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At least 955 records · Page 53Linked to original sources
PubMed2003

Small-molecule HIV-1 integrase inhibitors: the 2001-2002 update.

Integration of viral DNA into host cell chromosomal DNA to form a provirus is an essential step in the viral life cycle. This process is mediated by integrase (IN), a 32 KDa viral enzyme. The unique properties of IN makes it an ideal target for drug design. First, there are no cellular homologues to IN and the reactions catalyzed by IN are unique. Second, IN is absolutely required for viral replication and mutations in a number of key residues dramatically block viral replication. Third, IN has been validated as a legitimate target and the results from S-1360 (1) the only available IN inhibitor under clinical trials suggest synergistic effect with reverse transcriptase (RT) and protease (PR) inhibitors. During the past 10 years a plethora of inhibitors have been identified and some were shown to be selective against IN and block viral replication. The two most predominant classes of inhibitors have been the catechol containing hydroxylated aromatics and more recently the diketoacid containing aromatics. Herein, we review all small molecule compounds reported to inhibit recombinant HIV-1 IN with IC(50) values < 20 M during the past two years. It is important to bear in mind that the true mechanism of action and antiviral activities of many of the compounds are currently not established. However, based on the growing body of literature certain classes of compounds can be easily excluded as bona fide IN inhibitors.

Drug Design↗
PubMed2006

[Identification of the novel host factor that interacts with HIV-1 integrase and analysis of the expression profile of the interactor in vivo].

Human immunodeficiency virus type 1 (HIV-1) is one of the lentiviruses, and unlike other retroviruses, HIV-1 is capable of infecting not only dividing cells but also non-dividing cells. It has been strongly suggested that this property is due to the viral mechanism which facilitates the integration of the viral genome cDNA into the host chromosome more efficiently than other retroviruses. HIV-1 integrase (IN) is a viral protein which catalyzes insertion of the viral genome cDNA into the host cell chromosome. However, it has been suggested that HIV-1 IN also plays putative roles at the steps prior to integration such as uncoating, reverse transcription and nuclear transport of the viral cDNA. In this study, we tried to identify the novel host factor which interacted with HIV-1 IN using two different kinds of yeast two hybrid methods: the conventional yeast two hybrid method and the mating method. First, the full-length cDNA fragment of HIV-1 IN was amplified using polymerase chain reaction (PCR), and the amplified products were ligated into pGBT 9 vector as bait. Plasmid vectors expressing human lymphocytes cDNA library and HeLa cDNA library were used as prey plasmid in the conventional yeast two hybrid method and the mating method, respectively. These plasmids were transformed into the corresponding yeast strain cells, and several positive clones were isolated. As a result, a known gene product was identified as a candidate. Further analysis revealed that this protein was expressed in HeLa, 293 T cells, primary macrophages and activated T lymphocytes, and that suppression of this protein expression affected HIV-1 replication.

Cyclic AMP Response Element-Binding Protein↗
PubMed2001

Analysis of the DNA substrate structure and number of the processing sites on the activities of HIV-1 integrase in vitro.

A series of DNA substrates were synthesized to analyze the 3'-processing, integration and disintegration reactions taking place concurrently on the same DNA molecules and to evaluate the potential effects of various structural modifications of these molecules on the activities of HIV-1 integrase (IN). Our results indicate that DNA substrates containing multiple recognition sites for IN can produce efficiently the three activities of the enzyme. The 3'-processing and disintegration sites are recognized and processed by IN, both reactions being carried out in a competitive manner by the enzyme on the same DNA molecule. The presence of the gaps and unpaired nucleotides in the region surrounding the disintegration site had major deleterious effects on enzymes disintegration activity. Analysis of a different conformation at the base of the DNA hairpin has revealed a significant improvement of IN disintegration activity in the presence of double-stranded DNA on the 3' side of the disintegration site, suggesting that this region plays an important role in the stability of the enzyme-substrate complex. Interestingly, the efficiency of disintegration was strongly diminished in the presence of an unpaired nucleotide located immediately at the 3' end of the cleavage site. Overall, our results underline the extreme sensitivity of the HIV-1 IN to its substrates structure and conformation, especially for its disintegration activity, and the considerable importance of the disintegration activity in the reactions carried out in vitro by the purified enzyme.

DNA↗
PubMed1992

Retroviral integrase functions as a multimer and can turn over catalytically.

