Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Integrase”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,531 records · Page 85Linked to original sources
PubMed2005

The endonuclease domain of bacteriophage terminases belongs to the resolvase/integrase/ribonuclease H superfamily: a bioinformatics analysis validated by a functional study on bacteriophage T5.

Bacteriophage terminases are essential molecular motors involved in the encapsidation of viral DNA. They are hetero-multimers whose large subunit encodes both ATPase and endonuclease activities. Although the ATPase domain is well characterized from sequence and functional analysis, the C-terminal region remains poorly defined. We describe sequence-structure comparisons of the endonuclease region of various bacteriophages that revealed new sequence similarities shared by this region and the Holliday junction resolvase RuvC and to a lesser extent the HIV integrase and the ribonuclease H. Extensive sequence comparison and motif refinement led to a common signature of terminases and resolvases with three conserved acidic residues engaged in catalytic activity. Sequence analyses were validated by in vivo and in vitro functional assays showing that the nuclease activity of the endonuclease domain of bacteriophage T5 terminase was abolished by mutation of any of the three predicted catalytic aspartates. Overall, these data suggest that the endonuclease domains of terminases operate autonomously and that they adopt a fold similar to that of resolvases and share the same divalent cation-dependent enzymatic mechanism.

Amino Acid Sequence↗
PubMedDate not supplied

Synthesis of oligonucleotide-intercalator conjugates capable to inhibit HIV-1 DNA integration.

This investigation is devoted to design of short "switch" oligonucleotides mono- or bi-functionnalized with intercalating agents capable to form a stable triplex with HIV integrase-cognate sequences and inhibit selectively HIV integration. Methods of intercalator incorporation at 5'- and/or 3'-terminal positions or one of the pyrimidine heterocyclic bases are developed.

Base Sequence↗
PubMed1999

Multidrug-resistant human and animal Salmonella typhimurium isolates in France belong predominantly to a DT104 clone with the chromosome- and integron-encoded beta-lactamase PSE-1.

Epidemiologic relationships were investigated in 187 ampicillin-resistant Salmonella typhimurium strains (86 human, 101 animal) from >2000 strains isolated in 1994. Of 23 resistance patterns, the most frequent (ampicillin [Am], chloramphenicol [Cm], tetracycline [Tc], streptomycin and spectinomycin [Sm], and sulfonamides [Su]) was found in 69.5% of human and 64.8% of animal isolates. Four beta-lactamase genes were identified, blaTEM (24%), blaPSE-1 (78%), and blaSHV and oxa-2 (each <3%). blaPSE-1 and the integrase gene, intI1, but not blaTEM, blaSHV or oxa-2, were chromosomeborne and found almost exclusively in the AmCmTcSmSu strains. In these, polymerase chain reaction mapping revealed two distinct integrons carrying blaPSE-1 or aadA2. Lysotypes, plasmid profiles, and restriction fragment length polymorphisms (IS200) were determined for 50 representative isolates and for 3 DT104 strains from the United Kingdom (UK). The phage type of the PSE-1-producing AmCmTcSmSu strains was 12 atypic, indistinguishable from that of the DT104 strains. The combined data indicate that the same multiresistant clone has spread through human and animal ecosystems in the UK and France.

Animals↗
PubMed2002

Gene therapy using a simian virus 40-derived vector inhibits the development of in vivo human immunodeficiency virus type 1 infection of severe combined immunodeficiency mice implanted with human fetal thymic and liver tissue.

To evaluate the in vivo efficacy of gene therapy for treating human immunodeficiency virus type 1 (HIV-1) infection, a novel simian virus (SV) 40-derived vector gene delivery system that efficiently transduces human leukocytes was combined with a model using severe combined immunodeficiency mice infected with HIV-1 and implanted with human fetal thymic and liver tissue (thy/liv-SCID-hu mice). The SV40-derived vector, SV(Aw), which encodes a variable fragment antibody recognizing HIV-1 integrase (IN#33),was injected into the human thymic grafts of thy/liv-SCID-hu mice and induced IN#33 expression in most of the thymocytes in the graft. After in vivo challenge with HIV-1, IN#33 expression inhibited in vivo HIV-1 infection, as evidenced by the markedly lower number of HIV-1-infected thymocytes detected in human thymic grafts injected with the SV(Aw) vector, compared with those injected with a control SV40-derived vector. Thus, these findings demonstrate the utility of this new mouse model system for assessing the in vivo efficacy of HIV-1-specific gene therapy. In addition, these data indicate that SV40-derived vectors may provide a system capable of efficient in vivo gene delivery.

