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PubMed2000

Integrons: an antibiotic resistance gene capture and expression system.

Bacteria can transfer genetic information to provide themselves with protection against most antibiotics. The acquisition of resistance gene arrays involves genetic mobile elements like plasmids and transposons. Another class of genetic structures, termed integrons, have been described and contain one or more gene cassettes located at a specific site. Integrons are defined by an intl gene encoding an integrase, a recombination site attl and a strong promoter. At least six classes of integrons have been determined according to their intl gene. Classes 1, 2 and 3 are the most studied and are largely implicated in the dissemination of antibiotic resistance. A gene cassette includes an open reading frame and, at the 3'-end, a recombination site attC. Integration or excision of cassettes occur by a site-specific recombination mechanism catalyzed by the integrase. However, insertion can occur, albeit rarely, at non-specific sites leading to a stable situation for the cassette. Cassettes are transcribed from the common promoter located in the 5'-conserved segment and expression of distal genes is reduced by the presence of upstream cassettes. Most gene cassettes encode antibiotic resistant determinants but antiseptic resistant genes have also been described. Integrons seem to have a major role in the spread of multidrug resistance in gram-negative bacteria but integrons in gram-positive bacteria were described recently. Moreover, the finding of super-integrons with gene-cassettes coding for other determinants (biochemical functions, virulence factors) in Vibrio isolates dating from 1888 suggests the likely implication of this multicomponent cassette-integron system in bacterial genome evolution before the antibiotic era and to a greater extent than initially believed.

Animals↗
PubMed2004

Lamellarins, from A to Z: a family of anticancer marine pyrrole alkaloids.

The lamellarins form a group of more than 30 polyaromatic pyrrole alkaloids, isolated from diverse marine organisms, mainly but not exclusively ascidians and sponges. These molecules fall in three structural groups, with the central pyrrole ring fused or unfused (lamellarins O-R) to adjacent aromatic rings and with the quinoline moiety containing a 5, 6-single--as in lamellarins I-L--or a double bond, as it is the case for lamellarins D and M which are both potent cytotoxic agents. The family also includes sulphated members, such as the integrase inhibitor lamellarin alpha 20-sulfate. This review presents the origin and structure of the lamellarins and summarizes the various chemical pathways which have been proposed to synthesize all lamellarins and different structurally related marine pyrrole alkaloids, including ningalins, storniamides and lukianols. The mechanisms of actions of these marine products are also discussed. Inhibition of HIV-1 integrase by lamellarin alpha 20-sulfate and human topoisomerase I by lamellarin D and Molluscum contagiosum virus topoisomerase by lamellarin H, along with other effects on nuclear proteins, provide an experimental basis indicating that DNA manipulating enzymes are important targets for the lamellarins. Some of these marine compounds exhibit cytotoxic activities against tumor cells in vitro and are insensitive to Pgp-mediated drug efflux. The structure-activity relationships are discussed. Other compounds in the series, without being strongly cytotoxic, can reverse the multidrug resistance phenotype and thus may be useful to promote the therapeutic activity of conventional cytotoxic drugs toward chemoresistant tumors. A complete description of the chemistry and pharmacological profiles of the lamellarins is presented here to shed light on this undervalued family of marine alkaloids.

Alkaloids↗
PubMedDate not supplied

[The role of integrons in dissemination of antibiotic resistance].

Bacteria can transfer genetic information to get protection against most antibiotics. The acquisition of resistance genes involves genetic mobile elements such as plasmids and transposons. Another genetic structures, named integrons, have been described and contain one or more gene cassettes located at a specific site. Integrons contain an intI gene encoding a site-specific recombinase belonging to the integrase family and a recombination site attI. A gene cassette includes an open reading frame and, at the 3'-end, a recombination site attC. Integration or excision of cassettes occurs by a site-specific recombination mechanism catalyzed by the integrase. However, insertion can rarely occur, at non-specific sites leading to a stable situation for the cassette. Cassettes are transcribed from a common promoter located in the 5'-conserved segment and expression of distal genes is reduced by the presence of upstream cassettes. Most gene cassettes encode antibiotic resistant determinants but antiseptic resistant genes have also been described. Integrons seem to have a major role in the spread of multidrug resistance in Gram-negative bacteria but integrons in Gram-positive bacteria have been recently described. Moreover, the finding of super-integrons with gene cassettes coding for other determinants (biochemical functions, virulence factors) in different Gram negative bacteria suggests that integrons are probably implied in bacterial genome evolution.

