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Sample records for asv integrase proteins

  1. Biochemical Characterization of Novel Retroviral Integrase Proteins

    PubMed Central

    Ballandras-Colas, Allison; Naraharisetty, Hema; Li, Xiang; Serrao, Erik; Engelman, Alan

    2013-01-01

    Integrase is an essential retroviral enzyme, catalyzing the stable integration of reverse transcribed DNA into cellular DNA. Several aspects of the integration mechanism, including the length of host DNA sequence duplication flanking the integrated provirus, which can be from 4 to 6 bp, and the nucleotide preferences at the site of integration, are thought to cluster among the different retroviral genera. To date only the spumavirus prototype foamy virus integrase has provided diffractable crystals of integrase-DNA complexes, revealing unprecedented details on the molecular mechanisms of DNA integration. Here, we characterize five previously unstudied integrase proteins, including those derived from the alpharetrovirus lymphoproliferative disease virus (LPDV), betaretroviruses Jaagsiekte sheep retrovirus (JSRV), and mouse mammary tumor virus (MMTV), epsilonretrovirus walleye dermal sarcoma virus (WDSV), and gammaretrovirus reticuloendotheliosis virus strain A (Rev-A) to identify potential novel structural biology candidates. Integrase expressed in bacterial cells was analyzed for solubility, stability during purification, and, once purified, 3′ processing and DNA strand transfer activities in vitro. We show that while we were unable to extract or purify accountable amounts of WDSV, JRSV, or LPDV integrase, purified MMTV and Rev-A integrase each preferentially support the concerted integration of two viral DNA ends into target DNA. The sequencing of concerted Rev-A integration products indicates high fidelity cleavage of target DNA strands separated by 5 bp during integration, which contrasts with the 4 bp duplication generated by a separate gammaretrovirus, the Moloney murine leukemia virus (MLV). By comparing Rev-A in vitro integration sites to those generated by MLV in cells, we concordantly conclude that the spacing of target DNA cleavage is more evolutionarily flexible than are the target DNA base contacts made by integrase during integration. Given their

  2. Interactions of Host Proteins with the Murine Leukemia Virus Integrase

    PubMed Central

    Studamire, Barbara; Goff, Stephen P.

    2010-01-01

    Retroviral infections cause a variety of cancers in animals and a number of diverse diseases in humans such as leukemia and acquired immune deficiency syndrome. Productive and efficient proviral integration is critical for retroviral function and is the key step in establishing a stable and productive infection, as well as the mechanism by which host genes are activated in leukemogenesis. Host factors are widely anticipated to be involved in all stages of the retroviral life cycle, and the identification of integrase interacting factors has the potential to increase our understanding of mechanisms by which the incoming virus might appropriate cellular proteins to target and capture host DNA sequences. Identification of MoMLV integrase interacting host factors may be key to designing efficient and benign retroviral-based gene therapy vectors; key to understanding the basic mechanism of integration; and key in designing efficient integrase inhibitors. In this review, we discuss current progress in the field of MoMLV integrase interacting proteins and possible roles for these proteins in integration. PMID:21637732

  3. Retroviral Integrase Proteins and HIV-1 DNA Integration*

    PubMed Central

    Krishnan, Lavanya; Engelman, Alan

    2012-01-01

    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. PMID:23043109

  4. Retroviral integrase protein and intasome nucleoprotein complex structures

    PubMed Central

    Grawenhoff, Julia; Engelman, Alan N

    2017-01-01

    Retroviral replication proceeds through the integration of a DNA copy of the viral RNA genome into the host cellular genome, a process that is mediated by the viral integrase (IN) protein. IN catalyzes two distinct chemical reactions: 3’-processing, whereby the viral DNA is recessed by a di- or trinucleotide at its 3’-ends, and strand transfer, in which the processed viral DNA ends are inserted into host chromosomal DNA. Although IN has been studied as a recombinant protein since the 1980s, detailed structural understanding of its catalytic functions awaited high resolution structures of functional IN-DNA complexes or intasomes, initially obtained in 2010 for the spumavirus prototype foamy virus (PFV). Since then, two additional retroviral intasome structures, from the α-retrovirus Rous sarcoma virus (RSV) and β-retrovirus mouse mammary tumor virus (MMTV), have emerged. Here, we briefly review the history of IN structural biology prior to the intasome era, and then compare the intasome structures of PFV, MMTV and RSV in detail. Whereas the PFV intasome is characterized by a tetrameric assembly of IN around the viral DNA ends, the newer structures harbor octameric IN assemblies. Although the higher order architectures of MMTV and RSV intasomes differ from that of the PFV intasome, they possess remarkably similar intasomal core structures. Thus, retroviral integration machineries have adapted evolutionarily to utilize disparate IN elements to construct convergent intasome core structures for catalytic function. PMID:28289517

  5. Similarities in the HIV-1 and ASV Integrease Active Site Upon Metal Binding

    SciTech Connect

    Lins, Roberto D.; Straatsma, TP; Briggs, J. M.

    2000-04-05

    The HIV-1 integrase, which is essential for viral replication, catalyzes the insertion of viral DNA into the host chromosome thereby recruiting host cell machinery into making viral proteins. It represents the third main HIV enzyme target for inhibitor design, the first two being the reverse transcriptase and the protease. We report here a fully hydrated 2 ns molecular dynamics simulation performed using parallel NWChem3.2.1 with the AMBER95 force field. The HIV-1 integrase catalytic domain previously determined by crystallography (1B9D) and modeling including two Mg2+ ions placed into the active site based on an alignment against an ASV integrase structure containing two divalent metals (1VSH), was used as the starting structure. The simulation reveals a high degree of flexibility in the region of residues 140-149 even in the presence of a second divalent metal ion and a dramatic conformational change of the side chain of E152 when the second metal ion is present. This study shows similarities in the behavior of the catalytic residues in the HIV-1 and ASV integrases upon metal binding. The present simulation also provides support to the hypothesis that the second metal ion is likely to be carried into the HIV-1 integrase active site by the substrate, a strand of DNA.

  6. von Hippel–Lindau binding protein 1-mediated degradation of integrase affects HIV-1 gene expression at a postintegration step

    PubMed Central

    Mousnier, Aurélie; Kubat, Nicole; Massias-Simon, Aurélie; Ségéral, Emmanuel; Rain, Jean-Christophe; Benarous, Richard; Emiliani, Stéphane; Dargemont, Catherine

    2007-01-01

    HIV-1 integrase, the viral enzyme responsible for provirus integration into the host genome, can be actively degraded by the ubiquitin–proteasome pathway. Here, we identify von Hippel–Lindau binding protein 1(VBP1), a subunit of the prefoldin chaperone, as an integrase cellular binding protein that bridges interaction between integrase and the cullin2 (Cul2)-based von Hippel–Lindau (VHL) ubiquitin ligase. We demonstrate that VBP1 and Cul2/VHL are required for proper HIV-1 expression at a step between integrase-dependent proviral integration into the host genome and transcription of viral genes. Using both an siRNA approach and Cul2/VHL mutant cells, we show that VBP1 and the Cul2/VHL ligase cooperate in the efficient polyubiquitylation of integrase and its subsequent proteasome-mediated degradation. Results presented here support a role for integrase degradation by the prefoldin–VHL–proteasome pathway in the integration–transcription transition of the viral replication cycle. PMID:17698809

  7. IN VITRO EVOLUTION OF AN HIV INTEGRASE BINDING PROTEIN FROM A LIBRARY OF C-TERMINAL DOMAIN γS-CRYSTALLIN VARIANTS

    PubMed Central

    Moody, Issa S.; Verde, Shawn C.; Overstreet, Cathie M.; Robinson, W. Edward; Weiss, Gregory A.

    2014-01-01

    A protein without natural binding functions was engineered to bind HIV-1 integrase. Phage display selections applied a library of variants based on the C-terminal domain of the eye lens protein human γS-crystallin. Multiple loop regions were altered to encode libraries with ≈3.6×1011 different variants. A crystallin variant, termed Integrase-Binding Protein-10 (IBP-10), inhibits integrase catalysis with nanomolar Ki values. IBP-10 interacts with the integrase C-terminal domain and inhibits integrase substrate affinity. This allosteric mechanism allows IBP-10 to inhibit drug resistant integrase variants. The results demonstrate the applicability of the crystallin scaffold for the discovery of binding partners and enzyme inhibitors. PMID:22858140

  8. Phage integrases: biology and applications.

    PubMed

    Groth, Amy C; Calos, Michele P

    2004-01-16

    Phage integrases are enzymes that mediate unidirectional site-specific recombination between two DNA recognition sequences, the phage attachment site, attP, and the bacterial attachment site, attB. Integrases may be grouped into two major families, the tyrosine recombinases and the serine recombinases, based on their mode of catalysis. Tyrosine family integrases, such as lambda integrase, utilize a catalytic tyrosine to mediate strand cleavage, tend to recognize longer attP sequences, and require other proteins encoded by the phage or the host bacteria. Phage integrases from the serine family are larger, use a catalytic serine for strand cleavage, recognize shorter attP sequences, and do not require host cofactors. Phage integrases mediate efficient site-specific recombination between two different sequences that are relatively short, yet long enough to be specific on a genomic scale. These properties give phage integrases growing importance for the genetic manipulation of living eukaryotic cells, especially those with large genomes such as mammals and most plants, for which there are few tools for precise manipulation of the genome. Integrases of the serine family have been shown to work efficiently in mammalian cells, mediating efficient integration at introduced att sites or native sequences that have partial identity to att sites. This reaction has applications in areas such as gene therapy, construction of transgenic organisms, and manipulation of cell lines. Directed evolution can be used to increase further the affinity of an integrase for a particular native sequence, opening up additional applications for genomic modification.

  9. Use of Patient-Derived Human Immunodeficiency Virus Type 1 Integrases To Identify a Protein Residue That Affects Target Site Selection

    PubMed Central

    Harper, Amy L.; Skinner, Lynn M.; Sudol, Malgorzata; Katzman, Michael

    2001-01-01

    To identify parts of retroviral integrase that interact with cellular DNA, we tested patient-derived human immunodeficiency virus type 1 (HIV-1) integrases for alterations in the choice of nonviral target DNA sites. This strategy took advantage of the genetic diversity of HIV-1, which provided 75 integrase variants that differed by a small number of amino acids. Moreover, our hypothesis that biological pressures on the choice of nonviral sites would be minimal was validated when most of the proteins that catalyzed DNA joining exhibited altered target site preferences. Comparison of the sequences of proteins with the same preferences then guided mutagenesis of a laboratory integrase. The results showed that single amino acid substitutions at one particular residue yielded the same target site patterns as naturally occurring integrases that included these substitutions. Similar results were found with DNA joining reactions conducted with Mn2+ or with Mg2+ and were confirmed with a nonspecific alcoholysis assay. Other amino acid changes at this position also affected target site preferences. Thus, this novel approach has identified a residue in the central domain of HIV-1 integrase that interacts with or influences interactions with cellular DNA. The data also support a model in which integrase has distinct sites for viral and cellular DNA. PMID:11462051

  10. Coevolutionary Analysis Identifies Protein–Protein Interaction Sites between HIV-1 Reverse Transcriptase and Integrase

    PubMed Central

    Hetti Arachchilage, Madara; Piontkivska, Helen

    2016-01-01

    The replication of human immunodeficiency virus-1 (HIV-1) requires reverse transcription of the viral RNA genome and integration of newly synthesized pro-viral DNA into the host genome. This is mediated by the viral proteins reverse transcriptase (RT) and integrase (IN). The formation and stabilization of the pre-integration complex (PIC), which is an essential step for reverse transcription, nuclear import, chromatin targeting, and subsequent integration, involves direct and indirect modes of interaction between RT and IN proteins. While epitope-based treatments targeting IN–viral DNA and IN–RT complexes appear to be a promising combination for an anti-HIV treatment, the mechanisms of IN-RT interactions within the PIC are not well understood due to the transient nature of the protein complex and the intrinsic flexibility of its components. Here, we identify potentially interacting regions between the IN and RT proteins within the PIC through the coevolutionary analysis of amino acid sequences of the two proteins. Our results show that specific regions in the two proteins have strong coevolutionary signatures, suggesting that these regions either experience direct and prolonged interactions between them that require high affinity and/or specificity or that the regions are involved in interactions mediated by dynamic conformational changes and, hence, may involve both direct and indirect interactions. Other regions were found to exhibit weak, but positive correlations, implying interactions that are likely transient and/or have low affinity. We identified a series of specific regions of potential interactions between the IN and RT proteins (e.g., specific peptide regions within the C-terminal domain of IN were identified as potentially interacting with the Connection domain of RT). Coevolutionary analysis can serve as an important step in predicting potential interactions, thus informing experimental studies. These studies can be integrated with structural data

  11. Allosteric inhibition of HIV-1 integrase activity

    PubMed Central

    Engelman, Alan; Kessl, Jacques J.; Kvaratskhelia, Mamuka

    2013-01-01

    HIV-1 integrase is an important therapeutic target in the fight against HIV/AIDS. Integrase strand transfer inhibitors (INSTIs), which target the enzyme active site, have witnessed clinical success over the past 5 years, but the generation of drug resistance poses challenges to INSTI-based therapies moving forward. Integrase is a dynamic protein, and its ordered multimerization is critical to enzyme activity. The integrase tetramer, bound to viral DNA, interacts with host LEDGF/p75 protein to tether integration to active genes. Allosteric integrase inhibitors (ALLINIs) that compete with LEDGF/p75 for binding to integrase disrupt integrase assembly with viral DNA and allosterically inhibit enzyme function. ALLINIs display steep dose response curves and synergize with INSTIs ex vivo, highlighting this novel inhibitor class for clinical development. PMID:23647983

  12. Structural basis for the potent inhibition of the HIV integrase-LEDGF/p75 protein-protein interaction.

    PubMed

    Ribone, Sergio R; Quevedo, Mario A

    2017-08-01

    Integrase (IN) constitutes one of the key enzymes involved in the lifecycle of the Human Immunodeficiency Virus (HIV), the etiological agent of AIDS. The biological role of IN strongly depends on the recognition and binding of cellular cofactors belonging to the infected host cell. Thus, the inhibition of the protein-protein interaction (PPI) between IN and cellular cofactors has been envisioned as a promising therapeutic target. In the present work we explore a structure-activity relationship for a set of 14 compounds reported as inhibitors of the PPI between IN and the lens epithelium-derived growth factor (LEDGF/p75). Our results demonstrate that the possibility to adopt the bioactive conformation capable of interacting with the hotspots IN-LEDGF/p75 hotspots residues constitutes a critical feature to obtain a potent inhibition. A ligand efficiency (|Lig-Eff|) quantitative descriptor combining both interaction energetics and conformational requirements was developed and correlated with the reported biological activity. Our results contribute to the rational development of IN-LEDGF/p75 interaction inhibitors providing a solid quantitative structure-activity relationship aimed for the screening of new IN-LEDGF/p75 interaction inhibitors. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. The core and carboxyl-terminal domains of the integrase protein of human immunodeficiency virus type 1 each contribute to nonspecific DNA binding.