Animals↗
PubMed2005

Unnoticed spread of integron-carrying Enterobacteriaceae in intensive care units.

BACKGROUND: Integrons are strongly associated with multidrug resistance in Enterobacteriaceae. Little is known about the natural history of integron-associated resistance in hospitals during nonoutbreak periods. The prevalence of integrons and the incidence of cross-transmission and horizontal gene transfer in Enterobacteriaceae with reduced susceptibility to cephalosporins (ERSC) were determined for 2 intensive care units (ICUs). METHODS: Microbiological surveillance using rectal swab samples obtained 2 times per week and genotyping using amplified fragment-length polymorphism (AFLP) were used to determine colonization with and genetic relatedness of ERSC. IntI1 integrase polymerase chain reaction (PCR), conserved-segment PCR, restriction fragment-length polymorphism, and DNA sequencing were used to determine the prevalence and contents of integrons. RESULTS: Of 457 patients, 121 patients were colonized with ERSC, and 174 isolates underwent AFLP and PCR. In 34 isolates obtained from 31 patients, 11 different integrons were identified; these integrons encoded resistance to streptomycin/spectinomycin, gentamicin/tobramycin/kanamycin, chloramphenicol, and trimethoprim. Integrons could be divided into 7 clusters of > or =2 isolates each. Compared with isolates that were negative for integrons, isolates that were positive for integrons were associated with resistance to piperacillin, cephalosporins, aminoglycosides, and quinolones. Acquisition rates of integron-carrying ERSC were 10 cases per 1000 patient-days in the first ICU and 8 cases per 1000 patient-days in the second ICU, with most cases (26 of 34) being acquired during the ICU stay. Nineteen episodes resulted from cross-transmission. In addition, 2 cases of interspecies transfer and 1 case of intraspecies transfer of integrons were recorded. Younger age was independently associated with acquisition of integron-carrying ERSC (hazard ratio, 0.953; 95% confidence interval, 0.926-0.987). CONCLUSION: Surveillance, genotyping, and integron analysis identified previously unnoticed outbreaks of integron-carrying ERSC. Cross-transmission appeared to be the dominant route of transmission. Therefore, barrier precautions are necessary to prevent further spread.

Adult↗
PubMed2001

A retroviral DNA vaccine vector.

A versatile DNA vaccine (pdIV3) was constructed by replacing the integrase, vif, vpx, and vpr genes of a pathogenic simian immunodeficiency virus (SIV) molecular clone with a linker containing unique cloning sites. The 5' long terminal repeat (LTR) is truncated and transcription is controlled by a cytomegalovirus (CMV) promoter. The construct expresses Gag and Env in vitro and noninfectious virus particles are produced from transfected cells. The ability of pdIV3 to promote cellular and humoral immune responses, along with the flexibility of the linker design to allow insertion of immunostimulatory genes in future constructs, makes this a useful base vector for immunization against primate lentiviruses. We present the construction of a retroviral plasmid designed to serve as a template for the development of safe and effective vaccines against primate immunodeficiency retroviruses. This vaccine component should facilitate the simultaneous induction of cellular and humoral immune responses that protect primates against infection with SIV and human immunodeficiency virus (HIV) and the development of acquired immune deficiency syndrome (AIDS). This plasmid could induce the appropriate immune response required to attack both cell-free and cell-associated viruses. The lack of infectivity, the inability to integrate, and the SIV origin make this construct a safe alternative to attenuated vaccines based on HIV. In addition, we intend to develop this construct as an immunotherapeutic approach to lower the viremia in AIDS patients.

