protein, adopting a conformation consistent with extensive intermolecular contacts. These results support a unified model in which the genomes of diverse HIV-1 strains adopt an ED conformation. 相似文献
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构建以人类免疫缺陷病毒(HIV-1)5'LTR为靶点的药物筛选细胞模型,用于体外筛选潜在的对HIV-1启动子具有抑制作用的药物,或用于筛选与HIV-1复制相关的宿主因子。通过PCR扩增HIV-1 5'LTR片段和胸苷激酶基因(thymidine kinase gene,TK基因),以这2片段为模板进行重叠PCR将两者连接起来,连接产物经酶切后与pcDNA3.1载体连接;将连接正确的质粒转染HEK293细胞同时用G418加压筛选获得稳定细胞系;加入药物更昔洛韦(GCV)检测TK基因的表达。成功构建了HIV-1 5'LTR调控TK基因表达的稳定细胞系,在其培养过程中加入药物更昔洛韦时,细胞逐渐死亡。构建了以HIV-1 5'LTR为靶点的药物筛选细胞模型,该模型利用TK基因作为报告基因方便灵敏,可用于筛选针对HIV-1 5'LTR的潜在抗HIV药物。 相似文献
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《Nucleosides, nucleotides & nucleic acids》2013,32(4-7):909-914
A series of oligonucleotides conjugated to intercalators, as well as fluorescent and lipophilic substances, minor groove binders and photoactive molecules were synthesized for studies of their ability to form a stable triple helix. Purine-rich short double stranded DNA fragments from HIV-1 genome and pyrimidine 16-mer oligodeoxyribonucleotide were used as models. A conjugate of a dipyrido[3,2-a:2′,3′-c]phenazine-ruthenium (II) complex and a triple helix-forming oligonucleotide was constructed. Upon sequence-specific duplex and triplex formation of the conjugate, the ruthenium complex becomes highly fluorescent. The attached ruthenium complex induces a stabilization of the DNA triple helix and a significant increase of the time of residence of the third strand on the duplex. 相似文献
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Sequence-specific RNase H cleavage of gag mRNA from HIV-1 infected cells by an antisense oligonucleotide in vitro.
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We have used a ribonuclease protection assay to investigate RNase H cleavage of HIV-1 mRNA mediated by phosphorothioate antisense oligonucleotides complementary to the gag region of the HIV-1 genome in vitro. Cell lysate experiments in H9 and U937 cells chronically infected with HIV-1 IIIB showed RNase H cleavage of unspliced gag message but no cleavage of spliced message which did not contain the target gag region. RNase H cleavage products were detected at oligonucleotide concentrations as low as 0.01 microM and the RNase H activity was seen to be concentration dependent. Similar experiments with 1-, 3- and 5-mismatch oligonucleotides demonstrated sequence specificity at low concentrations, with cleavage of gag mRNA correlating with the predicted activities of the parent and mismatch oligonucleotides based on their hybridization melting temperatures. Experiments in living cells suggested that RNase H-specific antisense activity was largely determined by the amount of oligonucleotide taken up by the different cell lines studied. RNase H cleavage products were detected in antisense oligonucleotide treated MT-4 cells acutely infected with HIV-1 IIIB, but not in infected H9 cells treated with oligonucleotide under the same conditions. The data presented demonstrate potent and specific RNase H cleavage of HIV-1 mRNA mediated by an antisense oligonucleotide targeted to HIV-1 gag mRNA, and are in agreement with previous reports that the major obstacle to demonstrating antisense activity in living cells remains the lack of penetration of these agents into the desired cellular compartment. 相似文献