A number of studies have demonstrated that the retroviral protein integrase (IN) alone is sufficient to carry out two discrete steps required for retroviral integration: the endonucleolytic processing of viral DNA ends and the cleavage and joining of host DNA to the processed viral DNA termini. Little is known about the biochemical and biophysical mechanisms involved in these reactions. Here, we employ in vitro assays of Rous sarcoma virus IN to demonstrate for the first time that IN is capable of multiple turnover in both the processing and joining reactions. The turnover number calculated for the processing reaction is 0.26 cleavages/min/mol of IN. Our steady state kinetic studies indicate that both the processing and joining activities require a multimeric form of IN. Ultracentrifugation analyses reveal a substrate-independent reversible equilibrium among the monomeric, dimeric, and tetrameric forms of this protein. From these results we conclude that the minimal functional unit for both the processing and joining of each viral DNA end is an IN dimer.

Animals↗
PubMed1992

Structural implications of spectroscopic characterization of a putative zinc finger peptide from HIV-1 integrase.

The N-terminal domain of human immunodeficiency virus (HIV-1) integrase (IN) contains the sequence motif His-Xaa3-His-Xaa23-Cys-Xaa2-Cys, which is strongly conserved in all retroviral and retrotransposon IN proteins. This structural motif constitutes a putative zinc finger in which a metal ion may be coordinately bound by the His and Cys residues. A recombinant peptide, IN(1-55), composed of the N-terminal 55 amino acids of HIV-1 IN was expressed in Escherichia coli and purified. Utilizing a combination of techniques including UV-visible absorption, circular dichroism, Fourier transform infrared, and fluorescence spectroscopies, we have demonstrated that metal ions (Zn2+, Co2+, and Cd2+) are bound with equimolar stoichiometry by IN(1-55). The liganded peptide assumes a highly ordered structure with increased alpha-helical content and exhibits remarkable thermal stability. UV-visible difference spectra of the peptide-Co2+ complexes directly implicate thiols in metal coordination, and Co2+ d-d transitions in the visible range indicate that Co2+ is tetrahedrally coordinated. Mutant peptides containing conservative substitutions of one of the conserved His or either of the Cys residues displayed no significant Zn(2+)-induced conformational changes as monitored by CD and fluorescence spectra. We conclude that the N terminus of HIV-1 IN contains a metal-binding domain whose structure is stabilized by tetrahedral coordination of metal by histidines 12 and 16 and cysteines 40 and 43. A preliminary structural model for this zinc finger is presented.

Amino Acid Sequence↗
PubMed2004

Analogues of the styrylquinoline and styrylquinazoline HIV-1 integrase inhibitors: design and synthetic problems.

In our work, leading to new styrylquinoline and styrylquinazoline inhibitors of HIV integrase, we analyzed virtual combinatorial library that includes these compounds. Using this method we were able to find interesting synthetic targets. We optimized synthetic procedure yielding such compounds and obtained a couple of new analogues. Their activity will be evaluated in the near future.

Combinatorial Chemistry Techniques↗
PubMed2006

Predictive biomarkers for drug-resistant Acinetobacter baumannii isolates with bla(TEM-1), AmpC-type bla and integrase 1 genotypes.

BACKGROUND AND PURPOSE: We tested whether antibiotic susceptibilities of drug-resistant Acinetobacter baumannii isolates could be used to predict the clinically important genotypes bla(TEM-1), AmpC-type bla, and integrase 1 gene (IntI1). METHODS: We analyzed 401 A. baumannii isolates obtained at Changhua Christian Hospital between April 2001 and March 2002. The isolates were all from blood cultures, and identification of A. baumannii was confirmed by API-20NE. Antibiotic susceptibility testing (phenotype) was performed by disk diffusion method. Polymerase chain reaction was used to detect the genes bla(TEM-1), AmpC-type bla and IntI1. RESULTS: Of 32 A. baumannii isolates, 10 possessed bla(TEM-1), 21 AmpC-type bla, and 26 IntI1. Resistance to ceftazidime (CAZ) predicted bla(TEM-1) genotype with 63.6% sensitivity, 100% specificity, 55.6% positive predictive value (PPV) and 0% negative predictive value (NPV). Trimethoprim-sulfamethoxazole (SXT) and gentamicin (GM) resistance predicted IntI1 genotype with 83.3% sensitivity, 71.4% specificity, 95.2% PPV and 45.4% NPV. No resistance phenotype could predict the AmpC-type bla genotype. CONCLUSIONS: CAZ resistance predicted the bla(TEM-1) genotype with 100% specificity, and SXT and GM resistance predicted the IntI1 genotype with 92.5% PPV. Therefore, antibiotic susceptibilities to CAZ, SXT, and GM can be utilized clinically to detect critical genotypes in A. baumannii.

