A supramolecular assembly mediates lentiviral DNA integration
Allison Ballandras-ColasDaniel P. MaskellErik SerraoJulia LockePaolo SwuecS. JönssonAbhay KotechaNicola CookValerie E. PyeIan A. TaylorValgerður AndrésdóttirAlan EngelmanAlessandro CostaPeter Cherepanov
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Abstract:
Retroviral integrase (IN) functions within the intasome nucleoprotein complex to catalyze insertion of viral DNA into cellular chromatin. Using cryo-electron microscopy, we now visualize the functional maedi-visna lentivirus intasome at 4.9 angstrom resolution. The intasome comprises a homo-hexadecamer of IN with a tetramer-of-tetramers architecture featuring eight structurally distinct types of IN protomers supporting two catalytically competent subunits. The conserved intasomal core, previously observed in simpler retroviral systems, is formed between two IN tetramers, with a pair of C-terminal domains from flanking tetramers completing the synaptic interface. Our results explain how HIV-1 IN, which self-associates into higher-order multimers, can form a functional intasome, reconcile the bulk of early HIV-1 IN biochemical and structural data, and provide a lentiviral platform for design of HIV-1 IN inhibitors.Keywords:
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Integration of the HIV genome by integrase is absolutely required for productive infection.To determine the role of natural selection on HIV integrase biology.To study the activities of HIV integrases from a limited panel of North American clinical isolates from HIV-infected patients and to compare these proteins with integrases from two laboratory adapted reference strains (HI(VIIIRF) and HIV(NL4--3)).HIV was isolated and the particle-associated RNA was reverse transcribed and sequenced. Replication kinetics of molecularly cloned viruses containing each variant integrase were studied in tissue culture. The mutant integrase proteins were expressed, purified and specific activities of the enzymes were derived for both 3' end-processing and disintegration reactions.Despite 3--5% variability in integrase at the amino acid level, viruses showed no statistically significant differences in growth kinetics compared with the reference HIV(NL4--3) virus and only minor differences were observed in 3' end-processing and disintegration activities. All integrase proteins demonstrated similar sensitivity to an integrase inhibitor l-chicoric acid.These results demonstrate that integrase genes derived from HIV-infected individuals can differ from reference sequences but these mutations do not result in loss of function, including susceptibility to an integrase inhibitor; therefore, integrase remains an attractive target for antiviral drug design, as mutability appears to be restricted by function.
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A new type of multifunctional reagent creates a specific connection within and between two hemoglobin tetramers, resulting in a cross-linked bis-tetramer ("CLBT"). The tetrafunctional reagent (N,N'-5,5'-bis[bis(3,5-dibromosalicyl)isophthalyl]terephthalamide, DBIT) was prepared by conversion of the corresponding tetraacid to the tetrakis(3,5-dibromosalicylate). Deoxy hemoglobin reacts with DBIT to give a cross-linked bis-tetramer as the major product. Patterns in tryptic digests reveal that there are modifications of amino groups at β-Lys-82 and β-Val-1, indicative of a structure in which each tetramer is cross-linked between these positions. The cross-linked bis-tetramer has a decreased affinity for oxygen and very low cooperativity in oxygen binding. The properties of the material provide insights into the nature of protein−protein interactions.
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Objective To provide reference for our studies in designing new inhibitors of human immunodeficiency virus type-1(HIV-1)integrase.Methods According to the literature gathered,this article reviewed the structure,the function,and the catalytic mechanism of HIV-1 integrase,besides,the effect of inhibitors to the HIV-1 integrase in the different steps of the whole process was also reviewed.Results The HIV-1 integrase,a kind of protein,coded by the pol gene was composed of 288 amino acid resides and its relative molecular mass was 32 000.The inhibitors effectively influenced the HIV-1 integrase by interfering with the oligomerization of HIV-1 integrase,competitively binding with the DNA long terminal repeat,and intercepting the 3′-processing and strand transfer in the whole process.Conclusions Utilizing advanced means of computer aided drug design,it is possible to gradually elucidate the mechanism of action of the inhibitors by studying the structure-activity relationship of the existing inhibitors and the action mode between the inhibitors and the macromolecule.Thus,emphasis should be laid to search for new integrase inhibitors with high activities to provide a new way of treating AIDS.
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Abstract A soluble cyclic porphyrin oligomer (CPO) consisting of four 5,10‐diarylporphyrins linked by alternating azo and butadiyne bridges has been synthesised via an aminated dinickel(II) butadiyne dimer. This is the first cyclic tetramer that combines both azo and butadiyne bridges and extends the azoporphyrin family, which comprises only a very few examples. The electronic absorption spectrum of the tetramer is more similar to spectra of azoporphyrins than to those of butadiyne‐linked dimers or tetramer, exhibiting a two‐component Soret band with a splitting of 4190 cm –1 and a strongly red‐shifted Q band maximum at 735 nm.
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Drug design against human immunodeficiency virus type 1 (HIV-1) integrase through its mechanistic study is of great interest in the area in biological research. The main obstacle in this area is the absence of the full-length crystal structure for HIV-1 integrase to be used as a model. A complete structure, similar to HIV-1 of a prototype foamy virus integrase in complex with DNA, including all conservative residues, is available and has been extensively used in recent investigations.The aim of this study was to determine whether the above model is precisely representative of HIV-1 integrase. This would critically determine the success of any designed drug using the model in deactivation of integrase and AIDS treatment.Primarily, a new structure for HIV-1 was constructed, using a crystal structure of prototype foamy virus as the starting structure. The constructed structure of HIV-1 integrase was simultaneously simulated with a prototype foamy virus integrase on a separate occasion.Our results indicate that the HIV-1 system behaves differently from the prototype foamy virus in terms of folding, hydration, hydrophobicity of binding site and stability.Based on our findings, we can conclude that HIV-1 integrase is vastly different from the prototype foamy virus integrase and does not resemble it, and the modeling output of the prototype foamy virus simulations could not be simply generalized to HIV-1 integrase. Therefore, our HIV-1 model seems to be more representative and more useful for future research.
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The time course of the fragmentation of calf thymus chromatin by DNase II (deoxyribonucleate 3'-oligonucleotidohydrolase, EC 3.1.4.6) has been examined by sedimentation of chromatin digests through linear (5-20%) sucrose gradients. The action of nuclease is decidedly nonrandom, ultimately producing roughly equal amounts of acid-soluble oligonucleotides and 11S nucleoprotein particles. The 11S particles contain double-stranded DNA that is approximately 400 A or 120 base-pairs long, as measured by electron microscopic examination of deproteinized samples, and is maintained in a compact conformation within the intact particles. In addition, 15S nucleoprotein particles containing predominantly 800-A lengths of DNA have been isolated from less extensively digested chromatin. Evidence is presented which indicates that the 11S particles are fundamental structural units that are arranged in tandem along certain regions of chromatin fibrils. Preliminary experiments with different nucleases and with chromatin from different mammalian species indicate that these results are a natural consequence of the arrangement of DNA and proteins in mammalian chromatin and are not peculiar to the system described in detail.
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