Anti-Bacterial Agents↗
PubMed2000

Different classes of retrotransposons in coniferous spruce species.

We have used the conservation of reverse transcriptase and integrase domains among retroelements to PCR-amplify three well-known types of these mobile genetic elements. Reverse transcriptase sequences from Ty1-copia were identified in spruce in this way, as well as integrase sequences from the Ty3-gypsy group. Using these sequences as probes against a Picea glauca genomic bank, individual members from the LTR (long terminal direct repeat) groups were obtained. A partial Ty1-copia-type element named Spcl was isolated along with a Ty3-gypsy-type element named Spdl. Genomic Southern hybridizations revealed the complexity and high copy number of LTR retrotransposons in black and white spruce.

Amino Acid Sequence↗
PubMed2005

Characterization of Ty3-gypsy-like elements in Hibiscus syriacus.

Southern blot analysis revealed a ubiquitous distribu-tion and high copy number of Ty3-gypsy-like elements in the genome of Hibiscus syriacus. Comparative phylogenetic analysis of the large subunit of Rubisco and the integrase region of Ty3-gypsy elements in various plant species indicated that the retrotransposon-like sequences have different evolutionary histories and their own unique polymorphism in the H. syriacus population. Sequence-tagged site-restriction fragment length polymorphisms (STS-RFLP) analysis also indicated great variability in the numbers and sequences of Ty3-gypsy-like elements within H. syriacus varieties. Ty3-gypsy-like elements may still be active within H. syriacus, since Northern analysis of wounded leaves of H. syriacus variety Saehan with a probe for the integrase domain gave strong hybridization signals. The sequence heterogeneity and ubiquity of the Ty3-gypsy-like elements in H. syriacus genomes could provide reliable DNA markers for line identification as well for the analysis of genetic diversity in H. syriacus.

Amino Acid Sequence↗
PubMed1997

Equivalent inhibition of half-site and full-site retroviral strand transfer reactions by structurally diverse compounds.

In vitro assay systems which use recombinant retroviral integrase (IN) and short DNA oligonucleotides fail to recapitulate the full-site integration reaction as it is known to occur in vivo. The relevance of using such circumscribed in vitro assays to define inhibitors of retroviral integration has not been formerly demonstrated. Therefore, we analyzed a series of structurally diverse inhibitors with respect to inhibition of both half-site and full-site strand transfer reactions with either recombinant or virion-produced IN. Half-site and full-site reactions catalyzed by avian myeloblastosis virus and human immunodeficiency virus type 1 (HIV-1) IN from virions are shown to be equivalently sensitive to inhibition by compounds which inhibit half-site reactions catalyzed by the recombinant HIV-1 IN. These studies therefore support the utility of using in vitro assays employing either recombinant or virion-derived IN to identify inhibitors of integration.

Animals↗
PubMed2006

New peptide inhibitors of type IB topoisomerases: similarities and differences vis-a-vis inhibitors of tyrosine recombinases.

Topoisomerases relieve topological tension in DNA by breaking and rejoining DNA phosphodiester bonds. Type IB topoisomerases such as vaccinia topoisomerase (vTopo) and human topoisomerase I are structurally and mechanistically similar to the tyrosine recombinase family of enzymes, which includes bacteriophage lambda Integrase (Int). Previously, our laboratory identified peptide inhibitors of Int from a synthetic peptide combinatorial library. The most potent of these peptides also inhibit vTopo. Here, we used the same mixture-based screening procedure to identify peptide inhibitors directly against vTopo using a plasmid relaxation assay. The two most potent new peptides identified, WYCRCK and KCCRCK, inhibit plasmid relaxation, DNA cleavage and Holliday junction (HJ) resolution mediated by vTopo. The peptides tested bind double-stranded DNA at high concentrations but do not appear to displace the enzyme from its DNA substrate. WYCRCK binds specifically to HJ and perturbs the central base-pairing. This peptide also accumulates HJ intermediates when it inhibits Int-mediated recombination, whereas KCCRCK does not. Interestingly, WYCRCK shares four amino acids with a peptide identified against Int, WRWYCR. The octapeptide WRWYCRCK, containing amino acids from both hexapeptides, is more potent than either against vTopo. All peptides are less potent against the type IA Escherichia coli topoisomerase I or against restriction endonucleases. Like the Int-inhibitory peptide WRWYCR, WYCRCK binds to HJs, and both inhibit junction resolution by vTopo. Our results suggest that the newly identified WYCRCK and peptide WRWYCR interact with a distorted DNA intermediate arising during vTopo-mediated catalysis, or interfere with specific interactions between vTopo and DNA.