Animals↗
PubMed2003

HIV type 1 infection in Pygmy hunter gatherers is from contact with Bantu rather than from nonhuman primates.

To investigate the route of zoonotic transmission of HIV-1, we isolated three and seven HIV-1 strains from 449 Pygmy hunter gatherers and 169 neighboring Bantu, respectively, in southern Cameroon. Phylogenetic analysis based on pol-integrase and env-C2V3 sequences revealed that strains from Pygmies were 1CRF02_AG/CRF02_AG, 1 subtype G/CRF02 AG (pol/env), and 1 CRFll_cpx/CRF11_cpx, and that those from Bantu were 2 CRF02_AG/CRF02_AG, 1 CRF02_AG/CRF01_AE/A, 1 CRF02_AG/subtype A, 1 G/A, 1G/CRF02_AG, and 1 unclassified fH. CRF02_AG and CRF11_cpx have been identified in Cameroon. The results suggest that HIV-1 has been introduced into Pygmies through their neighboring Bantu rather than directly from nonhuman primates.

Adult↗
PubMed2004

Multiprotein HIV type 1 clade B DNA and MVA vaccines: construction, expression, and immunogenicity in rodents of the MVA component.

Recombinant modified vaccinia virus Ankara (MVA) expressing SIV or SHIV Gag-Pol and Env, alone or in conjunction with a related DNA vaccine, effectively controls immunodeficiency virus infections in nonhuman primates. Here we describe the construction, characterization, and immunogenicity of MVA/HIV 48, a candidate HIV-1 clade B Gag-Pol-Env vaccine. A novel transfer vector was designed to allow the incorporation of HIV genes regulated by vaccinia virus promoters together with a reporter gene into a single site in the MVA genome and to automatically delete the reporter after the initial isolation of the recombinant MVA. MVA/HIV 48 contains chimeric HIV-1 HXB-2/BH10 gag-pol sequences, a deletion of integrase, inactivating point mutations in reverse transcriptase, and HIV-1 ADA env sequences with a truncation of most of the cytoplasmic domain to enhance expression on the plasma membrane. Cells infected with MVA/HIV 48 expressed HIV proteins, which were processed to the expected size. The Env was inserted into the plasma membrane and was functional in a CCR5 coreceptor-dependent cell fusion assay. Moreover, virus-like particles were released into the medium and budding particles containing Env were visualized by immunoelectron microscopy. Rodents that were immunized with MVA/HIV 48 produced antibodies, which neutralized a heterologous HIV-MN strain, and Gag-specific CD8 T cells. In the accompanying paper, we show that MVA/HIV 48 provided efficient boosting of an HIV DNA vaccine.

AIDS Vaccines↗
PubMed2001

Virion-targeted viral inactivation: new therapy against viral infection.