    PubMed Central

    Engelman, A; Hickman, A B; Craigie, R

    1994-01-01

    The integrase protein of human immunodeficiency virus type 1 removes two nucleotides from the 3' ends of reverse-transcribed human immunodeficiency virus type 1 DNA (3' processing) and covalently inserts the processed ends into a target DNA (DNA strand transfer). Mutant integrase proteins that lack the amino-and/or carboxyl-terminal domains are incapable of catalyzing 3' processing and DNA strand transfer but are competent for an apparent reversal of the DNA strand transfer reaction (disintegration) in vitro. Here, we investigate the binding of integrase to DNA by UV cross-linking. Cross-linked complexes form with a variety of DNA substrates independent of the presence of divalent metal ion. Analysis with amino- and carboxyl-terminal deletion mutant proteins shows that residues 213 to 266 of the 288-residue protein are required for efficient cross-linking in the absence of divalent metal ion. Carboxyl-terminal deletion mutants that lack this region efficiently cross-link only to the branched disintegration DNA substrate, and this reaction is dependent on the presence of metal ion. Both the core and C-terminal domains of integrase therefore contribute to nonspecific DNA binding. Images PMID:8057470

  14. Inhibition of the integrase of human immunodeficiency virus (HIV) type 1 by anti-HIV plant proteins MAP30 and GAP31.

    PubMed Central

    Lee-Huang, S; Huang, P L; Huang, P L; Bourinbaiar, A S; Chen, H C; Kung, H F

    1995-01-01

    MAP30 (Momordica anti-HIV protein of 30 kDa) and GAP31 (Gelonium anti-HIV protein of 31 kDa) are anti-HIV plant proteins that we have identified, purified, and cloned from the medicinal plants Momordica charantia and Gelonium multiflorum. These antiviral agents are capable of inhibiting infection of HIV type 1 (HIV-1) in T lymphocytes and monocytes as well as replication of the virus in already-infected cells. They are not toxic to normal uninfected cells because they are unable to enter healthy cells. MAP30 and GAP31 also possess an N-glycosidase activity on 28S ribosomal RNA and a topological activity on plasmid and viral DNAs including HIV-1 long terminal repeats (LTRs). LTRs are essential sites for integration of viral DNA into the host genome by viral integrase. We therefore investigated the effect of MAP30 and GAP31 on HIV-1 integrase. We report that both of these antiviral agents exhibit dose-dependent inhibition of HIV-1 integrase. Inhibition was observed in all of the three specific reactions catalyzed by the integrase, namely, 3' processing (specific cleavage of the dinucleotide GT from the viral substrate), strand transfer (integration), and "disintegration" (the reversal of strand transfer). Inhibition was studied by using oligonucleotide substrates with sequences corresponding to the U3 and U5 regions of HIV LTR. In the presence of 20 ng of viral substrate, 50 ng of target substrate, and 4 microM integrase, total inhibition was achieved at equimolar concentrations of the integrase and the antiviral proteins, with EC50 values of about 1 microM. Integration of viral DNA into the host chromosome is a vital step in the replicative cycle of retroviruses, including the AIDS virus. The inhibition of HIV-1 integrase by MAP30 and GAP31 suggests that impediment of viral DNA integration may play a key role in the anti-HIV activity of these plant proteins. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7568024

  15. Production of Uninfectious Human Immunodeficiency Virus Type 1 Containing Viral Protein R Fused to a Single-Chain Antibody against Viral Integrase

    PubMed Central

    Okui, Nobuo; Kobayashi, Noriko; Kitamura, Yoshihiro

    1998-01-01

    A single-chain antibody (scAb) against human immunodeficiency virus type 1 (HIV-1) integrase was expressed as a fusion protein of scAb and HIV-1 viral protein R (Vpr), together with the HIV-1 genome, in human 293T cells. The expression did not affect virion production much but markedly reduced the infectivity of progeny virions. The fusion protein was found to be incorporated into the virions. The incorporation appears to account for the reduced infectivity. PMID:9658154

  16. Bacteriophage φC31 Integrase Mediated Transgenesis in Xenopus laevis for Protein Expression at Endogenous Levels

    NASA Astrophysics Data System (ADS)

    Allen, Bryan G.; Weeks, Daniel L.

    Bacteriophage φC31 inserts its genome into that of its host bacterium via the integrase enzyme which catalyzes recombination between a phage attachment site (attP) and a bacterial attachment site (attB). Integrase requires no accessory factors, has a high efficiency of recombination, and does not need perfect sequence fidelity for recognition and recombination between these attachment sites. These imperfect attachment sites, or pseudo-attachment sites, are present in many organisms and have been used to insert transgenes in a variety of species. Here we describe the φC31 integrase approach to make transgenic Xenopus laevis embryos.

  17. Identification of a hexapeptide inhibitor of the human immunodeficiency virus integrase protein by using a combinatorial chemical library.

    PubMed Central

    Puras Lutzke, R A; Eppens, N A; Weber, P A; Houghten, R A; Plasterk, R H

    1995-01-01

    Integration of human immunodeficiency virus (HIV) DNA into the human genome requires the virus-encoded integrase (IN) protein, and therefore the IN protein is a suitable target for antiviral strategies. To find a potent HIV IN inhibitor, we screened a "synthetic peptide combinatorial library." We identified a hexapeptide with the sequence HCKFWW that inhibits IN-mediated 3'-processing and integration with an IC50 of 2 microM. The peptide is active on IN proteins from other retroviruses such as HIV-2, feline immunodeficiency virus, and Moloney murine leukemia virus, supporting the notion that a conserved region of IN is targeted. The hexapeptide was also tested in the disintegration reaction. This phosphoryl-transfer reaction can be carried out by the catalytic core of IN alone, and the peptide HCKFWW was found to inhibit this reaction, suggesting that the hexapeptide acts at or near the catalytic site of IN. Identification of an IN hexapeptide inhibitor provides proof of concept for the approach, and, moreover, this peptide may be useful for structure-function analysis of IN. Images Fig. 4 Fig. 5 PMID:8524782

  18. Mapping Viral DNA Specificity to the Central Region of Integrase by Using Functional Human Immunodeficiency Virus Type 1/Visna Virus Chimeric Proteins

    PubMed Central

    Katzman, Michael; Sudol, Malgorzata

    1998-01-01

    We previously described the construction and analysis of the first set of functional chimeric lentivirus integrases, involving exchange of the N-terminal, central, and C-terminal regions of the human immunodeficiency virus type 1 (HIV-1) and visna virus integrase (IN) proteins. Based on those results, additional HIV-1/visna virus chimeric integrases were designed and purified. Each of the chimeric enzymes was functional in at least one oligonucleotide-based IN assay. Of a total of 12 chimeric IN proteins, 3 exhibit specific viral DNA processing, 9 catalyze insertion of viral DNA ends, 12 can reverse that reaction, and 11 are active for nonspecific alcoholysis. Functional data obtained with the processing assay indicate that the central region of the protein is responsible for viral DNA specificity. Target site selection for nonspecific alcoholysis again mapped to the central domain of IN, confirming our previous data indicating that this region can position nonviral DNA for nucleophilic attack. However, the chimeric proteins created patterns of viral DNA insertion distinct from that of either wild-type IN, suggesting that interactions between regions of IN influence target site selection for viral DNA integration. The results support a new model for the functional organization of IN in which viral DNA initially binds nonspecifically to the C-terminal portion of IN but the catalytic central region of the enzyme has a prominent role both in specific recognition of viral DNA ends and in positioning the host DNA for viral DNA integration. PMID:9499023

  19. [Analysis on bioactivity of HIV-1 integrase by ELISA method].

    PubMed

    Feng, Wei-Hong; Huang, Jian-Song; Zhan, Jin-Biao

    2007-03-01

    To develop an ELISA-based method for analyzing biologic activities of HIV-1 integrase and for high throughput screening of integrase inhibitors. After expression, renaturation and purification of integrase, the bioactivity of integrase and the inhibition of luffin-a were evaluated with an in vitro assay based on biotin-avidin EILSA and chemiluminescent substrates. (1) The specific activity of the purified integrase was 54.92 units/mg of protein. (2)IC(50) (concentration causing 50% inhibition of integrase) of luffin-a was (0.63 +/- 0.026) micromol/L. The non-radioactive assay can be used for analysis of bioactivities and high throughput screening of inhibitors of HIV-1 integrase.

  20. Exploring the binding of d(GGGT)4 to the HIV-1 integrase: An approach to investigate G-quadruplex aptamer/target protein interactions.

    PubMed

    Esposito, Veronica; Pirone, Luciano; Mayol, Luciano; Pedone, Emilia; Virgilio, Antonella; Galeone, Aldo

    2016-08-01

    The aptamer d(GGGT)4 (T30923 or T30695) forms a 5'-5' dimer of two stacked parallel G-quadruplexes, each characterized by three G-tetrads and three single-thymidine reversed-chain loops. This aptamer has been reported to exhibit anti-HIV activity by targeting the HIV integrase, a viral enzyme responsible for the integration of viral DNA into the host-cell genome. However, information concerning the aptamer/target interaction is still rather limited. In this communication we report microscale thermophoresis investigations on the interaction between the HIV-1 integrase and d(GGGT)4 aptamer analogues containing abasic sites singly replacing thymidines in the original sequence. This approach has allowed the identification of which part of the aptamer G-quadruplex structure is mainly involved in the interaction with the protein.

  1. Host Protein Ku70 Binds and Protects HIV-1 Integrase from Proteasomal Degradation and Is Required for HIV Replication*

    PubMed Central

    Zheng, Yingfeng; Ao, Zhujun; Wang, Binchen; Jayappa, Kallesh Danappa; Yao, Xiaojian

    2011-01-01

    HIV-1 integrase (IN) is a key viral enzymatic protein acting in several viral replication steps, including integration. IN has been shown to be an unstable protein degraded by the N-end rule pathway through the host ubiquitin-proteasome machinery. However, it is still not fully understood how this viral protein is protected from the host ubiquitin-proteasome system within cells during HIV replication. In the present study, we provide evidence that the host protein Ku70 interacts with HIV-1 IN and protects it from the Lys48-linked polyubiquitination proteasomal pathway. Moreover, Ku70 is able to down-regulate the overall protein polyubiquitination level within the host cells and to specifically deubiquitinate IN through their interaction. Mutagenic studies revealed that the C terminus of IN (residues 230–288) is required for IN binding to the N-terminal part of Ku70 (Ku70(1–430)), and their interaction is independent of Ku70/80 heterodimerization. Finally, knockdown of Ku70 expression in both virus-producing and target CD4+ T cells significantly disrupted HIV-1 replication and rendered two-long terminal repeat circles and integration undetectable, indicating that Ku70 is required for both the early and the late stages of the HIV-1 life cycle. Interestingly, Ku70 was incorporated into the progeny virus in an IN-dependent way. We proposed that Ku70 may interact with IN during viral assembly and accompany HIV-1 IN upon entry into the new target cells, acting to 1) protect IN from the host defense system and 2) assist IN integration activity. Overall, this report provides another example of how HIV-1 hijacks host cellular machinery to protect the virus itself and to facilitate its replication. PMID:21454661

  2. Structural and functional analysis of Tn4430: identification of an integrase-like protein involved in the co-integrate-resolution process.

