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1.
HIV Integrase (IN) is an enzyme that is responsible for the integration of the proviral genome into the human genome, and this integration step is the first step of the virus hijacking the human cell machinery for its propagation and replication. 10-23 DNAzyme has the potential to suppress gene expressions through sequence-specific mRNA cleavage. We have designed three novel DNAzymes, DIN54, DIN116, and DIN152, against HIV-1 Integrase gene using Mfold software and evaluated them for target site cleavage activity on the in vitro transcribed mRNA. All DNAzymes were tested for its inhibition of expression of HIV Integrase protein in the transiently transfected cell lines. DIN116 and DIN152 inhibited IN-EGFP expression by 80 percent and 70 percent respectively. 相似文献
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Retroviral integrases catalyze two reactions, 3′-processing of viral DNA ends, followed by integration of the processed ends into chromosomal DNA. X-ray crystal structures of integrase-DNA complexes from prototype foamy virus, a member of the Spumavirus genus of Retroviridae, have revealed the structural basis of integration and how clinically relevant integrase strand transfer inhibitors work. Underscoring the translational potential of targeting virus-host interactions, small molecules that bind at the host factor lens epithelium-derived growth factor/p75-binding site on HIV-1 integrase promote dimerization and inhibit integrase-viral DNA assembly and catalysis. Here, we review recent advances in our knowledge of HIV-1 DNA integration, as well as future research directions. 相似文献
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人类免疫缺陷病毒(HIV-1)整合酶抑制剂筛选及其活性测定 总被引:1,自引:0,他引:1
整合酶作用的病毒DNA整合进宿主DNA的过程是反转录病毒在宿主细胞中增殖的关键步骤.由于在正常人类细胞中不存在相似的功能蛋白,其抑制剂对人体的副作用可能很小,相对于经典AIDS治疗药物的毒副作用,整合酶抑制剂理论上要具有优势.在线性七肽库中筛选与整合酶有特异结合作用的噬菌体展示肽,选取TPSHSSR和HPERATL 2条肽,它们可以竞争性地抑制展示相应肽段的噬菌体与整合酶的结合,同时它们对整合酶的整合活性也有一定程度的抑制,半数抑制率分别为IC50=(54.56±5.18)μmol/L,IC50=(28.29±1.32)μmol/L.这些多肽可用于治疗艾滋病新药的开发应用及整合酶结构及作用机制的研究. 相似文献
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The time and mode of entry, and development of Ditylenchus destructor in peanut were studied in field and greenhouse experiments. Few nematodes were present in the cortex of the roots. At 90-120 days after planting, D. destructor was observed in the exocarp at the base of the pod near the point of connection with the peg. The peg was invaded from this primary infection site. The endocarp of the hull was usually penetrated through openings at the base of the mesocarp and sometimes at the pod apex. Numerous D. destructor were present in the testa and the vascular bundles. Nematodes were found in the embryo but not in the cotyledons. The histopathology of D. destructor closely resembles that of the peanut testa nematode, Aphelenchoides arachidis Bos. 相似文献
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Sp100是核颗粒ND10的组成蛋白,在哺乳动物细胞中广泛存在.Sp100参与多种细胞生理病理过程,如转录调控、细胞内抗病毒免疫等.利用酵母双杂交系统,我们发现了Sp100的互作蛋白HIV-1整合酶,免疫共沉淀实验进一步证实了Sp100与 HIV-1整合酶的互作,细胞内荧光共定位实验也证实了二者在细胞内部分共定位.此外,突变体实验表明,Sp100的C端300~480氨基酸和HIV-1的催化结构域是两个蛋白质的互作区域.利用siRNA降低细胞内Sp100的表达量,可以增加HIV-1整合酶介导的病毒的整合,反之,细胞内过表达Sp100则会降低HIV-1整合酶介导的病毒的整合.这是首次发现Sp100可以和HIV-1整合酶发生相互作用,并进而抑制病毒的整合.我们发现了Sp100作为HIV-1整合酶互作蛋白的新功能,并扩展了细胞防御病毒感染的相关研究. 相似文献
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Nicholas C. Fitzkee James E. Masse Yang Shen David R. Davies Ad Bax 《The Journal of biological chemistry》2010,285(23):18072-18084
The human immunodeficiency virus type 1 (HIV-1) integrase (IN) is a critical enzyme involved in infection. It catalyzes two reactions to integrate the viral cDNA into the host genome, 3′ processing and strand transfer, but the dynamic behavior of the active site during catalysis of these two processes remains poorly characterized. NMR spectroscopy can reveal important structural details about enzyme mechanisms, but to date the IN catalytic core domain has proven resistant to such an analysis. Here, we present the first NMR studies of a soluble variant of the catalytic core domain. The NMR chemical shifts are found to corroborate structures observed in crystals, and confirm prior studies suggesting that the α4 helix extends toward the active site. We also observe a dramatic improvement in NMR spectra with increasing MgCl2 concentration. This improvement suggests a structural transition not only near the active site residues but also throughout the entire molecule as IN binds Mg2+. In particular, the stability of the core domain is linked to the conformation of its C-terminal helix, which has implications for relative domain orientation in the full-length enzyme. 15N relaxation experiments further show that, although conformationally flexible, the catalytic loop of IN is not fully disordered in the absence of DNA. Indeed, automated chemical shift-based modeling of the active site loop reveals several stable clusters that show striking similarity to a recent crystal structure of prototype foamy virus IN bound to DNA. 相似文献