BACKGROUND: Acquired immune deficiency syndrome (AIDS) is resistant to all current therapy. Gene therapy is an attractive alternative or additive to current, unsatisfactory AIDS therapy. MATERIALS AND METHODS: To develop an antiviral molecule targeting viral integrase (HIV IN), we generated a single-chain antibody, termed scAb, which interacted with human immunodeficiency virus type 1 (HIV-1) IN and inhibited virus replication at the integration step when expressed intracellularly. To reduce infectivity from within the virus particles, we made expression plasmids (pC-scAbE-Vpr, pC-scAbE-CA, and pC-scAbE-WXXF), which expressed the anti-HIV IN scAb fused to the N-terminus of HIV-1-associated accessory protein R (Vpr), capsid protein (CA), and specific binding motif to Vpr (WXXF), respectively. All fusion proteins were tagged with a nine-amino acid peptide derived from influenza virus hemagglutinin (HA) at the C terminus. RESULTS: The fusion molecules, termed scAbE-Vpr, scAbE-CA, and scAbE-WXXF, interacted specifically with HIV IN immobilized on a nitrocellulose membrane. Immunoblot analysis showed that scAbE-Vpr, scAbE-CA, and scAbE-WXXF were incorporated into the virions produced by cotransfection of 293T cells with HIV-1 infectious clone DNA (pLAI) and pC-scAbE-Vpr, pC-scAbE-WXXF. A multinuclear activation galactosidase indicator (MAGI) assay revealed that the virions released from 293T cells cotransfected with pLAI and pC-scAbE-Vpr, pC-scAbE-WXXF had as little 1000-fold of the infectivity of the control wild-type virions, which were produced from the 293T cells transfected with pLAI alone. Furthermore, the virions produced from the 293T cells cotransfected with pLAI and an scAb expression vector (pC-scAb) showed only 1% of the infectivity of the control HIV-1 in a MAGI assay, although scAb was not incorporated into the virions. In either instance, the total quantity of the progeny virions released from the transfected 293T cells and the patterns of the virion proteins were hardly affected by the presence of scAb, scAbE-Vpr, or scAbE-WXXF, as determined by virion-associated reverse transcriptase assay and by immunoblot analysis, respectively. Because G418-selected HeLa clones carrying the expression plasmid for scAbE-WXXF were obtained much more frequently than those for scAbE-Vpr, scAbE-WXXF was inferred to be less toxic to cells than scAbE-Vpr. The result that scAbE-WXXF with viral incorporation achieved more than a 10-fold reduction in infectivity of the progeny virions than scAb without incorporation suggests that scAbE-WXXF is a potential antiviral molecule, inhibiting replication by neutralization of HIV IN activity both within cells and within virions. Moreover, it is nontoxic to human cells. We termed this gene therapy "virion-"targeted-viral inactivation" and these molecules "packageable antiviral therapeutics." CONCLUSION: This new gene therapy has the potential for wide application in many viral infectious diseases.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Characterization of HIV replication complexes early after cell-to-cell infection.

In this study, we have characterized the HIV DNA-containing replication complexes present in cells early after cell-to-cell infection, using sucrose gradient sedimentation and immunoprecipitation. Six hours after cell-to-cell infection, a cytoplasmic HIV replication complex sedimented as a large structure (320S). This replication complex was precipitated by antisera to three virus-coded enzymes (reverse transcriptase, integrase, protease), to the matrix protein (p17), and to cellular histones but not to the major capsid protein (p24). This replication complex was not associated with cell membranes and could not be dissociated into smaller discrete subunits, using detergents. Nuclear extracts from the same cell-to-cell infection contained a smaller (80S) complex that lacked reverse transcriptase and matrix protein (p17). Cytoplasmic replication complexes from a cell-free virus infection sedimented as 160S structures under identical conditions, as previously reported. Our results indicate that, following cell-to-cell transmission of HIV, all the HIV pol gene products, the matrix protein p17, and cellular histones are present in cytoplasmic replication complexes that are taking part in or have completed reverse transcription. Transportation of the cytoplasmic replication complex to the nucleus is associated with structural changes, including a reduction in size and altered protein composition.

Base Sequence↗
PubMed1994

HIV type 2 proviral load measured by quantitative polymerase chain reaction correlates with CD4+ lymphopenia in HIV type 2-infected individuals.

The efficiency of detection of 2 sets of primer pairs from putatively conserved regions of the human immunodeficiency virus type 2 (HIV-2) genome were assessed in 86 seropositive individuals from The Gambia by nested polymerase chain reaction (PCR). HIV-2 long terminal repeat (LTR) target sequences were detected in DNA extracted from peripheral blood mononuclear cells (PBMCs) in 84 of 86 (97%) individuals whereas HIV-2 integrase (pol) gene sequences were detected in 39 of 41 (95%) individuals. The use of LTR target sequences and recombinant Pfu DNA polymerase, rather than Taq polymerase, in a modified secondary amplification reaction mediated the incorporation of 35S-labeled nucleotides in a quantitative radiometric assay. This sensitive assay was used to quantify HIV-2 proviral DNA in clinical samples and compared well with estimations by limiting end-point dilution of target molecules. A linear response between counts and the number of copies amplified from serial dilutions of pROD10 plasmid DNA (3-2000 copies) yielded a standard curve to allow extrapolation to clinical data. Increased levels of HIV-2 proviral DNA, expressed as copies per 10(5) CD4-positive lymphocytes, were associated with declining CD4 count in 63 adult patients (Spearman rank correlation, r = -0.71, n = 63, p < 0.001) and with the occurrence of HIV-related clinical disease. Kruskall-Wallis analysis of variance analysis showed the mean proviral copy number (log10) to be significantly different between groups (p < 0.001) where CD4 counts were grouped as < 200/mm3 (3.4 +/- 1.05 copies), 200-500/mm3 (2.84 +/- 0.93 copies), and > 500/mm3 (1.88 +/- 0.43 copies).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed2006