    PubMed Central

    Mahillon, J; Lereclus, D

    1988-01-01

    The 4149-bp transposon Tn4430 from Bacillus thuringiensis is delineated by 38-bp inverted repeats and codes for a 113-kd protein that shares homology with the transposases (TnpA) of Tn3, Tn21 and Tn501. Through transpositional recombination, this protein generates the formation of co-integrates between both donor and target replicons, with duplication of Tn4430 molecules. These features are characteristic of transposons of the Tn3 family (class II elements). The second step of the transposition process, the co-integrate resolution, is mediated by a 32-kd protein. This protein (TnpI) displays regional similarities with site-specific recombinases of the integrase family, such as Int of bacteriophage lambda, Cre of bacteriophage P1 or TnpA and TnpB of the Tn554 transposon. Moreover, the 250-bp sequence upstream to the tnpI gene contains several structural features that are reminiscent of the attP attachment site of phage lambda. This unique association between the integrase-like TnpI recombinase and the TnpA transposase qualifies Tn4430 as a member of a new group within the class II mobile genetic elements. Images PMID:2842151

  3. Dynamic Modulation of HIV-1 Integrase Structure and Function by Cellular Lens Epithelium-derived Growth Factor (LEDGF) Protein*S⃞

    PubMed Central

    McKee, Christopher J.; Kessl, Jacques J.; Shkriabai, Nikolozi; Dar, Mohd Jamal; Engelman, Alan; Kvaratskhelia, Mamuka

    2008-01-01

    The mandatory integration of the reverse-transcribed HIV-1 genome into host chromatin is catalyzed by the viral protein integrase (IN), and IN activity can be regulated by numerous viral and cellular proteins. Among these, LEDGF has been identified as a cellular cofactor critical for effective HIV-1 integration. The x-ray crystal structure of the catalytic core domain (CCD) of IN in complex with the IN binding domain (IBD) of LEDGF has furthermore revealed essential protein-protein contacts. However, mutagenic studies indicated that interactions between the full-length proteins were more extensive than the contacts observed in the co-crystal structure of the isolated domains. Therefore, we have conducted detailed biochemical characterization of the interactions between full-length IN and LEDGF. Our results reveal a highly dynamic nature of IN subunit-subunit interactions. LEDGF strongly stabilized these interactions and promoted IN tetramerization. Mass spectrometric protein footprinting and molecular modeling experiments uncovered novel intra- and inter-protein-protein contacts in the full-length IN-LEDGF complex that lay outside of the observable IBD-CCD structure. In particular, our studies defined the IN tetramer interface important for enzymatic activities and high affinity LEDGF binding. These findings provide new insight into how LEDGF modulates HIV-1 IN structure and function, and highlight the potential for exploiting the highly dynamic structure of multimeric IN as a novel therapeutic target. PMID:18801737

  4. Human Immunodeficiency Virus Type 1 Employs the Cellular Dynein Light Chain 1 Protein for Reverse Transcription through Interaction with Its Integrase Protein

    PubMed Central

    Jayappa, Kallesh Danappa; Ao, Zhujun; Wang, Xiaoxia; Mouland, Andrew J.; Shekhar, Sudhanshu; Yang, Xi

    2015-01-01

    ABSTRACT In this study, we examined the requirement for host dynein adapter proteins such as dynein light chain 1 (DYNLL1), dynein light chain Tctex-type 1 (DYNLT1), and p150Glued in early steps of human immunodeficiency virus type 1 (HIV-1) replication. We found that the knockdown (KD) of DYNLL1, but not DYNLT1 or p150Glued, resulted in significantly lower levels of HIV-1 reverse transcription in cells. Following an attempt to determine how DYNLL1 could impact HIV-1 reverse transcription, we detected the DYNLL1 interaction with HIV-1 integrase (IN) but not with capsid (CA), matrix (MA), or reverse transcriptase (RT) protein. Furthermore, by mutational analysis of putative DYNLL1 interaction motifs in IN, we identified the motifs 52GQVD and 250VIQD in IN as essential for DYNLL1 interaction. The DYNLL1 interaction-defective IN mutant HIV-1 (HIV-1INQ53A/Q252A) exhibited impaired reverse transcription. Through further investigations, we have also detected relatively smaller amounts of particulate CA in DYNLL1-KD cells or in infections with HIV-1INQ53A/Q252A mutant virus. Overall, our study demonstrates the novel interaction between HIV-1 IN and cellular DYNLL1 proteins and suggests the requirement of this virus-cell interaction for proper uncoating and efficient reverse transcription of HIV-1. IMPORTANCE Host cellular DYNLL1, DYNLT1, and p150Glued proteins have been implicated in the replication of several viruses. However, their roles in HIV-1 replication have not been investigated. For the first time, we demonstrated that during viral infection, HIV-1 IN interacts with DYNLL1, and their interaction was found to have a role in proper uncoating and efficient reverse transcription of HIV-1. Thus, interaction of IN and DYNLL1 may be a potential target for future anti-HIV therapy. Moreover, while our study has evaluated the involvement of IN in HIV-1 uncoating and reverse transcription, it also predicts a possible mechanism by which IN contributes to these early viral

  5. The role of flexibility and hydration on the sequence-specific DNA recognition by the Tn916 integrase protein: a molecular dynamics analysis.

    PubMed

    Gorfe, Alemayehu A; Caflisch, Amedeo; Jelesarov, Ilian

    2004-01-01

    The N-terminal domain of the Tn916 integrase protein (INT-DBD) is responsible for DNA binding in the process of strand cleavage and joining reactions required for transposition of the Tn916 conjugative transposon. Site-specific association is facilitated by numerous protein-DNA contacts from the face of a three-stranded beta-sheet inserted into the major groove. The protein undergoes a subtle conformational transition and is slightly unfolded in the protein-DNA complex. The conformation of many charged residues is poorly defined by NMR data but mutational studies have indicated that removal of polar side chains decreases binding affinity, while non-polar contacts are malleable. Based on analysis of the binding enthalpy and binding heat capacity, we have reasoned that dehydration of the protein-DNA interface is incomplete. This study presents results from a molecular dynamics investigation of the INT-DBD-DNA complex aimed at a more detailed understanding of the role of conformational dynamics and hydration in site-specific binding. Comparison of simulations (total of 13 ns) of the free protein and of the bound protein conformation (in isolation or DNA-bound) reveals intrinsic flexibility in certain parts of the molecule. Conformational adaptation linked to partial unfolding appears to be induced by protein-DNA contacts. The protein-DNA hydrogen-bonding network is highly dynamic. The simulation identifies protein-DNA interactions that are poorly resolved or only surmised from the NMR ensemble. Single water molecules and water clusters dynamically optimize the complementarity of polar interactions at the 'wet' protein-DNA interface. The simulation results are useful to establish a qualitative link between experimental data on individual residue's contribution to binding affinity and thermodynamic properties of INT-DBD alone and in complex with DNA.

  6. New Applications for Phage Integrases

    PubMed Central

    Fogg, Paul C.M.; Colloms, Sean; Rosser, Susan; Stark, Marshall; Smith, Margaret C.M.

    2014-01-01

    Within the last 25 years, bacteriophage integrases have rapidly risen to prominence as genetic tools for a wide range of applications from basic cloning to genome engineering. Serine integrases such as that from ϕC31 and its relatives have found an especially wide range of applications within diverse micro-organisms right through to multi-cellular eukaryotes. Here, we review the mechanisms of the two major families of integrases, the tyrosine and serine integrases, and the advantages and disadvantages of each type as they are applied in genome engineering and synthetic biology. In particular, we focus on the new areas of metabolic pathway construction and optimization, biocomputing, heterologous expression and multiplexed assembly techniques. Integrases are versatile and efficient tools that can be used in conjunction with the various extant molecular biology tools to streamline the synthetic biology production line. PMID:24857859

  7. HIV-1 Group O Integrase Displays Lower Enzymatic Efficiency and Higher Susceptibility to Raltegravir than HIV-1 Group M Subtype B Integrase

    PubMed Central

    Depatureaux, Agnès; Quashie, Peter K.; Mesplède, Thibault; Han, Yingshan; Koubi, Hannah; Plantier, Jean-Christophe; Oliveira, Maureen; Moisi, Daniela; Brenner, Bluma

    2014-01-01

    HIV-1 group O (HIV-O) is a rare HIV-1 variant characterized by a high number of polymorphisms, especially in the integrase coding region. As HIV-O integrase enzymes have not previously been studied, our aim was to assess the impact of HIV-O integrase polymorphisms on enzyme function and susceptibility to integrase inhibitors. Accordingly, we cloned and purified integrase proteins from each of HIV-1 group O clades A and B, an HIV-O divergent strain, and HIV-1 group M (HIV-M, subtype B), used as a reference. To assess enzymatic function of HIV-O integrase, we carried out strand transfer and 3′ processing assays with various concentrations of substrate (DNA target and long terminal repeats [LTR], respectively) and characterized these enzymes for susceptibility to integrase strand transfer inhibitors (INSTIs) in cell-free assays and in tissue culture, in the absence or presence of various concentrations of several INSTIs. The inhibition constant (Ki) and 50% effective concentration (EC50) values were calculated for HIV-O integrases and HIV-O viruses, respectively, and compared with those of HIV-M. The results showed that HIV-O integrase displayed lower activity in strand transfer assays than did HIV-M enzyme, whereas 3′ processing activities were similar to those of HIV-M. HIV-O integrases were more susceptible to raltegravir (RAL) in competitive inhibition assays and in tissue culture than were HIV-M enzymes and viruses, respectively. Molecular modeling suggests that two key polymorphic residues that are close to the integrase catalytic site, 74I and 153A, may play a role in these differences. PMID:25224008

  8. HIV-1 group O integrase displays lower enzymatic efficiency and higher susceptibility to raltegravir than HIV-1 group M subtype B integrase.

    PubMed

    Depatureaux, Agnès; Quashie, Peter K; Mesplède, Thibault; Han, Yingshan; Koubi, Hannah; Plantier, Jean-Christophe; Oliveira, Maureen; Moisi, Daniela; Brenner, Bluma; Wainberg, Mark A

    2014-12-01

    HIV-1 group O (HIV-O) is a rare HIV-1 variant characterized by a high number of polymorphisms, especially in the integrase coding region. As HIV-O integrase enzymes have not previously been studied, our aim was to assess the impact of HIV-O integrase polymorphisms on enzyme function and susceptibility to integrase inhibitors. Accordingly, we cloned and purified integrase proteins from each of HIV-1 group O clades A and B, an HIV-O divergent strain, and HIV-1 group M (HIV-M, subtype B), used as a reference. To assess enzymatic function of HIV-O integrase, we carried out strand transfer and 3' processing assays with various concentrations of substrate (DNA target and long terminal repeats [LTR], respectively) and characterized these enzymes for susceptibility to integrase strand transfer inhibitors (INSTIs) in cell-free assays and in tissue culture, in the absence or presence of various concentrations of several INSTIs. The inhibition constant (Ki) and 50% effective concentration (EC50) values were calculated for HIV-O integrases and HIV-O viruses, respectively, and compared with those of HIV-M. The results showed that HIV-O integrase displayed lower activity in strand transfer assays than did HIV-M enzyme, whereas 3' processing activities were similar to those of HIV-M. HIV-O integrases were more susceptible to raltegravir (RAL) in competitive inhibition assays and in tissue culture than were HIV-M enzymes and viruses, respectively. Molecular modeling suggests that two key polymorphic residues that are close to the integrase catalytic site, 74I and 153A, may play a role in these differences. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  9. Control of Recombination Directionality by the Listeria Phage A118 Protein Gp44 and the Coiled-Coil Motif of Its Serine Integrase.

    PubMed

    Mandali, Sridhar; Gupta, Kushol; Dawson, Anthony R; Van Duyne, Gregory D; Johnson, Reid C

    2017-06-01

    The serine integrase of phage A118 catalyzes integrative recombination between attP on the phage and a specific attB locus on the chromosome of Listeria monocytogenes, but it is unable to promote excisive recombination between the hybrid attL and attR sites found on the integrated prophage without assistance by a recombination directionality factor (RDF). We have identified and characterized the phage-encoded RDF Gp44, which activates the A118 integrase for excision and inhibits integration. Gp44 binds to the C-terminal DNA binding domain of integrase, and we have localized the primary binding site to be within the mobile coiled-coil (CC) motif but distinct from the distal tip of the CC that is required for recombination. This interaction is sufficient to inhibit integration, but a second interaction involving the N-terminal end of Gp44 is also required to activate excision. We provide evidence that these two contacts modulate the trajectory of the CC motifs as they extend out from the integrase core in a manner dependent upon the identities of the four att sites. Our results support a model whereby Gp44 shapes the Int-bound complexes to control which att sites can synapse and recombine.IMPORTANCE Serine integrases mediate directional recombination between bacteriophage and bacterial chromosomes. These highly regulated site-specific recombination reactions are integral to the life cycle of temperate phage and, in the case of Listeria monocytogenes lysogenized by A118 family phage, are an essential virulence determinant. Serine integrases are also utilized as tools for genetic engineering and synthetic biology because of their exquisite unidirectional control of the DNA exchange reaction. Here, we identify and characterize the recombination directionality factor (RDF) that activates excision and inhibits integration reactions by the phage A118 integrase. We provide evidence that the A118 RDF binds to and modulates the trajectory of the long coiled-coil motif that

  10. Characterization of DNA binding property of the HIV-1 host factor and tumor suppressor protein Integrase Interactor 1 (INI1/hSNF5).