Bacteriophage lambda↗
PubMed2006

A statistical method for comparing viral growth curves.

Viral replication is often analyzed by growth curves, in which viral multiplication in the presence of host cells is measured as a function of time. Comparing growth curves is one of the most sensitive ways of comparing viral growth under different conditions or for comparing replication of different viral mutants. However, such experiments are rarely analyzed in a statistically rigorous fashion. Here a statistical method is described for comparing curves, using replication of HIV in the presence of an integrase inhibitor as an example. A complication in the analysis arises due to the fact that sequential measurements of virus accumulation are not independent, which constrains the choice of statistical method. In the recommended approach, the values for virus accumulation over time are fitted to an exponential equation, then the means of the extracted growth rates compared using a nonparametric test, either the Mann-Whitney U-test for two samples or the Kruskal-Wallis test for multiple samples. A web-based tutorial for implementing this method is available at http://microb230.med.upenn.edu/tutorials/wangTutorial.

Data Interpretation, Statistical↗
PubMed2004

Prediction of HIV-1 integrase/viral DNA interactions in the catalytic domain by fast molecular docking.

This study details the separate analyses of binding specificity of HIV-1 integrase (IN) and viral B-DNA forms through ligand-receptor docking studies by means of a fast molecular docking method. The application of solvated electrostatics with the University of Houston Brownian Dynamics Program (UHBD) and configurational sampling by the Daughter of Turnip (DOT) docking program resulted in the computation of energies of more than 113 billion configurations for each ligand-receptor docking study, a procedure considered computationally intractable a few years ago. A specific binding pattern of viral DNA to the IN catalytic domain region has been predicted as a result of these calculations. In a representative docked configuration, we observe the 3'-hydroxyl of the conserved deoxyadenosine to be close to one of the two divalent metal ions that are necessary for catalysis. A superimposition of our energy-minimized docked complex on representative structures from a molecular dynamics (MD) simulation of a crystallographically resolved IN/inhibitor complex revealed an overlap of viral DNA with the inhibitor, indicating that the bound inhibitor might operate by blocking substrate binding. The DOT docking calculation also identified a second, adjacent DNA-binding site, which we believe is the nonspecific host DNA binding site. The binding pattern predicted by DOT complements previous electrostatics, MD simulation, photo-cross-linking, and mutagenesis studies and also provides a further refinement of the IN/viral DNA binding interaction as a basis for new structure-based design efforts.

Catalytic Domain↗
PubMed2000

Basis of HTLV type 1 target site selection.

Sequencing integration sites from >/=200 proviruses isolated from infected individuals revealed that HTLV-1 integration is not random at the level of the nucleotide sequence. The virus was found to integrate in A/T-rich regions with a weak consensus sequence at positions within and without the hexameric repeat generated during integration. These features were not associated with a preference for integration near active regions or repeat elements of the host chromosomes. However, about 6% of HTLV-1 proviruses were found to be integrated into transcription units, suggesting that in some cells, HTLV-1 integration may alter gene expression in vivo. Therefore, the target choice in vivo seems to be determined by local features rather than by the accessibility of specific regions. This led us subsequently to analyze the role of the DNA structure in HTLV-1 integration in vitro. Double-strand HTLV-1 or HIV-1 3' LTR extremities were used as substrates for in vitro strand transfer reactions using highly purified HTLV-1 and HIV-1 integrases (INs) expressed in Escherichia coli, and two synthetic naked 50-bp double-strand DNA molecules harboring different structures were used as targets. A fluorometric quantitative analysis of integration products was designed to assess the reaction efficiency for both target sequences. As suggested for HTLV-1 in vivo (present results), and, as previously described for other retroviruses in vitro, the structure of the target was found to greatly influence the site and the efficiency of integration. Both HIV-1 and HTLV-1 INs underwent the same target structural constraint, i.e., a strong preference for curved DNA. Altogether these results indicate that if most or all the regions of the genome appear to be accessible to HTLV-1 integration, local DNA curvature seems to confer a kinetic advantage for both in vitro and in vivo HTLV-1 integration.

Base Sequence↗
PubMed2002

Inhibition of human immunodeficiency virus type 1 integration by diketo derivatives.