Phylogenetic analysis of Brazilian HIV type 1 subtype D strains: tracing the origin of this subtype in Brazil.

HIV-1 Subtype D occurs mainly in East and Central African countries, especially Uganda, where the prevalence of HIV-1 infection is among the highest in the world. We present the phylogenetic analysis of one nonautochthonous and four autochthonous (including a near full-length genome) Brazilian HIV-1 subtype D strains identified in Rio de Janeiro State, where subtypes B, F1, and BF1 recombinants predominate. Phylogenetic inferences using maximum likelihood were applied on a near-full length genome and on concatenated gag, protease, reverse transcriptase, integrase, C2V3/env, gp41, and nef segments. Sequences from an Angolan immigrant showed close genetic similarity with a strain described in Finland, from an HIV patient of African origin, whereas all four autochthonous Brazilian sequences clustered with South African strains, where subtype D occurs only in isolated cases. Our results suggest the successful introduction and circulation in Brazil of closely related HIV-1 subtype D strains, possibly of South African origin.

Brazil↗
PubMed2006

Molecular analysis of chromosomal rearrangements in mammalian cells after phiC31-mediated integration.

Reports on insertional mutagenesis due to integration of gene therapy vectors into the host genome have raised concerns about the genetic manipulation of somatic cells. Previously, it was demonstrated that integrase phiC31 derived from a Streptomyces phage mediates site-specific integration into the host genome of mammalian cells in vitro and in vivo by recombining the attB recognition site in an episomal plasmid and one or more pseudoattP sites in the host chromosomes. In the present study we investigated whether cryptic phiC31 recognition sites in the host genome may result in chromosomal rearrangements. Of 69 independent integration events analyzed in human cells, 6 (8.7%) integrated into human chromosome 19 (19q13.31) and 10 (14.49%) integrated into human chromosome 12 (12q22). Most importantly, of all integration sites analyzed, 15% were found to contain an integrated transgene that was flanked by DNA sequences originating from two different chromosomes. To confirm chromosomal translocations we performed a polymerase chain reaction analysis of chromosomal DNA flanking the transgene and also performed limited studies to determine the genotype of single-cell clones. Although the mechanism responsible for chromosomal translocations needs to be further characterized, we speculate that cryptic phiC31 attachment sites flanking the transgene and cryptic phiC31 attachment sites in the host genome recombine with each other.

Animals↗
PubMed2005

Integrons in Escherichia coli from food-producing animals in The Netherlands.

The presence and character of class 1 integrons in multidrug-resistant Escherichia coli from slaughter animals and meat was determined by integrase-specific PCR and conserved segment PCR-restriction fragment length polymorphism (RFLP). At least five different class 1 integron types were found and three types were shared between hospitalized patients, humans in the community, meat, and slaughter animals. Common integron types indicate that antibiotic resistance genes are exchanged via the food chain between different reservoirs of both human and animal origin.

Animals↗
PubMed1997

Functional RT and IN incorporated into HIV-1 particles independently of the Gag/Pol precursor protein.