    PubMed

    Das, Supratik; Banerjee, Baisakhi; Hossain, Maidul; Thangamuniyandi, Muruganandan; Dasgupta, Saumya; Chongdar, Nipa; Kumar, Gopinatha Suresh; Basu, Gautam

    2013-01-01

    Integrase Interactor 1 (INI1/hSNF5) is a component of the hSWI/SNF chromatin remodeling complex. The INI1 gene is either deleted or mutated in rhabdoid cancers like ATRT (Atypical terratoid and rhabdoid tumor). INI1 is also a host factor for HIV-1 replication. INI1 binds DNA non-specifically. However, the mechanism of DNA binding and its biological role are unknown. From agarose gel retardation assay (AGRA), Ni-NTA pull-down and atomic force microscopy (AFM) studies we show that amino acids 105-183 of INI1 comprise the minimal DNA binding domain (DBD). The INI1 DBD is absent in plants and in yeast SNF5. It is present in Caenorhabditis elegans SNF5, Drosophila melanogaster homologue SNR1 and is a highly conserved domain in vertebrates. The DNA binding property of this domain in SNR1, that is only 58% identical to INI1/hSNF5, is conserved. Analytical ultracentrifugation studies of INI1 DBD and INI1 DBD:DNA complexes at different concentrations show that the DBD exists as a monomer at low protein concentration and two molecules of monomer binds one molecule of DNA. At high protein concentration, it exists as a dimer and binds two DNA molecules. Furthermore, isothermal calorimetry (ITC) experiments demonstrate that the DBD monomer binds DNA with a stoichiometry (N) of ∼0.5 and Kd  = 0.94 µM whereas the DBD dimer binds two DNA molecules sequentially with K'd1 = 222 µM and K'd2 = 1.16 µM. Monomeric DBD binding to DNA is enthalpy driven (ΔH = -29.9 KJ/mole). Dimeric DBD binding to DNA is sequential with the first binding event driven by positive entropy (ΔH'1 = 115.7 KJ/mole, TΔS'1 = 136.8 KJ/mole) and the second binding event driven by negative enthalpy (ΔH'2 = -106.3 KJ/mole, TΔS'2 = -75.7 KJ/mole). Our model for INI1 DBD binding to DNA provides new insights into the mechanism of DNA binding by INI1.

  11. Prospective strategies for targeting HIV-1 integrase function

    PubMed Central

    Luo, Yang; Muesing, Mark A

    2011-01-01

    Integration is a key step in the HIV-1 life cycle in which the ends of linear viral DNA are covalently joined with host chromosomal DNA. Integrase is the highly conserved and essential viral protein that performs two catalytically related reactions that ultimately lead to the insertion of the viral genome into that of the host cell. The only chemotherapeutic agents against integrase currently available for HIV-1 infected individuals are those that interrupt strand transfer, the second step of catalysis. Accordingly, this article outlines possible future strategies targeting the first catalytic step, 3′ processing, as well as other nonenzymatic, yet indispensible, functions thought to be coordinated by integrase. Importantly, the interruption of irremediable recombination between viral and host DNAs represents the last step after viral entry at which an otherwise irreversible infection can be prevented. PMID:21359091

  12. Integrase and integration: biochemical activities of HIV-1 integrase

    PubMed Central

    Delelis, Olivier; Carayon, Kevin; Saïb, Ali; Deprez, Eric; Mouscadet, Jean-François

    2008-01-01

    Integration of retroviral DNA is an obligatory step of retrovirus replication because proviral DNA is the template for productive infection. Integrase, a retroviral enzyme, catalyses integration. The process of integration can be divided into two sequential reactions. The first one, named 3'-processing, corresponds to a specific endonucleolytic reaction which prepares the viral DNA extremities to be competent for the subsequent covalent insertion, named strand transfer, into the host cell genome by a trans-esterification reaction. Recently, a novel specific activity of the full length integrase was reported, in vitro, by our group for two retroviral integrases (HIV-1 and PFV-1). This activity of internal cleavage occurs at a specific palindromic sequence mimicking the LTR-LTR junction described into the 2-LTR circles which are peculiar viral DNA forms found during viral infection. Moreover, recent studies demonstrated the existence of a weak palindromic consensus found at the integration sites. Taken together, these data underline the propensity of retroviral integrases for binding symmetrical sequences and give perspectives for targeting specific sequences used for gene therapy. PMID:19091057

  13. The prototype foamy virus protease is active independently of the integrase domain

    PubMed Central

    2012-01-01

    Background Recently, contradictory results on foamy virus protease activity were published. While our own results indicated that protease activity is regulated by the viral RNA, others suggested that the integrase is involved in the regulation of the protease. Results To solve this discrepancy we performed additional experiments showing that the protease-reverse transcriptase (PR-RT) exhibits protease activity in vitro and in vivo, which is independent of the integrase domain. In contrast, Pol incorporation, and therefore PR activity in the viral context, is dependent on the integrase domain. To further analyse the regulation of the protease, we incorporated Pol in viruses by expressing a GagPol fusion protein, which supported near wild-type like infectivity. A GagPR-RT fusion, lacking the integrase domain, also resulted in wild-type like Gag processing, indicating that the integrase is dispensable for viral Gag maturation. Furthermore, we demonstrate with a trans-complementation assays that the PR in the context of the PR-RT protein supports in trans both, viral maturation and infectivity. Conclusion We provide evidence that the FV integrase is required for Pol encapsidation and that the FV PR activity is integrase independent. We show that an active PR can be encapsidated in trans as a GagPR-RT fusion protein. PMID:22574974

  14. Identification of discrete functional domains of HIV-1 integrase and their organization within an active multimeric complex.

    PubMed Central

    Engelman, A; Bushman, F D; Craigie, R

    1993-01-01

    HIV-1 integrase protein possesses the 3' processing and DNA strand transfer activities that are required to integrate HIV DNA into a host chromosome. The N-, C-terminal and core domains of integrase are necessary for both activities in vitro. We find that certain pairs of mutant integrase proteins, which are inactive when each protein is assayed alone, can support near wild type levels of activity when both proteins are present together in the reaction mixture. This complementation implies that HIV-1 integrase functions as a multimer and has enabled us to probe the organization of the functional domains within active mixed multimers. We have identified a minimal set of functional integrase domains that are sufficient for 3' processing and DNA strand transfer and find that some domains are contributed in trans by separate monomers within the functional complex. Images PMID:8344264

  15. RETROVIRAL INTEGRASE: THEN AND NOW

    PubMed Central

    Andrake, Mark D.; Skalka, Anna Marie

    2016-01-01

    The retroviral integrases are virally encoded, specialized recombinases that catalyze the insertion of viral DNA into the host cell’s DNA, a process that is essential for virus propagation. We have learned a great deal since the existence of an integrated form of retroviral DNA (the provirus) was first proposed by Howard Temin in 1964. Initial studies focused on the genetics and biochemistry of avian and murine virus DNA integration, but the pace of discovery increased substantially with advances in technology, and an influx of investigators focused on the human immunodeficiency virus (HIV). We begin with a brief account of the scientific landscape in which some of the earliest discoveries were made, and summarize research that led to our current understanding of the biochemistry of integration. A more detailed account of recent analyses of integrase structure follows, as they have provided valuable insights into enzyme function and raised important new questions. PMID:26958915

  16. Pharmacology of HIV Integrase Inhibitors

    PubMed Central

    Adams, Jessica L; Greener, Benjamin N; Kashuba, Angela DM

    2013-01-01

    Purpose of Review The purpose of this paper is to review recent and relevant pharmacology data for three HIV integrase inhibitors: raltegravir (marketed), dolutegravir and elvitegravir (both in Phase III drug development). Recent Findings Data from January 2011 to April 2012 were evaluated. These data better characterized integrase inhibitor pharmacokinetics, assessed dosing regimens and investigated previously undescribed drug-drug interactions. Due to formulation challenges, raltegravir inter- and intra-patient pharmacokinetic variability is high. Twice daily 400mg dosing has been shown to be clinically superior to 800mg once daily dosing. A pediatric formulation of raltegravir with less variable pharmacokinetics and greater bioavailability was FDA approved in December 2011. Cobicistat-boosted elvitegravir, and the second generation integrase inhibitor dolutegravir, have lower pharmacokinetic variability and are dosed once daily. Dolutegravir drug interactions are similar to raltegravir, while boosted elvitegravir participates in additional CYP3A mediated interactions. Summary Raltegravir’s potent antiretroviral activity has resulted in widespread use in both treatment naïve and experienced patients. Dolutegravir and cobicistat-boosted elvitegravir have some pharmacokinetic advantages. Pharmacokinetic data in special populations (pregnancy, pediatrics) to optimize dosing are still required. PMID:22789987

  17. Developing a dynamic pharmacophore model for HIV-1 integrase.

    PubMed

    Carlson, H A; Masukawa, K M; Rubins, K; Bushman, F D; Jorgensen, W L; Lins, R D; Briggs, J M; McCammon, J A

    2000-06-01

    We present the first receptor-based pharmacophore model for HIV-1 integrase. The development of "dynamic" pharmacophore models is a new method that accounts for the inherent flexibility of the active site and aims to reduce the entropic penalties associated with binding a ligand. Furthermore, this new drug discovery method overcomes the limitation of an incomplete crystal structure of the target protein. A molecular dynamics (MD) simulation describes the flexibility of the uncomplexed protein. Many conformational models of the protein are saved from the MD simulations and used in a series of multi-unit search for interacting conformers (MUSIC) simulations. MUSIC is a multiple-copy minimization method, available in the BOSS program; it is used to determine binding regions for probe molecules containing functional groups that complement the active site. All protein conformations from the MD are overlaid, and conserved binding regions for the probe molecules are identified. Those conserved binding regions define the dynamic pharmacophore model. Here, the dynamic model is compared to known inhibitors of the integrase as well as a three-point, ligand-based pharmacophore model from the literature. Also, a "static" pharmacophore model was determined in the standard fashion, using a single crystal structure. Inhibitors thought to bind in the active site of HIV-1 integrase fit the dynamic model but not the static model. Finally, we have identified a set of compounds from the Available Chemicals Directory that fit the dynamic pharmacophore model, and experimental testing of the compounds has confirmed several new inhibitors.

  18. Developing a Dynamic Pharmacophore Model for HIV-1 Integrase

    SciTech Connect

    Carlson, Heather A.; Masukawa, Keven M.; Rubins, Kathleen; Bushman, Frederic; Jorgensen, William L.; Lins, Roberto; Briggs, James; Mccammon, Andy

    2000-05-11

    We present the first receptor-based pharmacophore model for HIV-1 integrase. The development of ''dynamic'' pharmacophore models is a new method that accounts for the inherent flexibility of the active site and aims to reduce the entropic penalties associated with binding a ligand. Furthermore, this new drug discovery method overcomes the limitation of an incomplete crystal structure of the target protein. A molecular dynamics (MD) simulation describes the flexibility of the uncomplexed protein. Many conformational models of the protein are saved from the MD simulations and used in a series of multi-unit search for interacting conformers (MUSIC) simulations. MUSIC is a multiple-copy minimization method, available in the BOSS program; it is used to determine binding regions for probe molecules containing functional groups that complement the active site. All protein conformations from the MD are overlaid, and conserved binding regions for the probe molecules are identified. Those conserved binding regions define the dynamic pharmacophore model. Here, the dynamic model is compared to known inhibitors of the integrase as well as a three-point, ligand-based pharmacophore model from the literature. Also, a ''static'' pharmacophore model was determined in the standard fashion, using a single crystal structure. Inhibitors thought to bind in the active site of HIV-1 integrase fit the dynamic model but not the static model. Finally, we have identified a set of compounds from the Available Chemicals Directory that fit the dynamic pharmacophore model, and experimental testing of the compounds has confirmed several new inhibitors.

  19. In vivo and in vitro characterization of site-specific recombination of actinophage R4 integrase.

    PubMed

    Miura, Takamasa; Hosaka, Yayoi; Yan-Zhuo, Yang; Nishizawa, Tomoyasu; Asayama, Munehiko; Takahashi, Hideo; Shirai, Makoto

    2011-01-01

    The site-specific integrase of actinophage R4 belongs to the serine recombinase family. During the lysogenization process, it catalyzes site-specific recombination between the phage genome and the chromosome of Streptomyces parvulus 2297. An in vivo assay using Escherichia coli cells revealed that the minimum lengths of the recombination sites attB and attP are 50-bp and 49-bp, respectively, for efficient intramolecular recombination. The in vitro assay using overproduced R4 integrases as a hexahistidine (His(6))-glutathione-S-transferase (GST)-R4 integrase fusion protein, showed that the purified protein preparation retains the site-specific recombination activity which catalyzes the site-specific recombination between attP and attB in the intermolecular reaction. It also revealed that the inverted repeat within attP is essential for efficient in vitro intermolecular recombination. In addition, a gel shift assay showed that His(6)-GST-R4 integrase bound to the 50-bp attB and 49-bp attP specifically. Moreover, based on a detailed comparison analysis of amino acid sequences of serine integrases, we found the DNA binding region that is conserved in the serine recombinase containing the large C-terminal domain. Based on the results presented on this report, attachment sites needed in vitro and in vivo for site-specific recombination by the R4 integrase have been defined more precisely. This knowledge is useful for developing new genetic manipulation tools in the future.

  20. Human T-cell leukemia virus type 1 reverse transcriptase (RT) originates from the pro and pol open reading frames and requires the presence of RT-RNase H (RH) and RT-RH-integrase proteins for its activity.