A series of diketo derivatives was found to inhibit human immunodeficiency virus type 1 (HIV-1) integrase activity. Only L-708,906 inhibited the replication of HIV-1(III(B)) (50% effective concentration, 12 micro M), HIV-1 clinical strains, HIV-1 strains resistant to reverse transcriptase or fusion inhibitors, HIV-2 (ROD strain) and simian immunodeficiency virus (MAC(251)). The combinations of L-708,906 with zidovudine, nevirapine, or nelfinavir proved to be subsynergistic. In cell culture, addition of L-708,906 could be postponed for 7 h after infection, a moment coinciding with HIV integration. Inhibition of integration in cell culture was confirmed by quantitative Alu-PCR.

Acetoacetates↗
PubMed2003

New anti-human immunodeficiency virus type 1 6-aminoquinolones: mechanism of action.

A 6-aminoquinolone derivative, WM5, which bears a methyl substituent at the N-1 position and a 4-(2-pyridyl)-1-piperazine moiety at position 7 of the bicyclic quinolone ring system, was previously shown to exhibit potent activity against replication of human immunodeficiency virus type 1 (HIV-1) in de novo-infected human lymphoblastoid cells (V. Cecchetti et al., J. Med. Chem. 43:3799-3802, 2000). In this report, we further investigated WM5's mechanism of antiviral activity. WM5 inhibited HIV-1 replication in acutely infected cells as well as in chronically infected cells. The 50% inhibitory concentrations were 0.60 +/- 0.06 and 0.85 +/- 0.05 micro M, respectively. When the effects of WM5 on different steps of the virus life cycle were analyzed, the reverse transcriptase activity and the integrase and protease activities were not impaired. By using a transient trans-complementation assay to examine the activity of WM5 on the replicative potential of HIV-1 in a single round of infection, a sustained inhibition of Tat-mediated long terminal repeat (LTR)-driven transcription (>80% of controls) was obtained in the presence of 5 micro M WM5. Interestingly, the aminoquinolone was found to efficiently complex TAR RNA, with a dissociation constant in the nanomolar range (19 +/- 0.6 nM). These data indicate that WM5 is a promising lead compound for the development of a new class of HIV-1 transcription inhibitors characterized by recognition of viral RNA target(s).

Aminoquinolines↗
PubMed1998

Application of electrospray ionization mass spectrometry for studying human immunodeficiency virus protein complexes.

Mass spectrometry (MS) with electrospray ionization (ESI) has shown utility for studying noncovalent protein complexes, as it offers advantages in sensitivity, speed, and mass accuracy. The stoichiometry of the binding partners can be easily deduced from the molecular weight measurement. In many examples of protein complexes, the gas phase-based measurement is consistent with the expected solution phase binding characteristics. This quality suggests the utility of ESI-MS for investigating solution phase molecular interactions. Complexes composed of proteins from the human immunodeficiency virus (HIV) have been studied using ESI-MS. Multiply charged protein dimers from HIV integrase catalytic core (F185K) and HIV protease have been observed. Furthermore, the ternary complex between HIV protease dimer and inhibitor pepstatin A was studied as a function of solution pH. Zinc binding to zinc finger-containing nucleocapsid protein (NCp7) and the NCp7-psi RNA 1:1 stoichiometry complex was also studied by ESI-MS. No protein-RNA complex was observed in the absence of zinc, consistent with the role of the zinc finger motifs for RNA binding.

Amino Acid Sequence↗
PubMed2003

Mutational analysis of loxP sites for efficient Cre-mediated insertion into genomic DNA.

The Cre/loxP system has been used in transgenic models primarily to excise DNA flanked by loxP sites for gene deletion. However, the insertion reaction is more difficult to control since the excision event is kinetically favored. Mutant loxP sites favoring integration were identified using a novel, bacterial screening system. Utilizing lambda integrase, mutant loxP sites were placed at the E. coli attB site and the excision-insertion ratios of incoming DNA plasmids carrying a second, complementary mutant loxP site were determined. Comparison of 50 mutant loxP sites combinations to the native loxP site revealed that mutations to the inner 6 bp of the Cre binding domain severely inhibited recombination, while those in the outer 8 bps were more tolerated. The most efficient loxP combinations resulted in 1421-fold and 1529-fold increases in relative integration rates over wild-type loxP sites. These loxP mutants could be exploited for site-directed "tag and insert" recombination experiments.