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Structural and Dynamical Properties of a Full-length HIV-1 Integrase: Molecular Dynamics Simulations
Atchara Wijitkosoom Somsak Tonmunphean Thanh N. Truong Supot Hannongbua 《Journal of biomolecular structure & dynamics》2013,31(6):613-624
Abstract The structural and dynamical properties of the complete full-length structure of HIV-1 integrase were investigated using Molecular Dynamics approach. Simulations were carried out for the three systems, core domain only (CORE), full-length structure without (FULL) and with a Mg2+ (FULL+ION) in its active site, aimed to investigate the difference in the molecular properties of the full-length models due to their different construction procedures as well as the effects of the two ends, C- and N-terminal, on those properties in the core domain. The full-length structure was prepared from the two experimental structures of two-domain fragment. The following properties were observed to differ significantly from the previous reports: (i) relative topology formed by an angle between the three domains; (ii) the cavity size defined by the catalytic triad, Asp64, Asp116, and Glul52; (iii) distances and solvation of the Mg2+; and (iv) conformation of the catalytic residues. In addition, the presence of the two terminal domains decreases the mobility of the central core domain significantly. 相似文献
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Agapkina J. J. Tashlitskii V. N. Deprez E. Brochon J.-C. Shugalii A. V. Mouscadet J.-F. Gottikh M. B. 《Molecular Biology》2004,38(5):718-727
Integrase (IN) is responsible for one of the key stages in the replication cycle of human immunodeficiency virus type 1, namely, integration of a DNA copy of the viral RNA into the infected cell genome. IN recognizes the nucleotide sequences located at the ends of the U3 and U5 regions of long terminal repeats (LTRs) of the viral DNA and sequentially catalyzes the 3-end processing and strand transfer reactions. Analogs of U5 regions containing non-nucleoside insertions have been used to study the interaction between IN and viral DNA. Substrate modification has been demonstrated to have almost no effect on the rate of DNA binding by IN. However, the removal of heterocyclic bases from positions 5 and 6 of the substrate molecule and from position 3 of the processed strand almost completely inhibits IN enzymatic activity, which indicates the importance of these bases for the formation of an active enzyme–substrate complex. By contrast, modification of the third base of the nonprocessed strand stimulates 3-processing. Since the base removal disturbs the complementary and stacking interactions in DNA, these results indicate that double-helix destabilization near the cleaved bond promotes 3-end processing. 相似文献
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A tetramer model for human immunodeficiency virus type 1 (HIV-1) integrase (IN) with DNA representing long terminal repeat (LTR) termini was previously assembled to predict the IN residues that interact with the LTR termini; these predictions were experimentally verified for nine amino acid residues [Chen, A., Weber, I. T., Harrison, R. W. & Leis, J. (2006). Identification of amino acids in HIV-1 and avian sarcoma virus integrase subsites required for specific recognition of the long terminal repeat ends. J. Biol. Chem., 281, 4173-4182]. In a similar strategy, the unique amino acids found in avian sarcoma virus IN, rather than HIV-1 or Mason-Pfizer monkey virus IN, were substituted into the structurally related positions of HIV-1 IN. Substitutions of six additional residues (Q44, L68, E69, D229, S230, and D253) showed changes in the 3′ processing specificity of the enzyme, verifying their predicted interaction with the LTR DNA. The newly identified residues extend interactions along a 16-bp length of the LTR termini and are consistent with known LTR DNA/HIV-1 IN cross-links. The tetramer model for HIV-1 IN with LTR termini was modified to include two IN binding domains for lens-epithelium-derived growth factor/p75. The target DNA was predicted to bind in a surface trench perpendicular to the plane of the LTR DNA binding sites of HIV-1 IN and extending alongside lens-epithelium-derived growth factor. This hypothesis is supported by the in vitro activity phenotype of HIV-1 IN mutant, with a K219S substitution showing loss in strand transfer activity while maintaining 3′ processing on an HIV-1 substrate. Mutations at seven other residues reported in the literature have the same phenotype, and all eight residues align along the length of the putative target DNA binding trench. 相似文献