    PubMed

    Trentin, B; Rebeyrotte, N; Mamoun, R Z

    1998-08-01

    The first description of an active form of a recombinant human T-cell leukemia virus type 1 (HTLV-1) reverse transcriptase (RT) and subsequent predictions of its amino acid sequence and quaternary structure are reported here. By using amino acid alignment methods, the NH2 and COOH termini of the RT, RNase H (RH), and integrase (IN) domains of the Pol polyprotein were determined. The HTLV-1 RT seems to be unique since its NH2 terminus is probably encoded by the pro open reading frame (ORF) fused downstream, via a transframe peptide, to the polypeptide encoded by the pol ORF. The HTLV-1 Pol amino acid sequence was revealed to be highly similar to that of Rous sarcoma virus (RSV), particularly at the RT-RH hinge region. These two domains remain linked for RSV; this may also be the case for HTLV-1. In light of these results, RT, RT-RH, and RT-RH-IN genes were constructed and introduced into His-tagged protein expression vectors. The corresponding proteins were synthesized in vitro, and the DNA polymerase activities of different protein combinations were tested. Solely the RT-RH-RT-RH-IN combination was found to have a significant activity level. Velocity sedimentation analysis suggested that the HTLV-1 RT-RH and RT-RH-IN monomers are likely associated in an oligomeric structure, probably of the alpha3/beta type.

  1. Biochemical and virological analysis of the 18-residue C-terminal tail of HIV-1 integrase

    PubMed Central

    Dar, Mohd J; Monel, Blandine; Krishnan, Lavanya; Shun, Ming-Chieh; Di Nunzio, Francesca; Helland, Dag E; Engelman, Alan

    2009-01-01

    Background The 18 residue tail abutting the SH3 fold that comprises the heart of the C-terminal domain is the only part of HIV-1 integrase yet to be visualized by structural biology. To ascertain the role of the tail region in integrase function and HIV-1 replication, a set of deletion mutants that successively lacked three amino acids was constructed and analyzed in a variety of biochemical and virus infection assays. HIV-1/2 chimers, which harbored the analogous 23-mer HIV-2 tail in place of the HIV-1 sequence, were also studied. Because integrase mutations can affect steps in the replication cycle other than integration, defective mutant viruses were tested for integrase protein content and reverse transcription in addition to integration. The F185K core domain mutation, which increases integrase protein solubility, was furthermore analyzed in a subset of mutants. Results Purified proteins were assessed for in vitro levels of 3' processing and DNA strand transfer activities whereas HIV-1 infectivity was measured using luciferase reporter viruses. Deletions lacking up to 9 amino acids (1-285, 1-282, and 1-279) displayed near wild-type activities in vitro and during infection. Further deletion yielded two viruses, HIV-11-276 and HIV-11-273, that displayed approximately two and 5-fold infectivity defects, respectively, due to reduced integrase function. Deletion mutant HIV-11-270 and the HIV-1/2 chimera were non-infectious and displayed approximately 3 to 4-fold reverse transcription in addition to severe integration defects. Removal of four additional residues, which encompassed the C-terminal β strand of the SH3 fold, further compromised integrase incorporation into virions and reverse transcription. Conclusion HIV-11-270, HIV-11-266, and the HIV-1/2 chimera were typed as class II mutant viruses due to their pleiotropic replication defects. We speculate that residues 271-273 might play a role in mediating the known integrase-reverse transcriptase interaction, as

  2. A novel integrase targeting agent to explore the future prospective of HIV eradication: dolutegravir.

    PubMed

    Narang, Bawneet K; Grewal, Gurpreet K; Roy, Subhajit; Bariwal, Jitender; Gupta, Manish K; Rawal, Ravindra K

    2014-01-01

    Out of the 15 discrete proteins encoding the total amount of genetic information in the chromosomes of human immunodeficiency virus type 1; three perform the vital enzymatic functions i.e. a reverse transcription, an integration, and proteolysis. The HIV integrase is the new validated drug target against AIDS amongst all essential enzymes due to the lack of the human homologue. In last few years quite, a few but potent inhibitors inhibiting HIV-1 integrase have been recognized and hence have gained a state-of-the-art for treating the infection caused by HIV-1. The greater understanding of HIV-integrase biological structure has further lead to continuous efforts for the proposal of novel inhibitors targeting diverse steps in the progression of integration with the primary goal to overcome resistance due to the rapid occurrence of integrase mutations in the treated patients. This review is focused on various aspects of the recently approved HIV integrase inhibitor "dolutegravir", its efficacy, safety profiles with the clinical data and molecular modeling studies highlighting its importance over the already approved HIV-integrase inhibitors.

  3. Molecular Dynamics Studies on the HIV-1 Integrase Catalytic Domain

    SciTech Connect

    Lins, Roberto D.; Briggs, J. M.; Straatsma, TP; Carlson, Heather A.; Greenwald, Jason; Choe, Senyon; Mccammon, Andy

    1999-06-30

    The HIV-1 integrase, which is essential for viral replication, catalyzes the insertion of viral DNA into the host chromosome, thereby recruiting host cell machinery into making viral proteins. It represents the third main HIV enzyme target for inhibitor design, the first two being the reverse transcriptase and the protease. Two 1-ns molecular dynamics simulations have been carried out on completely hydrated models of the HIV-1 integrase catalytic domain, one with no metal ions and another with one magnesium ion in the catalytic site. The simulations predict that the region of the active site that is missing in the published crystal structures has (at the time of this work) more secondary structure than previously thought. The flexibility of this region has been discussed with respect to the mechanistic function of the enzyme. The results of these simulations will be used as part of inhibitor design projects directed against the catalytic domain of the enzyme.

  4. Intermolecular disintegration and intramolecular strand transfer activities of wild-type and mutant HIV-1 integrase.

    PubMed Central

    Mazumder, A; Engelman, A; Craigie, R; Fesen, M; Pommier, Y

    1994-01-01

    We report the activities of HIV integrase protein on a novel DNA substrate, consisting of a pair of gapped duplex molecules. Integrase catalyzed an intermolecular disintegration reaction that requires positioning of a pair of the gapped duplexes in a configuration that resembles the intgration intermediate. However, the major reaction resulted from an intramolecular reaction involving a single gapped duplex, giving rise to a hairpin. Surprisingly, a deletion mutant of integrase that lacks both the amino and carboxyl terminal regions still catalyzed the intermolecular disintegration reaction, but supported only a very low level of the intramolecular reaction. The central core region of integrase is therefore sufficient to both bind the gapped duplex DNA and juxtapose a pair of such molecules through protein-protein interactions. We suggest that the branched DNA structures of the previously reported disintegration substrate, and the intermolecular disintegration substrate described here, assist in stabilizing protein-protein interactions that otherwise require the amino and carboxy terminal regions of integrase. Images PMID:8152908

  5. Uneven Genetic Robustness of HIV-1 Integrase

    PubMed Central

    Rihn, Suzannah J.; Hughes, Joseph; Wilson, Sam J.

    2014-01-01

    ABSTRACT Genetic robustness (tolerance of mutation) may be a naturally selected property in some viruses, because it should enhance adaptability. Robustness should be especially beneficial to viruses like HIV-1 that exhibit high mutation rates and exist in immunologically hostile environments. Surprisingly, however, the HIV-1 capsid protein (CA) exhibits extreme fragility. To determine whether fragility is a general property of HIV-1 proteins, we created a large library of random, single-amino-acid mutants in HIV-1 integrase (IN), covering >40% of amino acid positions. Despite similar degrees of sequence variation in naturally occurring IN and CA sequences, we found that HIV-1 IN was significantly more robust than CA, with random nonsilent IN mutations only half as likely to cause lethal defects. Interestingly, IN and CA were similar in that a subset of mutations with high in vitro fitness were rare in natural populations. IN mutations of this type were more likely to occur in the buried interior of the modeled HIV-1 intasome, suggesting that even very subtle fitness effects suppress variation in natural HIV-1 populations. Lethal mutations, in particular those that perturbed particle production, proteolytic processing, and particle-associated IN levels, were strikingly localized at specific IN subunit interfaces. This observation strongly suggests that binding interactions between particular IN subunits regulate proteolysis during HIV-1 virion morphogenesis. Overall, use of the IN mutant library in conjunction with structural models demonstrates the overall robustness of IN and highlights particular regions of vulnerability that may be targeted in therapeutic interventions. IMPORTANCE The HIV-1 integrase (IN) protein is responsible for the integration of the viral genome into the host cell chromosome. To measure the capacity of IN to maintain function in the face of mutation, and to probe structure/function relationships, we created a library of random single

  6. Authentic HIV-1 integrase inhibitors

    PubMed Central

    Liao, Chenzhong; Marchand, Christophe; Burke, Terrence R; Pommier, Yves; Nicklaus, Marc C

    2010-01-01

    HIV-1 integrase (IN) is indispensable for HIV-1 replication and has become a validated target for developing anti-AIDS agents. In two decades of development of IN inhibition-based anti-HIV therapeutics, a significant number of compounds were identified as IN inhibitors, but only some of them showed antiviral activity. This article reviews a number of patented HIV-1 IN inhibitors, especially those that possess high selectivity for the strand transfer reaction. These compounds generally have a polar coplanar moiety, which is assumed to chelate two magnesium ions in the binding site. Resistance to those compounds, when given to patients, can develop as a result of IN mutations. We refer to those compounds as authentic IN inhibitors. Continued drug development has so far delivered one authentic IN inhibitor to the market (raltegravir in 2007). Current and future attention will be focused on the development of novel authentic IN inhibitors with the goal of overcoming viral resistance. PMID:21426159

  7. Mutations Located outside the Integrase Gene Can Confer Resistance to HIV-1 Integrase Strand Transfer Inhibitors.

    PubMed

    Malet, Isabelle; Subra, Frédéric; Charpentier, Charlotte; Collin, Gilles; Descamps, Diane; Calvez, Vincent; Marcelin, Anne-Geneviève; Delelis, Olivier

    2017-09-26

    Resistance to the integrase strand transfer inhibitors raltegravir and elvitegravir is often due to well-identified mutations in the integrase gene. However, the situation is less clear for patients who fail dolutegravir treatment. Furthermore, most in vitro experiments to select resistance to dolutegravir have resulted in few mutations of the integrase gene. We performed an in vitro dolutegravir resistance selection experiment by using a breakthrough method. First, MT4 cells were infected with human immunodeficiency virus type 1 (HIV-1) Lai. After integration into the host cell genome, cells were washed to remove unbound virus and 500 nM dolutegravir was added to the cell medium. This high concentration of the drug was maintained throughout selection. At day 80, we detected a virus highly resistant to dolutegravir, raltegravir, and elvitegravir that remained susceptible to zidovudine. Sequencing of the virus showed no mutations in the integrase gene but highlighted the emergence of five mutations, all located in the nef region, of which four were clustered in the 3' polypurine tract (PPT). Mutations selected in vitro by dolutegravir, located outside the integrase gene, can confer a high level of resistance to all integrase inhibitors. Thus, HIV-1 can use an alternative mechanism to develop resistance to integrase inhibitors by selecting mutations in the 3' PPT region. Further studies are required to determine to what extent these mutations may explain virological failure during integrase inhibitor therapy.IMPORTANCE Integrase strand transfer inhibitors (INSTIs) are increasingly used both as first-line drugs and in rescue therapy because of their low toxicity and high efficacy in both treatment-naive and treatment-experienced patients. Until now, resistance mutations selected by INSTI exposure have either been described in patients or selected in vitro and involve the integrase gene. Most mutations selected by raltegravir, elvitegravir, or dolutegravir exposure

  8. N-terminal Half of Transportin SR2 Interacts with HIV Integrase.

    PubMed

    Tsirkone, Vicky G; Blokken, Jolien; De Wit, Flore; Breemans, Jolien; De Houwer, Stéphanie; Debyser, Zeger; Christ, Frauke; Strelkov, Sergei V

    2017-03-29

    The karyopherin transportin SR2 (TRN-SR2, TNPO3) is responsible for shuttling specific cargoes such as serine/arginine-rich splicing factors from the cytoplasm to the nucleus. This protein plays a key role in HIV infection by facilitating the nuclear import of the pre-integration complex (PIC) which contains the viral DNA as well as several cellular and HIV proteins including the integrase. The process of nuclear import is considered to be the bottleneck of the viral replication cycle and therefore represents a promising target for anti-HIV drug design. Previous studies have demonstrated that the direct interaction between TRN-SR2 and HIV integrase predominantly involves the catalytic core domain (CCD) and the C-terminal domain (CTD) of the latter. We aimed at providing a detailed molecular view of this interaction through a biochemical characterization of the respective protein complex. Size-exclusion chromatography was used to characterize the interaction of TRN-SR2 with a truncated variant of HIV-1 integrase including both the CCD and CTD. These experiments indicate that one TRN-SR2 molecule can specifically bind one CCD-CTD dimer. Next, the regions of the solenoid-like TRN-SR2 molecule that are involved in the interaction with integrase were identified using AlphaScreen binding assays, revealing that the integrase interacts with the N-terminal half of TRN-SR2 principally through the HEAT repeats 4, 10 and 11. Combining these results with small-angle X-ray scattering data for the complex of TRN-SR2 with truncated integrase we propose a molecular model of the complex. We speculate that nuclear import of the PIC may proceed concurrently with the normal nuclear transport.