The expression and incorporation of retroviral enzymes into virions in the form of Gag/Pol precursor polyproteins is believed to be important for the assembly of infectious viral particles. HIV-1 encodes a 160 kDa Gag/Pol precursor that includes Gag, protease (PR), reverse transcriptase (RT) and integrase (IN). We have developed the use of HIV accessory proteins (Vpr and Vpx) as vehicles to incorporate protein of both viral and non-viral origin into virions by expression in trans as heterologous fusion proteins (Wu et al., 1995, 1996a). To analyze the role of Gag/Pol in the formation of infectious virions, we incorporated RT and IN into HIV-1 particles in trans, as fusion partners of viral protein R (Vpr). Virions derived from an RT and IN minus proviral clone were infectious and replicated through a complete cycle of infection when RT and IN proteins were provided in trans. These results demonstrate that functional RT and IN proteins can be provided in trans, and that their expression and incorporation into virions as components of Gag/Pol are not required for the formation of infectious virions. Thus, for the first time, we have demonstrated for a human pathogenic retrovirus that processes of assembly and the function of critical viral enzymes can be unlinked. This finding will provide unique opportunities to explore retroviral RT/IN function and the role of Gag/Pol in the formation of infectious virions in the context of a replicating virus (in vivo).

Blotting, Western↗
PubMed1997

Crystal structure of the site-specific recombinase, XerD.

The structure of the site-specific recombinase, XerD, that functions in circular chromosome separation, has been solved at 2.5 A resolution and reveals that the protein comprises two domains. The C-terminal domain contains two conserved sequence motifs that are located in similar positions in the structures of XerD, lambda and HP1 integrases. However, the extreme C-terminal regions of the three proteins, containing the active site tyrosine, are very different. In XerD, the arrangement of active site residues supports a cis cleavage mechanism. Biochemical evidence for DNA bending is encompassed in a model that accommodates extensive biochemical and genetic data, and in which the DNA is wrapped around an alpha-helix in a manner similar to that observed for CAP complexed with DNA.

Amino Acid Sequence↗
PubMed1999

Wild-type Flp recombinase cleaves DNA in trans.

Site-specific recombinases of the Integrase family utilize a common chemical mechanism to break DNA strands during recombination. A conserved Arg-His-Arg triad activates the scissile phosphodiester bond, and an active-site tyrosine provides the nucleophile to effect DNA cleavage. Is the tyrosine residue for the cleavage event derived from the same recombinase monomer which provides the RHR triad (DNA cleavage in cis), or are the triad and tyrosine derived from two separate monomers (cleavage in trans)? Do all members of the family follow the same cleavage rule, cis or trans? Solution studies and available structural data have provided conflicting answers. Experimental results with the Flp recombinase which strongly support trans cleavage have been derived either by pairing two catalytic mutants of Flp or by pairing wild-type Flp and a catalytic mutant. The inclusion of the mutant has raised new concerns, especially because of the apparent contradictions in their cleavage modes posed by other Int family members. Here we test the cleavage mode of Flp using an experimental design which excludes the use of the mutant protein, and show that the outcome is still only trans DNA cleavage.

Amino Acid Sequence↗
PubMed1999

X-ray structure of T4 endonuclease VII: a DNA junction resolvase with a novel fold and unusual domain-swapped dimer architecture.

Phage T4 endonuclease VII (Endo VII), the first enzyme shown to resolve Holliday junctions, recognizes a broad spectrum of DNA substrates ranging from branched DNAs to single base mismatches. We have determined the crystal structures of the Ca2+-bound wild-type and the inactive N62D mutant enzymes at 2.4 and 2.1 A, respectively. The Endo VII monomers form an elongated, highly intertwined molecular dimer exhibiting extreme domain swapping. The major dimerization elements are two pairs of antiparallel helices forming a novel 'four-helix cross' motif. The unique monomer fold, almost completely lacking beta-sheet structure and containing a zinc ion tetrahedrally coordinated to four cysteines, does not resemble any of the known junction-resolving enzymes, including the Escherichia coli RuvC and lambda integrase-type recombinases. The S-shaped dimer has two 'binding bays' separated by approximately 25 A which are lined by positively charged residues and contain near their base residues known to be essential for activity. These include Asp40 and Asn62, which function as ligands for the bound calcium ions. A pronounced bipolar charge distribution suggests that branched DNA substrates bind to the positively charged face with the scissile phosphates located near the divalent cations. A model for the complex with a four-way DNA junction is presented.

Amino Acid Sequence↗