Base Sequence↗
PubMed1990

Genetic elements involved in Tn21 site-specific integration, a novel mechanism for the dissemination of antibiotic resistance genes.

Transposon Tn21 codes for a site-specific integration system, which is probably a novel recombination mechanism, responsible for the acquisition of resistance genes in this widespread family of transposons. Using insertion and deletion mutagenesis we have identified the genetic loci of the various recombination hot-spots (RHS) and of the gene product (the integrase) that catalyses the reaction. The site of recombination has been localized in two of the RHSs to the DNA sequence GTTAG, which is present at the 3' termini of a loosely conserved palindromic sequence of approximately 59 bp. This 59 bp sequence, which flanks the inserted genes in a number of naturally occurring transposons, is the only element required in cis for the recombination reaction.

Base Sequence↗
PubMed1990

HIV-1 replication is controlled at the level of T cell activation and proviral integration.

During progression of the Acquired Immune Deficiency Syndrome (AIDS), the human immunodeficiency virus type 1 (HIV-1) is harbored in CD4+ T cells, which act as the primary reservoir for the virus. In vitro, HIV-1 requires activated T cells for a productive infection; however, in vivo, the number of circulating T cells in the activated state that are potential targets for HIV-1 infection is low. We have investigated the ability of HIV-1 to infect resting T cells, and the consequences of such an infection. T cell activation was not required for HIV-1 infection; however, viral DNA was unable to integrate in resting T cells and was maintained extrachromosomally. Subsequent T cell activation allowed integration of extrachromosomal forms and led to a productive viral life cycle. Extrachromosomal forms of viral DNA were found to persist for several weeks after infection of resting T cells and, following T cell activation, these forms maintained their ability to integrate and act as a template for infectious virus. Several lines of evidence, including temporal analysis of HIV-1 replication and analysis of an HIV-1 integrase deletion mutant, indicated that extra-chromosomal HIV-1 DNA genomes were transcriptionally active. These results are compatible with a model whereby HIV-1 can persist in a non-productive extra-chromosomal state in resting T cells until subsequent antigen-induced or mitogen-induced T cell activation, virus integration and release. Thus agents that induce T cell activation may control the rate of HIV-1 replication and spread during AIDS progression.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Two defective forms of reverse transcriptase can complement to restore retroviral infectivity.

Retroviral DNA synthesis requires both the DNA polymerase and the RNaseH activities of reverse transcriptase (RT). To test whether two defective RTs--one carrying a mutation in the RNaseH domain and the other with a mutation in DNA polymerase--could work together to complete viral DNA synthesis, we generated phenotypically mixed virions of Moloney murine leukemia virus (M-MuLV) that contained two kinds of mutant RTs. One RNaseH catalytic site mutant complemented both tested DNA polymerase mutants and small amounts of intact viral DNA were generated. This demonstrates that retroviral DNA synthesis can be completed--albeit inefficiently--when DNA polymerase and RNaseH activities are provided by separate RT molecules. Other RNaseH mutants failed to complement, suggesting that some aspects of the RNaseH domain are essential to RT's DNA polymerase function. Phenotypically mixed virions were also used to demonstrate that RT and integrase (IN) can be provided by separate polyprotein precursors and complete the early stages of retroviral replication.

3T3 Cells↗
PubMed1994

DNA bending creates favored sites for retroviral integration: an explanation for preferred insertion sites in nucleosomes.

The choice of retroviral integration sites is strongly influenced by chromatin: integration in vitro occurs more efficiently into nucleosomal DNA than into naked DNA, and a characteristic pattern of preferred insertion sites with a 10 bp periodicity is observed at the outer face of the nucleosomal DNA. At least three features of nucleosomal DNA could be responsible for the creation of these favored sites: the presence of histones, attachment of the DNA to a protein surface, and DNA bending. To test each of these possibilities, we studied integration in vitro with human immunodeficiency virus and murine leukemia virus integrases into four model targets that mimic features of nucleosomal DNA: (i) catabolite activator protein-DNA complexes; (ii) lac repressor-operator complexes; (iii) lac repressor-induced loops; and (iv) intrinsically bent A-tract DNA. We found that bending of the target DNA can create favored integration sites at the outer face of the helix, irrespective of whether the bent DNA is attached to a protein surface. Our findings offer an explanation for the preferred usage of nucleosomes as integration targets. In addition, they suggest that bending of the target DNA might be an intrinsic feature of the integration reaction.

Base Sequence↗