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Greenhouse and laboratory experiments were performed to determine if an interaction exists between Meloidogyne incognita and Hoplolaimus columbus on Davis soybean. Greenhouse tests were performed with three population levels of M. incognita and H. columbus (0, 1,500, 6,000/1.5-liter pot) separately and in all combinations. Dry root weight (DRT) declined nonlinearly and dry shoot weight (DST) declined linearly with respect to increasing initial populations of M. incognita and H. columbus. When the two nematode species were added to the soil together, the amount of DRT and DST suppression by one species was dependent on the initial level of the concomitant species. The final root population of M. incognita or H. columbus declined linearly with increasing initial population density of the concomitant species. H. columbus suppressed M. incognita populations in the soil nonlinearly, but M. incognita had no effect on H. columbus. 相似文献
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Human immunodeficiency virus(HIV) attacks human immune system and causes life-threatening acquired immune deficiency syndrome(AIDS). Treatment with combination antiretroviral therapy(cART) could inhibit virus growth and slow progression of the disease, however, at the same time posing various adverse effects. Host ubiquitin-proteasome pathway(UPP) plays important roles in host immunity against pathogens including viruses by inducing degradation of viral proteins. Previously a series of methods for retargeting substrates for ubiquitin-proteasome degradation have been successfully established. In this study, we attempted to design and construct artificial chimeric ubiquitin ligases(E3 s) based on known human E3 s in order to manually target HIV-1 integrase for ubiquitin proteasome pathway-mediated degradation.Herein, a series of prototypical chimeric E3 s have been designed and constructed, and original substrate-binding domains of these E3 s were replaced with host protein domains which interacted with viral proteins. After functional assessment screening, 146 LI was identified as a functional chimeric E3 for HIV-1 NL4-3 integrase. 146 LI was then further optimized to generate 146 LIS(146 LI short) which has been shown to induce Lys48-specific polyubiquitination and reduce protein level of HIV-1 NL4-3 integrase more effectively in cells. Lymphocyte cells with 146 LIS knock-in generated by CRISPR/Cas-mediated homology-directed repair(HDR) showed remarkably decreased integration of HIV-1 NL4-3 viral DNAs and reduced viral replication without obvious cell cytotoxicity. Our study successfully obtained an artificial chimeric E3 which can induce Lys48-specific polyubiquitination and proteasome-mediated degradation of HIV-1 NL4-3 integrase, thus effectively inhibiting viral DNA integration and viral replication upon virus infection. 相似文献
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HIV-1复制需要HIV-1整合酶将其环状DNA整合进宿主DNA中,这其中包括2个重要反应,即“3′-加工”和“链转移”,两者均由HIV-1整合酶催化完成.阻断其中的任一反应,都能达到抑制HIV-1复制的目的.因此,了解HIV-1整合酶的完整结构和聚合状态,对深入探讨其作用机理及设计新型抑制剂具有重要的指导作用.然而,迄今为止仅有HIV-1整合酶单独结构域的晶体结构可供参考,而其全酶晶体结构尚未获得解析.本研究利用分子模拟技术,通过蛋白质 蛋白质/DNA分子对接、动力学模拟等方法,构建了全长整合酶四聚体的结构模型、HIV-1 DNA与整合酶复合物的结构模型,进一步从理论上证实HIV-1整合酶是以四聚体形态发挥催化作用,明确“3′-加工”和“链转移”在HIV-1整合酶上的催化位点.同时,通过与作用机理相似的细菌转座子Tn5转座酶等的结构比对,推测HIV-1整合酶的核心结构域中应有第2个Mg2+存在,其位置螯合于Asp64与Glu152之间.在HIV-1整合酶结构研究的基础上,有望进一步设计出新的抗艾滋病药物. 相似文献
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Wolfgang Weber Hagop Demirdjian Roberto D. Lins James M. Briggs Ricardo Ferreira J. Andrew McCammon 《Journal of biomolecular structure & dynamics》2013,31(3):733-745