  9. Retroviral Integrase Structure and DNA Recombination Mechanism

    PubMed Central

    Engelman, Alan; Cherepanov, Peter

    2015-01-01

    SUMMARY Due to the importance of human immunodeficiency virus type 1 (HIV-1) integrase as a drug target, the biochemistry and structural aspects of retroviral DNA integration have been the focus of intensive research during the past three decades. The retroviral integrase enzyme acts on the linear double-stranded viral DNA product of reverse transcription. Integrase cleaves specific phosphodiester bonds near the viral DNA ends during the 3′ processing reaction. The enzyme then uses the resulting viral DNA 3′-OH groups during strand transfer to cut chromosomal target DNA, which simultaneously joins both viral DNA ends to target DNA 5′-phosphates. Both reactions proceed via direct transesterification of scissile phosphodiester bonds by attacking nucleophiles: a water molecule for 3′ processing, and the viral DNA 3′-OH for strand transfer. X-ray crystal structures of prototype foamy virus integrase-DNA complexes revealed the architectures of the key nucleoprotein complexes that form sequentially during the integration process and explained the roles of active site metal ions in catalysis. X-ray crystallography furthermore elucidated the mechanism of action of HIV-1 integrase strand transfer inhibitors, which are currently used to treat AIDS patients, and provided valuable insights into the mechanisms of viral drug resistance. PMID:25705574

  10. Ty3 integrase mutants defective in reverse transcription or 3'-end processing of extrachromosomal Ty3 DNA.

    PubMed Central

    Kirchner, J; Sandmeyer, S B

    1996-01-01

    Ty3, a retroviruslike element in Saccharomyces cerevisiae, encodes an integrase (IN) which is essential for position-specific transposition. The Ty3 integrase contains the highly conserved His-Xaa(3-7)-His-Xaa(23-32)-Cys-Xaa(2)-Cys and Asp, Asp-Xaa(35)-Glu [D,D(35)E] motifs found in retroviral integrases. Mutations were introduced into the coding region for the Ty3 integrase to determine the effects in vivo of changes in conserved residues of the putative catalytic triad D,D(35)E and the nonconserved carboxyl-terminal region. Ty3 viruslike particles were found to be associated with significant amounts of linear DNA of the approximate size expected for a full-length reverse transcription product and with plus-strand strong-stop DNA. The full-length, preintegrative DNA has at each 3' end 2 bp that are removed prior to or during integration. Such 3'-end processing has not been observed for other retroviruslike elements. A mutation at either D-225 or E-261 of the Ty3 integrase blocked transposition and prevented processing of the 3' ends of Ty3 DNA in vivo, suggesting that the D,D(35)E region is part of the catalytic domain of Ty3 IN. Carboxyl-terminal deletions of integrase caused a dramatic reduction in the amount of Ty3 DNA in vivo and a decrease in reverse transcriptase activity in vitro but did not affect the apparent size or amount of the 55-kDa reverse transcriptase in viruslike particles. The 115-kDa viruslike particle protein, previously shown to react with antibodies to Ty3 integrase, was shown to be a reverse transcriptase-IN fusion protein. These results are consistent with a role for the integrase domain either in proper folding of reverse transcriptase or as part of a heterodimeric reverse transcriptase molecule. PMID:8676501

  11. Uncommon Pathways of Immune Escape Attenuate HIV-1 Integrase Replication Capacity

    PubMed Central

    Chopera, Denis R.; Olvera, Alex; Brumme, Chanson J.; Sela, Jennifer; Markle, Tristan J.; Martin, Eric; Carlson, Jonathan M.; Le, Anh Q.; McGovern, Rachel; Cheung, Peter K.; Kelleher, Anthony D.; Jessen, Heiko; Markowitz, Martin; Rosenberg, Eric; Frahm, Nicole; Sanchez, Jorge; Mallal, Simon; John, Mina; Harrigan, P. Richard; Heckerman, David; Brander, Christian; Walker, Bruce D.; Brumme, Zabrina L.

    2012-01-01

    An attenuation of the HIV-1 replication capacity (RC) has been observed for immune-mediated escape mutations in Gag restricted by protective HLA alleles. However, the extent to which escape mutations affect other viral proteins during natural infection is not well understood. We generated recombinant viruses encoding plasma HIV-1 RNA integrase sequences from antiretroviral-naïve individuals with early (n = 88) and chronic (n = 304) infections and measured the in vitro RC of each. In contrast to data from previous studies of Gag, we observed little evidence that host HLA allele expression was associated with integrase RC. A modest negative correlation was observed between the number of HLA-B-associated integrase polymorphisms and RC in chronic infection (R = −0.2; P = 0.003); however, this effect was not driven by mutations restricted by protective HLA alleles. Notably, the integrase variants S119R, G163E, and I220L, which represent uncommon polymorphisms associated with HLA-C*05, -A*33, and -B*52, respectively, correlated with lower RC (all q < 0.2). We identified a novel C*05-restricted epitope (HTDNGSNF114–121) that likely contributes to the selection of the S119R variant, the polymorphism most significantly associated with lower RC in patient sequences. An NL4-3 mutant encoding the S119R polymorphism displayed a ∼35%-reduced function that was rescued by a single compensatory mutation of A91E. Together, these data indicate that substantial HLA-driven attenuation of integrase is not a general phenomenon during HIV-1 adaptation to host immunity. However, uncommon polymorphisms selected by HLA alleles that are not conventionally regarded to be protective may be associated with impaired protein function. Vulnerable epitopes in integrase might therefore be considered for future vaccine strategies. PMID:22496233

  12. Different Pathways Leading to Integrase Inhibitors Resistance

    PubMed Central

    Thierry, Eloïse; Deprez, Eric; Delelis, Olivier

    2017-01-01

    Integrase strand-transfer inhibitors (INSTIs), such as raltegravir (RAL), elvitegravir, or dolutegravir (DTG), are efficient antiretroviral agents used in HIV treatment in order to inhibit retroviral integration. By contrast to RAL treatments leading to well-identified mutation resistance pathways at the integrase level, recent clinical studies report several cases of patients failing DTG treatment without clearly identified resistance mutation in the integrase gene raising questions for the mechanism behind the resistance. These compounds, by impairing the integration of HIV-1 viral DNA into the host DNA, lead to an accumulation of unintegrated circular viral DNA forms. This viral DNA could be at the origin of the INSTI resistance by two different ways. The first one, sustained by a recent report, involves 2-long terminal repeat circles integration and the second one involves expression of accumulated unintegrated viral DNA leading to a basal production of viral particles maintaining the viral information. PMID:28123383

  13. Making serine integrases work for us.

    PubMed

    Stark, W Marshall

    2017-06-06

    DNA site-specific recombinases are enzymes (often associated with mobile DNA elements) that catalyse breaking and rejoining of DNA strands at specific points, thereby bringing about precise genetic rearrangements. Serine integrases are a group of recombinases derived from bacteriophages. Their unusual properties, including directionality of recombination and simple site requirements, are leading to their development as efficient, versatile tools for applications in experimental biology, biotechnology, synthetic biology and gene therapy. This article summarizes our current knowledge of serine integrase structure and mechanism, then outlines key factors that affect the performance of these phage recombination systems. Recently published studies, that have expanded the repertoire of available systems and reveal system-specific characteristics, will help us to choose the best integrases for envisaged applications. Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.

  14. Prevalence of SOS-mediated control of integron integrase expression as an adaptive trait of chromosomal and mobile integrons

    PubMed Central

    2011-01-01

    Background Integrons are found in hundreds of environmental bacterial species, but are mainly known as the agents responsible for the capture and spread of antibiotic-resistance determinants between Gram-negative pathogens. The SOS response is a regulatory network under control of the repressor protein LexA targeted at addressing DNA damage, thus promoting genetic variation in times of stress. We recently reported a direct link between the SOS response and the expression of integron integrases in Vibrio cholerae and a plasmid-borne class 1 mobile integron. SOS regulation enhances cassette swapping and capture in stressful conditions, while freezing the integron in steady environments. We conducted a systematic study of available integron integrase promoter sequences to analyze the extent of this relationship across the Bacteria domain. Results Our results showed that LexA controls the expression of a large fraction of integron integrases by binding to Escherichia coli-like LexA binding sites. In addition, the results provide experimental validation of LexA control of the integrase gene for another Vibrio chromosomal integron and for a multiresistance plasmid harboring two integrons. There was a significant correlation between lack of LexA control and predicted inactivation of integrase genes, even though experimental evidence also indicates that LexA regulation may be lost to enhance expression of integron cassettes. Conclusions Ancestral-state reconstruction on an integron integrase phylogeny led us to conclude that the ancestral integron was already regulated by LexA. The data also indicated that SOS regulation has been actively preserved in mobile integrons and large chromosomal integrons, suggesting that unregulated integrase activity is selected against. Nonetheless, additional adaptations have probably arisen to cope with unregulated integrase activity. Identifying them may be fundamental in deciphering the uneven distribution of integrons in the Bacteria

  15. Regulated gene insertion by steroid-induced PhiC31 integrase.

    PubMed

    Sharma, Nynne; Moldt, Brian; Dalsgaard, Trine; Jensen, Thomas G; Mikkelsen, Jacob Giehm

    2008-06-01

    Nonviral integration systems are widely used genetic tools in transgenesis and play increasingly important roles in strategies for therapeutic gene transfer. Methods to efficiently regulate the activity of transposases and site-specific recombinases have important implications for their spatiotemporal regulation in live transgenic animals as well as for studies of their applicability as safe vectors for genetic therapy. In this report, strategies for posttranslational induction of a variety of gene-inserting proteins are investigated. An engineered hormone-binding domain, derived from the human progesterone receptor, hPR891, and specifically recognized by the synthetic steroid mifepristone, is fused to the Sleeping Beauty, Frog Prince, piggyBac and Tol2 transposases as well as to the Flp and PhiC31 recombinases. By analyzing mifepristone-directed inducibility of gene insertion in cultured human cells, efficient posttranslational regulation of the Flp recombinase and the PhiC31 integrase is documented. In addition, fusion of the PhiC31 integrase with the ER(T2) modified estrogen receptor hormone-binding domain results in a protein, which is inducible by a factor of 22-fold and retains 75% of the activity of the wild-type protein. These inducible PhiC31 integrase systems are important new tools in transgenesis and in safety studies of the PhiC31 integrase for gene therapy applications.

  16. Multiple effects of mutations in human immunodeficiency virus type 1 integrase on viral replication.

    PubMed Central

    Engelman, A; Englund, G; Orenstein, J M; Martin, M A; Craigie, R

    1995-01-01

    The integration of a DNA copy of the human immunodeficiency virus type 1 (HIV-1) genome into a chromosome of an infected cell is a pivotal step in virus replication. Integration requires the activity of the virus-encoded integrase, which enters the cell as a component of the virion. Results of numerous mutagenesis studies have identified amino acid residues and protein domains of HIV-1 integrase critical for in vitro activity, but only a few of these mutants have been studied for their effects on HIV replication. We have introduced site-directed changes into an infectious DNA clone of HIV-1 and show that integrase mutations can affect virus replication at a variety of steps. We identified mutations that altered virion morphology, levels of particle-associated integrase and reverse transcriptase, and viral DNA synthesis. One replication-defective mutant virus which had normal morphology and protein composition displayed increased levels of circular viral DNA following infection of a T-cell line. This virus also had a significant titer in a CD4-positive indicator cell assay, which requires the viral Tat protein. Although unintegrated viral DNA can serve as a template for Tat expression in infected indicator cells, this level of expression is insufficient to support a spreading viral infection in CD4-positive lymphocytes. PMID:7535863

  17. Virtual screening with AutoDock Vina and the common pharmacophore engine of a low diversity library of fragments and hits against the three allosteric sites of HIV integrase: participation in the SAMPL4 protein-ligand binding challenge.

    PubMed

    Perryman, Alexander L; Santiago, Daniel N; Forli, Stefano; Santos-Martins, Diogo; Olson, Arthur J

    2014-04-01

    To rigorously assess the tools and protocols that can be used to understand and predict macromolecular recognition, and to gain more structural insight into three newly discovered allosteric binding sites on a critical drug target involved in the treatment of HIV infections, the Olson and Levy labs collaborated on the SAMPL4 challenge. This computational blind challenge involved predicting protein-ligand binding against the three allosteric sites of HIV integrase (IN), a viral enzyme for which two drugs (that target the active site) have been approved by the FDA. Positive control cross-docking experiments were utilized to select 13 receptor models out of an initial ensemble of 41 different crystal structures of HIV IN. These 13 models of the targets were selected using our new "Rank Difference Ratio" metric. The first stage of SAMPL4 involved using virtual screens to identify 62 active, allosteric IN inhibitors out of a set of 321 compounds. The second stage involved predicting the binding site(s) and crystallographic binding mode(s) for 57 of these inhibitors. Our team submitted four entries for the first stage that utilized: (1) AutoDock Vina (AD Vina) plus visual inspection; (2) a new common pharmacophore engine; (3) BEDAM replica exchange free energy simulations, and a Consensus approach that combined the predictions of all three strategies. Even with the SAMPL4's very challenging compound library that displayed a significantly lower amount of structural diversity than most libraries that are conventionally employed in prospective virtual screens, these approaches produced hit rates of 24, 25, 34, and 27 %, respectively, on a set with 19 % declared binders. Our only entry for the second stage challenge was based on the results of AD Vina plus visual inspection, and it ranked third place overall according to several different metrics provided by the SAMPL4 organizers. The successful results displayed by these approaches highlight the utility of the computational

  18. Virtual screening with AutoDock Vina and the common pharmacophore engine of a low diversity library of fragments and hits against the three allosteric sites of HIV integrase: participation in the SAMPL4 protein-ligand binding challenge

    NASA Astrophysics Data System (ADS)

    Perryman, Alexander L.; Santiago, Daniel N.; Forli, Stefano; Santos-Martins, Diogo; Olson, Arthur J.