Acinetobacter Infections↗
PubMed1994

The influence of DNA and nucleosome structure on integration events directed by HIV integrase.

DNA copies of the human immunodeficiency virus (HIV) genome integrate nonrandomly into the chromosomal DNA of the host cell. In this report, we investigate the molecular basis of this selectivity using the virus-encoded HIV integrase to direct integration of a synthetic HIV long terminal repeat substrate into either DNA molecules of known structure or previously defined nucleosomal complexes. We find that the structure of the target greatly influences the site of integration, and, moreover, DNA curvature, flexibility, and rigidity in solution all influence the frequency of integration. Importantly, for DNA with all of these properties, the distortion of the double helix directed by association with the histone proteins promotes the integration reaction and alters the distribution of sites that are selected for integration. We suggest that both intrinsic DNA structure and the folding of DNA into chromosomal structures will exert a major influence on target site selection for integration of the viral genome.

Base Sequence↗
PubMed1993

Characterization of the forward and reverse integration reactions of the Moloney murine leukemia virus integrase protein purified from Escherichia coli.

The forward and reverse reactions for integration were characterized for the Moloney murine leukemia virus integrase (M-MuLV IN) protein. The M-MuLV IN was recombinantly produced in Escherichia coli, and was purified to greater than 90% homogeneity by a one-step affinity purification scheme. M-MuLV IN was highly active for integration as measured by in vitro cleavage and strand transfer assays. Furthermore, the integration of a model viral substrate into lambda concatamers by IN correctly produced the flanking 4-base pair duplications characteristic of M-MuLV IN. The reverse reaction of integration, disintegration, was also catalyzed by the recombinant M-MuLV IN. Two products were generated, a 3'-recessed long terminal repeat and a ligated target DNA, from a model integration-intermediate substrate in the presence of M-MuLV IN. The requirements and optimal conditions for maximal integration and disintegration activity for M-MuLV IN were determined. The forward and reverse reactions required different concentrations of manganese ion and reductant. Salt was also titrated for the forward and reverse reactions. Sodium chloride inhibited integration, but had little affect on disintegration. Low concentrations of potassium chloride enhanced integration, but had no affect on disintegration. The dinucleotide cleavage, strand transfer, and the disintegration reactions each had a unique pH profile of activity.

Base Sequence↗
PubMed1997

The genome of Moloney murine leukemia virus can be integrated by the integrase of human immunodeficiency virus type 1 expressed alone in vivo.

An in vivo integration assay using the expressed human immunodeficiency virus type 1 (HIV-1) integrase (IN) protein and plasmids carrying a copy of the infectious Moloney murine leukemia virus (MuLV) provirus genome as substrates is presented. The HIV-1 IN gene was taken from vector pINSD and cloned into vector pXT1 to give pXT1-IN. Two and three nucleotides from the circle junction on one pair of U3 and U5 attachment (att) sequences on an infectious MuLV provirus vector pMLV-K were changed by means of site-directed mutagenesis to that of the corresponding HIV-1 att sequences to generate vector pMLV*(U3U5). The MuLV IN sequence was partially deleted for vectors pMLV-K and pMLV*(U3U5) to generate vectors pMLV delta IN and pMLV*(U3U5) delta IN. Integration of these wild type and MuLV IN partially deleted or att mutated MuLV provirus vectors in the transfected cells by the expressed HIV-1 IN was monitored by means of a non-radioactive reverse transcriptase (RT) assay for released and collected virions. No RT activity was detected for the NIH/3T3 cell singly transfected with vector pMLV delta IN. However some RT activities were observed for the HIV-1 IN expressing cell transfected either with vectors pMLV delta IN or pMLV*(U3U5) delta IN. This indicated that in the absence of other HIV-1 proteins expressed the MuLV provirus genome was integrated by the expressed HIV-1 IN protein. The integration of these MuLV provirus genomes was further confirmed by polymerase chain reaction analysis on the genomic DNA extracted from the transfected cells using the MuLV IN sequence remained from partial deletion as a target.