Abstract The three-dimensional structure of the active site region of the enzyme HIV-1 integrase is not unambiguously known. This region includes a flexible peptide loop that cannot be well resolved in crystallographic determinations. Here we present two different computional approaches with different levels of resolution and on different time-scales to understand this flexibility and to analyze the dynamics of this part of the protein. We have used molecular dynamics simulations with an atomic model to simulate the region in a realistic and reliable way for 1 ns. It is found that parts of the loop wind up after 300 ps to extend an existing helix. This indicates that the helix is longer than in the earlier crystal structures that were used as basis for this study. Very recent crystal data confirms this finding, underlining the predictive value of accurate MD simulations. Essential dynamics analysis of the MD trajectory yields an anharmonic motion of this loop. We have supplemented the MD data with a much lower resolution Brownian dynamics simulation of 600 ns length. It provides ideas about the slow-motion dynamics of the loop. It is found that the loop explores a conformational space much larger than in the MD trajectory, leading to a “gating”-like motion with respect to the active site. 相似文献
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The population development of Ditylenchus destructor in the roots, pegs, hulls, and seeds of eight peanut (Arachis hypogaea) genotypes was studied in the greenhouse. Although all genotypes tested were good hosts for D. destructor, differences in host suitability were observed. Invasion of the plant parts by Ditylenchus destructor proceeded more slowly in genotypes with long growth periods. During the second half of the growth period of these genotypes, D. destructor populations emigrated from the hulls and seeds into the soil but reinfected the pods after a few weeks. The genotypes with the longest growth periods supported the highest number of nematodes when each genotype was harvested at its usual harvest time. The long-season genotypes supported low numbers of nematodes when harvested before crop maturity. 相似文献
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Custer and Hood soybean cultivars were inoculated with nine levels of Criconemoides simile ranging from 300 to 20,600 nematodes per plant. Rate of reproduction decreased as inoculum level was increased beyond 900-2,000 nematodes. Final population density was influenced by both composition and level of inoculum. There was an indication that substance(s). inhibitory to larvae, accumnlated in soil in which Hood was grown for 11 months, Significant reduction of fresh weight of roots of Hood, but not Custer, occurred at population densities of 37,000 and 44,700 nematodes per plant. 相似文献
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Amit Sharma Alison Slaughter Nivedita Jena Lei Feng Jacques J. Kessl Hind J. Fadel Nirav Malani Frances Male Li Wu Eric Poeschla Frederic D. Bushman James R. Fuchs Mamuka Kvaratskhelia 《PLoS pathogens》2014,10(5)
The quinoline-based allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are promising candidates for clinically useful antiviral agents. Studies using these compounds have highlighted the role of IN in both early and late stages of virus replication. However, dissecting the exact mechanism of action of the quinoline-based ALLINIs has been complicated by the multifunctional nature of these inhibitors because they both inhibit IN binding with its cofactor LEDGF/p75 and promote aberrant IN multimerization with similar potencies in vitro. Here we report design of small molecules that allowed us to probe the role of HIV-1 IN multimerization independently from IN-LEDGF/p75 interactions in infected cells. We altered the rigid quinoline moiety in ALLINIs and designed pyridine-based molecules with a rotatable single bond to allow these compounds to bridge between interacting IN subunits optimally and promote oligomerization. The most potent pyridine-based inhibitor, KF116, potently (EC50 of 0.024 µM) blocked HIV-1 replication by inducing aberrant IN multimerization in virus particles, whereas it was not effective when added to target cells. Furthermore, KF116 inhibited the HIV-1 IN variant with the A128T substitution, which confers resistance to the majority of quinoline-based ALLINIs. A genome-wide HIV-1 integration site analysis demonstrated that addition of KF116 to target or producer cells did not affect LEDGF/p75-dependent HIV-1 integration in host chromosomes, indicating that this compound is not detectably inhibiting IN-LEDGF/p75 binding. These findings delineate the significance of correctly ordered IN structure for HIV-1 particle morphogenesis and demonstrate feasibility of exploiting IN multimerization as a therapeutic target. Furthermore, pyridine-based compounds present a novel class of multimerization selective IN inhibitors as investigational probes for HIV-1 molecular biology. 相似文献