    2014-04-01

    To rigorously assess the tools and protocols that can be used to understand and predict macromolecular recognition, and to gain more structural insight into three newly discovered allosteric binding sites on a critical drug target involved in the treatment of HIV infections, the Olson and Levy labs collaborated on the SAMPL4 challenge. This computational blind challenge involved predicting protein-ligand binding against the three allosteric sites of HIV integrase (IN), a viral enzyme for which two drugs (that target the active site) have been approved by the FDA. Positive control cross-docking experiments were utilized to select 13 receptor models out of an initial ensemble of 41 different crystal structures of HIV IN. These 13 models of the targets were selected using our new "Rank Difference Ratio" metric. The first stage of SAMPL4 involved using virtual screens to identify 62 active, allosteric IN inhibitors out of a set of 321 compounds. The second stage involved predicting the binding site(s) and crystallographic binding mode(s) for 57 of these inhibitors. Our team submitted four entries for the first stage that utilized: (1) AutoDock Vina (AD Vina) plus visual inspection; (2) a new common pharmacophore engine; (3) BEDAM replica exchange free energy simulations, and a Consensus approach that combined the predictions of all three strategies. Even with the SAMPL4's very challenging compound library that displayed a significantly lower amount of structural diversity than most libraries that are conventionally employed in prospective virtual screens, these approaches produced hit rates of 24, 25, 34, and 27 %, respectively, on a set with 19 % declared binders. Our only entry for the second stage challenge was based on the results of AD Vina plus visual inspection, and it ranked third place overall according to several different metrics provided by the SAMPL4 organizers. The successful results displayed by these approaches highlight the utility of the computational

  19. Structural Basis for Inhibitor-Induced Aggregation of HIV Integrase

    PubMed Central

    Sherrill-Mix, Scott; Hwang, Young; Eilers, Grant; McDanal, Charlene; Wang, Ping; Temelkoff, David

    2016-01-01

    The allosteric inhibitors of integrase (termed ALLINIs) interfere with HIV replication by binding to the viral-encoded integrase (IN) protein. Surprisingly, ALLINIs interfere not with DNA integration but with viral particle assembly late during HIV replication. To investigate the ALLINI inhibitory mechanism, we crystallized full-length HIV-1 IN bound to the ALLINI GSK1264 and determined the structure of the complex at 4.4 Å resolution. The structure shows GSK1264 buried between the IN C-terminal domain (CTD) and the catalytic core domain. In the crystal lattice, the interacting domains are contributed by two different dimers so that IN forms an open polymer mediated by inhibitor-bridged contacts; the N-terminal domains do not participate and are structurally disordered. Engineered amino acid substitutions at the inhibitor interface blocked ALLINI-induced multimerization. HIV escape mutants with reduced sensitivity to ALLINIs commonly altered amino acids at or near the inhibitor-bound interface, and these substitutions also diminished IN multimerization. We propose that ALLINIs inhibit particle assembly by stimulating inappropriate polymerization of IN via interactions between the catalytic core domain and the CTD and that understanding the interface involved offers new routes to inhibitor optimization. PMID:27935939

  20. The evaluation of statins as potential inhibitors of the LEDGF/p75-HIV-1 integrase interaction.

    PubMed

    Harrison, Angela T; Kriel, Frederik H; Papathanasopoulos, Maria A; Mosebi, Salerwe; Abrahams, Shaakira; Hewer, Raymond

    2015-03-01

    Lovastatin was identified through virtual screening as a potential inhibitor of the LEDGF/p75-HIV-1 integrase interaction. In an AlphaScreen assay, lovastatin inhibited the purified recombinant protein-protein interaction (IC50 = 1.97 ± 0.45 μm) more effectively than seven other tested statins. None of the eight statins, however, yielded antiviral activity in vitro, while only pravastatin lactone yielded detectable inhibition of HIV-1 integrase strand transfer activity (31.65% at 100 μm). A correlation between lipophilicity and increased cellular toxicity of the statins was observed.

  1. HIV-1 integrase modulates the interaction of the HIV-1 cellular cofactor LEDGF/p75 with chromatin.

    PubMed

    Astiazaran, Paulina; Bueno, Murilo Td; Morales, Elisa; Kugelman, Jeffrey R; Garcia-Rivera, Jose A; Llano, Manuel

    2011-04-21

    Chromatin binding plays a central role in the molecular mechanism of LEDGF/p75 in HIV-1 DNA integration. Conflicting results have been reported in regards to the relevance of the LEDGF/p75 chromatin binding element PWWP domain in its HIV-1 cofactor activity. Here we present evidence that re-expression of a LEDGF/p75 mutant lacking the PWWP domain (ΔPWWP) rescued HIV-1 infection in cells verified to express background levels of endogenous LEDGF/p75 that do not support efficient HIV-1 infection. The HIV-1 cofactor activity of LEDGF/p75 ΔPWWP was similar to that of LEDGF/p75 wild type (WT). A possible molecular explanation for the nonessential role of PWWP domain in the HIV-1 cofactor activity of LEDGF/p75 comes from the fact that coexpression of HIV-1 integrase significantly restored the impaired chromatin binding activity of LEDGF/p75 ΔPWWP. However, integrase failed to promote chromatin binding of a non-chromatin bound LEDGF/p75 mutant that lacks both the PWWP domain and the AT hook motifs (ΔPWWP/AT) and that exhibits negligible HIV-1 cofactor activity. The effect of integrase on the chromatin binding of LEDGF/p75 requires the direct interaction of these two proteins. An HIV-1 integrase mutant, unable to interact with LEDGF/p75, failed to enhance chromatin binding, whereas integrase wild type did not increase the chromatin binding strength of a LEDGF/p75 mutant lacking the integrase binding domain (ΔIBD). Our data reveal that the PWWP domain of LEDGF/p75 is not essential for its HIV-1 cofactor activity, possibly due to an integrase-mediated increase of the chromatin binding strength of this LEDGF/p75 mutant.

  2. Structure based Virtual Screening toward the discovery of novel inhibitors for impeding the protein-protein interaction between HIV-1 integrase and human lens epithelium-derived growth factor (LEDGF/p75).

    PubMed

    Panwar, Umesh; Singh, Sanjeev Kumar

    2017-09-26

    HIV -1 integrase is a unique promising component of the viral replication cycle, catalyzing the integration of reverse transcribed viral cDNA into the host cell genome. Generally, IN activity requires both viral as well as a cellular co-factor in the processing replication cycle. Among them, the human lens epithelium-derived growth factor (LEDGF/p75) represented as promising cellular co-factor which supports the viral replication by tethering IN to the chromatin. Due to because of its major importance in the early steps of HIV replication, the interaction between IN and LEDGF/p75 has become a pleasing target for anti-HIV drug discovery. The present study involves the finding of novel inhibitor based on the information of dimeric CCD of IN in complex with known inhibitor, which were carried out by applying a structure based virtual screening concept with molecular docking. Additionally, Free binding energy, ADME properties, PAINS analysis, Density Functional Theory and Enrichment Calculations were performed on selected compounds for getting a best lead molecule. On the basis of these analyses, the current study proposes top 3 compounds: Enamine- Z742267384, Maybridge- HTS02400, and Specs- AE-848/37125099 with acceptable pharmacological properties and enhanced binding affinity to inhibit the interaction between IN and LEDGF/p75. Furthermore, Simulation studies were carried out on these molecules to expose their dynamics behaviour and stability. We expect that the findings obtained here could be future therapeutic agents and may provide an outline for the experimental studies to stimulate the innovative strategy for research community.

  3. Insight into the ERVK Integrase – Propensity for DNA Damage

    PubMed Central

    Bray, Samantha; Turnbull, Matthew; Hebert, Sherry; Douville, Renée N.

    2016-01-01

    Retroviruses create permanently integrated proviruses that exist in the host genome. Retroviral genomes encode for functionally conserved gag, pro, pol, and env regions, as well as integrase (IN), which is required for retroviral integration. IN mediates viral genome insertion through 3′ end processing of the viral DNA and the strand transfer reaction. This process requires the formation of a pre-integration complex, comprised of IN, viral DNA, and cellular proteins. Viral insertion causes DNA damage, leading to the requirement of host DNA repair mechanisms. Therefore, a failure of DNA repair pathways may result in genomic instability and potentially cause host cell death. Considering the numerous human diseases associated with genomic instability, the endogenous retrovirus-K (ERVK) IN should be considered as a putative contributor to DNA damage in human cells. Future research and drug discovery should focus on ERVK IN activity and its role in human conditions, such as neurological disease and cancers. PMID:27990140

  4. Allosteric HIV-1 Integrase Inhibitors Lead to Premature Degradation of the Viral RNA Genome and Integrase in Target Cells.

    PubMed

    Madison, Michaela K; Lawson, Dana Q; Elliott, Jennifer; Ozantürk, Ayşe Naz; Koneru, Pratibha C; Townsend, Dana; Errando, Manel; Kvaratskhelia, Mamuka; Kutluay, Sebla B

    2017-09-01

    Recent evidence indicates that inhibition of HIV-1 integrase (IN) binding to the viral RNA genome by allosteric integrase inhibitors (ALLINIs) or through mutations within IN yields aberrant particles in which the viral ribonucleoprotein complexes (vRNPs) are eccentrically localized outside the capsid lattice. These particles are noninfectious and are blocked at an early reverse transcription stage in target cells. However, the basis of this reverse transcription defect is unknown. Here, we show that the viral RNA genome and IN from ALLINI-treated virions are prematurely degraded in target cells, whereas reverse transcriptase remains active and stably associated with the capsid lattice. The aberrantly shaped cores in ALLINI-treated particles can efficiently saturate and be degraded by a restricting TRIM5 protein, indicating that they are still composed of capsid proteins arranged in a hexagonal lattice. Notably, the fates of viral core components follow a similar pattern in cells infected with eccentric particles generated by mutations within IN that inhibit its binding to the viral RNA genome. We propose that IN-RNA interactions allow packaging of both the viral RNA genome and IN within the protective capsid lattice to ensure subsequent reverse transcription and productive infection in target cells. Conversely, disruption of these interactions by ALLINIs or mutations in IN leads to premature degradation of both the viral RNA genome and IN, as well as the spatial separation of reverse transcriptase from the viral genome during early steps of infection.IMPORTANCE Recent evidence indicates that HIV-1 integrase (IN) plays a key role during particle maturation by binding to the viral RNA genome. Inhibition of IN-RNA interactions yields aberrant particles with the viral ribonucleoprotein complexes (vRNPs) eccentrically localized outside the conical capsid lattice. Although these particles contain all of the components necessary for reverse transcription, they are blocked

  5. Catalytically-active complex of HIV-1 integrase with a viral DNA substrate binds anti-integrase drugs.

    PubMed

    Alian, Akram; Griner, Sarah L; Chiang, Vicki; Tsiang, Manuel; Jones, Gregg; Birkus, Gabriel; Geleziunas, Romas; Leavitt, Andrew D; Stroud, Robert M

    2009-05-19

    HIV-1 integration into the host cell genome is a multistep process catalyzed by the virally-encoded integrase (IN) protein. In view of the difficulty of obtaining a stable DNA-bound IN at high concentration as required for structure determination, we selected IN-DNA complexes that form disulfide linkages between 5'-thiolated DNA and several single mutations to cysteine around the catalytic site of IN. Mild reducing conditions allowed for selection of the most thermodynamically-stable disulfide-linked species. The most stable complexes induce tetramer formation of IN, as happens during the physiological integration reaction, and are able to catalyze the strand transfer step of retroviral integration. One of these complexes also binds strand-transfer inhibitors of HIV antiviral drugs, making it uniquely valuable among the mutants of this set for understanding portions of the integration reaction. This novel complex may help define substrate interactions and delineate the mechanism of action of known integration inhibitors.

  6. HIV integrase inhibitors: from diketoacids to heterocyclic templates: a history of HIV integrase medicinal chemistry at Merck West Point and Merck Rome (IRBM).

    PubMed

    Egbertson, Melissa S

    2007-01-01

    Replication of the human immunodeficiency virus (HIV) is dependent upon the enzyme HIV integrase (IN), one of three essential enzymes encoded in the viral genome. HIV-1 IN catalyzes the insertion of the proviral DNA into the host genome (strand transfer). HIV-1 IN therefore presents an attractive chemotherapeutic target for the treatment of HIV infection and AIDS that could provide patients and physicians with an additional option for treatment. Assays were developed to identify inhibitors of IN strand transfer. Diketoacid lead compounds were explored and developed into a variety of heterocyclic templates that are potent inhibitors of integrase strand transfer with suitable medicinal chemical properties for treating HIV infection and AIDS. The 1,6-naphthyridine L-870810 (Antiviral activity in cells IC(95) NHS = 102 nM, n=237), was shown to be efficacious in reducing viral RNA by 1.7 log units after doses of 400mg BID to HIV infected patients. Optimization of physical properties led to L-900564, an inhibitor of HIV IN that has excellent cell potency in the presence of protein (Antiviral activity in cells IC(95) NHS = 16 nM, n=15), excellent activity against mutants raised to HIV integrase inhibitors, and a very good pharmacokinetic profile.