Cell Line↗
PubMed2003

Arm sequences contribute to the architecture and catalytic function of a lambda integrase-Holliday junction complex.

lambda integrase (Int) mediates recombination between attachment sites on lambda phage and E. coli DNAs. With the assistance of accessory proteins that induce DNA loops, Int bridges pairs of distinct arm- and core-type DNA binding sites to form synapsed recombination complexes, which then recombine via a Holliday junction (HJ) intermediate. We show that, in addition to promoting the proper positioning of Int protomers, the arm sequences facilitate the catalytic activities of the Int tetramer, independent of accessory proteins or physical continuity between the arm and core sites. We have determined the architecture of ternary complexes containing a HJ, Int, and P'1,2 arm-type DNA. These structures accommodate simultaneous binding of Int to direct-repeat arm sites and indirect-repeat core sites and afford a new view of the higher-order recombinogenic complexes.

Bacteriophage lambda↗
PubMed2000

Haploinsufficiency of Snf5 (integrase interactor 1) predisposes to malignant rhabdoid tumors in mice.

Malignant rhabdoid tumor (MRT) is an aggressive, highly lethal cancer of young children. Tumors occur in various locations, including kidney, brain, and soft tissues. Despite intensive therapy, 80% of affected children die, often within 1 year of diagnosis. The majority of MRT samples and cell lines have sustained biallelic inactivating mutations of the hSNF5 (integrase interactor 1) gene, suggesting that hSNF5 may act as a tumor suppressor. We sought to examine the role of Snf5 in development and cancer in a murine model. Here we report that Snf5 is widely expressed during embryogenesis with focal areas of high-level expression in the mandibular portion of the first branchial arch and central nervous system. Homozygous knockout of Snf5 results in embryonic lethality by embryonic day 7, whereas heterozygous mice are born at the expected frequency and appear normal. However, beginning as early as 5 weeks of age, heterozygous mice develop tumors consistent with MRT. The majority of tumors arise in soft tissues derived from the first branchial arch. Our findings constitute persuasive genetic evidence that Snf5, a core member of the Swi/Snf chromatin-remodeling complex, functions as a tumor suppressor gene, and, moreover, Snf5 heterozygotes provide a murine model of this lethal pediatric cancer.

Amino Acid Sequence↗
PubMed1999

Crystal structures of catalytic core domains of retroviral integrases and role of divalent cations in enzymatic activity.

Crystal structures of the enzymatically competent catalytic domains of HIV-1 and ASV IN have been solved in the last few years. The structure of HIV-1 IN has been described only for apoenzyme and for a complex with Mg2+, whereas the structure of ASV IN has been presented as the apoenzyme, in the presence of divalent cations (Mn2+, Mg2+, Ca2+, Zn2+, and Cd2+), and with an inhibitor. A single ion of Mn2+, Mg2+, or Ca2+ interacts with the two aspartate side chains of the D,D(35)E catalytic center in octahedral coordination with four water molecules. However, two ions of Zn2+ or Cd2+ bind to the active site of IN with tetrahedral and octahedral coordination, respectively. Only small adjustments take place in the active site of ASV IN on binding of the metal cofactor(s), which are absolutely required for the activity of this enzyme. The placement of the side chains and metal ions in the active site is very similar to that observed even in distant members of this superfamily of polynucleotidyltransferases. Here the role of divalent cations in the enzymatic activity of IN and the search for inhibitors of this enzyme are discussed.

Amino Acid Sequence↗
PubMed1999

Substrate recognition by retroviral integrases.

Substrate recognition by the retroviral IN enzyme is critical for retroviral integration. To catalyze this recombination event, IN must recognize and act on two types of substrates, viral DNA and host DNA, yet the necessary interactions exhibit markedly different degrees of specificity. Although particular sequences at the viral DNA termini are recognized by IN, many host DNA sequences can serve as the target for integration. Over the last decade, both in vitro and in vivo data have contributed to our understanding of how IN recognizes its substrates. This review provides an overview of the sequence and structure requirements for recognition of viral and host DNA by different retroviral INs and discusses recent progress in mapping protein domains involved in these interactions.

Animals↗
PubMed1999

Inhibitors of human immunodeficiency virus integrase.

Integration of the viral DNA into a host cell chromosome is an essential step for HIV replication and maintenance of persistent infection. Two viral factors are essential for integration: the viral DNA termini (the att sites) and IN. Accruing knowledge of the IN structure, catalytic mechanisms, and interactions with other proteins can be used to design strategies to block integration. A large number of inhibitors have been identified that can be used as leads for the development of potent and selective anti-IN drugs with antiviral activity.

Animals↗