  7. Modeling, analysis, and validation of a novel HIV integrase structure provide insights into the binding modes of potent integrase inhibitors.

    PubMed

    Chen, X; Tsiang, M; Yu, F; Hung, M; Jones, G S; Zeynalzadegan, A; Qi, X; Jin, H; Kim, C U; Swaminathan, S; Chen, J M

    2008-07-11

    It has been shown that L-731988, a potent integrase inhibitor, targets a conformation of the integrase enzyme formed when complexed to viral DNA, with the 3'-end dinucleotide already cleaved. It has also been shown that diketo acid inhibitors bind to the strand transfer complex of integrase and are competitive with the host target DNA. However, published X-ray structures of HIV integrase do not include the DNA; thus, there is a need to develop a model representing the strand transfer complex. In this study, we have constructed an active-site model of the HIV-1 integrase complexed with viral DNA using the crystal structure of DNA-bound transposase and have identified a binding mode for inhibitors. This proposed binding mechanism for integrase inhibitors involves interaction with a specific Mg(2+) in the active site, accentuated by a hydrophobic interaction in a cavity formed by a flexible loop upon DNA binding. We further validated the integrase active-site model by selectively mutating key residues predicted to play an important role in the binding of inhibitors. Thus, we have a binding model that is applicable to a wide range of potent integrase inhibitors and is consistent with the available resistant mutation data.

  8. Comparison of Newly Assembled Full Length HIV-1 Integrase With Prototype Foamy Virus Integrase: Structure-Function Prospective

    PubMed Central

    Dayer, Mohammad Reza

    2016-01-01

    Background 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. Objectives 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. Materials and Methods 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. Results 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. Conclusions 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. PMID:27540450

  9. Multimodal mechanism of action of allosteric HIV-1 integrase inhibitors

    PubMed Central

    Jurado, Kellie Ann; Engelman, Alan

    2013-01-01

    Integrase (IN) is required for lentivirus replication and is a proven drug target for the prevention of AIDS in HIV-1 infected patients. While clinical strand transfer inhibitors disarm the IN active site, allosteric inhibition of enzyme activity through the disruption of IN-IN protein interfaces holds great therapeutic potential. A promising class of allosteric IN inhibitors (ALLINIs), 2-(quinolin-3-yl) acetic acid derivatives, engage the IN catalytic core domain dimerization interface at the binding site for the host integration co-factor LEDGF/p75. ALLINIs promote IN multimerization and, independent of LEDGF/p75 protein, block the formation of the active IN-DNA complex, as well as inhibit the IN-LEDGF/p75 interaction in vitro. Yet, rather unexpectedly, the full inhibitory effect of these compounds is exerted during the late phase of HIV-1 replication. ALLINIs impair particle core maturation as well as reverse transcription and integration during the subsequent round of virus infection. Recapitulating the pleiotropic phenotypes observed with numerous IN mutant viruses, ALLINIs provide insight into underlying aspects of IN biology that extend beyond its catalytic activity. Therefore, in addition to the potential to expand our repertoire of HIV-1 antiretrovirals, ALLINIs afford important structural probes to dissect the multifaceted nature of the IN protein throughout the course of HIV-1 replication. PMID:24274067

  10. Rapid Optimization of Engineered Metabolic Pathways with Serine Integrase Recombinational Assembly (SIRA).

    PubMed

    Merrick, C A; Wardrope, C; Paget, J E; Colloms, S D; Rosser, S J

    2016-01-01

    Metabolic pathway engineering in microbial hosts for heterologous biosynthesis of commodity compounds and fine chemicals offers a cheaper, greener, and more reliable method of production than does chemical synthesis. However, engineering metabolic pathways within a microbe is a complicated process: levels of gene expression, protein stability, enzyme activity, and metabolic flux must be balanced for high productivity without compromising host cell viability. A major rate-limiting step in engineering microbes for optimum biosynthesis of a target compound is DNA assembly, as current methods can be cumbersome and costly. Serine integrase recombinational assembly (SIRA) is a rapid DNA assembly method that utilizes serine integrases, and is particularly applicable to rapid optimization of engineered metabolic pathways. Using six pairs of orthogonal attP and attB sites with different central dinucleotide sequences that follow SIRA design principles, we have demonstrated that ΦC31 integrase can be used to (1) insert a single piece of DNA into a substrate plasmid; (2) assemble three, four, and five DNA parts encoding the enzymes for functional metabolic pathways in a one-pot reaction; (3) generate combinatorial libraries of metabolic pathway constructs with varied ribosome binding site strengths or gene orders in a one-pot reaction; and (4) replace and add DNA parts within a construct through targeted postassembly modification. We explain the mechanism of SIRA and the principles behind designing a SIRA reaction. We also provide protocols for making SIRA reaction components and practical methods for applying SIRA to rapid optimization of metabolic pathways.

  11. phiC31 integrase-mediated site-specific recombination in barley.

    PubMed

    Kapusi, Eszter; Kempe, Katja; Rubtsova, Myroslava; Kumlehn, Jochen; Gils, Mario

    2012-01-01

    The Streptomyces phage phiC31 integrase was tested for its feasibility in excising transgenes from the barley genome through site-specific recombination. We produced transgenic barley plants expressing an active phiC31 integrase and crossed them with transgenic barley plants carrying a target locus for recombination. The target sequence involves a reporter gene encoding green fluorescent protein (GFP), which is flanked by the attB and attP recognition sites for the phiC31 integrase. This sequence disruptively separates a gusA coding sequence from an upstream rice actin promoter. We succeeded in producing site-specific recombination events in the hybrid progeny of 11 independent barley plants carrying the above target sequence after crossing with plants carrying a phiC31 expression cassette. Some of the hybrids displayed fully executed recombination. Excision of the GFP gene fostered activation of the gusA gene, as visualized in tissue of hybrid plants by histochemical staining. The recombinant loci were detected in progeny of selfed F(1), even in individuals lacking the phiC31 transgene, which provides evidence of stability and generative transmission of the recombination events. In several plants that displayed incomplete recombination, extrachromosomal excision circles were identified. Besides the technical advance achieved in this study, the generated phiC31 integrase-expressing barley plants provide foundational stock material for use in future approaches to barley genetic improvement, such as the production of marker-free transgenic plants or switching transgene activity.

  12. phiC31 Integrase-Mediated Site-Specific Recombination in Barley

    PubMed Central

    Rubtsova, Myroslava; Kumlehn, Jochen; Gils, Mario

    2012-01-01

    The Streptomyces phage phiC31 integrase was tested for its feasibility in excising transgenes from the barley genome through site-specific recombination. We produced transgenic barley plants expressing an active phiC31 integrase and crossed them with transgenic barley plants carrying a target locus for recombination. The target sequence involves a reporter gene encoding green fluorescent protein (GFP), which is flanked by the attB and attP recognition sites for the phiC31 integrase. This sequence disruptively separates a gusA coding sequence from an upstream rice actin promoter. We succeeded in producing site-specific recombination events in the hybrid progeny of 11 independent barley plants carrying the above target sequence after crossing with plants carrying a phiC31 expression cassette. Some of the hybrids displayed fully executed recombination. Excision of the GFP gene fostered activation of the gusA gene, as visualized in tissue of hybrid plants by histochemical staining. The recombinant loci were detected in progeny of selfed F1, even in individuals lacking the phiC31 transgene, which provides evidence of stability and generative transmission of the recombination events. In several plants that displayed incomplete recombination, extrachromosomal excision circles were identified. Besides the technical advance achieved in this study, the generated phiC31 integrase-expressing barley plants provide foundational stock material for use in future approaches to barley genetic improvement, such as the production of marker-free transgenic plants or switching transgene activity. PMID:23024817

  13. Discovery and analysis of inhibitors of the human immunodeficiency integrase.

    PubMed

    Hazuda, D; Felock, P J; Hastings, J C; Pramanik, B; Wolfe, A L

    1997-05-01

    An essential step in the replication of retroviruses is the integration of a DNA copy of the viral genome into the genome of the host cell. Integration encompasses a series of ordered endonucleolytic and DNA strand transfer reactions catalyzed by the viral enzyme, integrase. The requirement for integrase activity in the propagation of HIV-1 in cell culture defines the enzyme as a potential target for chemotherapeutic intervention. We have therefore developed a non-radioisotopic microtiter plate assay which can be used to identify novel inhibitors of integrase from random chemical screens and for the bioassay driven isolation of inhibitors from natural products. This assay uncouples various steps in the reaction pathway and therefore can be exploited to characterize inhibitors. In this monograph we describe a series of modifications to the method which facilitate such mechanistic studies using as an example a series of previously described integrase inhibitors.

  14. Genome-wide manipulations of Drosophila melanogaster with transposons, Flp recombinase, and ΦC31 integrase.

    PubMed

    Venken, Koen J T; Bellen, Hugo J

    2012-01-01

    Transposable elements, the Flp recombinase, and the ΦC31 integrase are used in Drosophila melanogaster for numerous genome-wide manipulations. Often, their use is combined in a synergistic fashion to alter and engineer the fruit fly genome. Transposons are the foundation for all transgenic technologies in flies and hence almost all innovations in the fruit fly. They have been instrumental in the generation of genome-wide collections of insertions for gene disruption and manipulation. Many important transgenic strains of these collections are available from public repositories. The Flp protein is the most widely used recombinase to induce mitotic clones to study individual gene function. However, Flp has also been used to generate chromosome- and genome-wide collections of precise deletions, inversions, and duplications. Similarly, transposons that contain attP attachment sites for the ΦC31 integrase can be used for numerous applications. This integrase was incorporated into a transgenesis system that allows the integration of small to very large DNA fragments that can be easily manipulated through recombineering. This system allowed the creation of genomic DNA libraries for genome-wide gene manipulations and X chromosome duplications. Moreover, the attP sites are being used to create libraries of tens of thousands of RNAi constructs and tissue-specific GAL4 lines. This chapter focuses on genome-wide applications of transposons, Flp recombinase, and ΦC31 integrase that greatly facilitate experimental manipulation of Drosophila.

  15. Identification of conserved amino acid residues critical for human immunodeficiency virus type 1 integrase function in vitro.

    PubMed Central

    Engelman, A; Craigie, R

    1992-01-01

    We have probed the structural organization of the human immunodeficiency virus type 1 integrase protein by limited proteolysis and the functional organization by site-directed mutagenesis of selected amino acid residues. A central region of the protein was relatively resistant to proteolysis. Proteins with altered amino acids in this region, or in the N-terminal part of the protein that includes a putative zinc-binding motif, were purified and assayed for 3' processing, DNA strand transfer, and disintegration activities in vitro. In general, these mutations had parallel effects on 3' processing and DNA strand transfer, suggesting that integrase may utilize a single active site for both reactions. The only proteins that were completely inactive in all three assays contained mutations at conserved amino acids in the central region, suggesting that this part of the protein may be involved in catalysis. In contrast, none of the mutations in the N-terminal region resulted in a protein that was inactive in all three assays, suggesting that this part of integrase may not be essential for catalysis. The disintegration reaction was particularly insensitive to these amino acid substitutions, indicating that some function that is important for 3' processing and DNA strand transfer may be dispensable for disintegration. Images PMID:1404595

  16. Allosteric Inhibition of Human Immunodeficiency Virus Integrase

    PubMed Central

    Gupta, Kushol; Brady, Troy; Dyer, Benjamin M.; Malani, Nirav; Hwang, Young; Male, Frances; Nolte, Robert T.; Wang, Liping; Velthuisen, Emile; Jeffrey, Jerry; Van Duyne, Gregory D.; Bushman, Frederic D.

    2014-01-01

    HIV-1 replication in the presence of antiviral agents results in evolution of drug-resistant variants, motivating the search for additional drug classes. Here we report studies of GSK1264, which was identified as a compound that disrupts the interaction between HIV-1 integrase (IN) and the cellular factor lens epithelium-derived growth factor (LEDGF)/p75. GSK1264 displayed potent antiviral activity and was found to bind at the site occupied by LEDGF/p75 on IN by x-ray crystallography. Assays of HIV replication in the presence of GSK1264 showed only modest inhibition of the early infection steps and little effect on integration targeting, which is guided by the LEDGF/p75·IN interaction. In contrast, inhibition of late replication steps was more potent. Particle production was normal, but particles showed reduced infectivity. GSK1264 promoted aggregation of IN and preformed LEDGF/p75·IN complexes, suggesting a mechanism of inhibition. LEDGF/p75 was not displaced from IN during aggregation, indicating trapping of LEDGF/p75 in aggregates. Aggregation assays with truncated IN variants revealed that a construct with catalytic and C-terminal domains of IN only formed an open polymer associated with efficient drug-induced aggregation. These data suggest that the allosteric inhibitors of IN are promising antiviral agents and provide new information on their mechanism of action. PMID:24904063

  17. Probing chelation motifs in HIV integrase inhibitors

    PubMed Central

    Agrawal, Arpita; DeSoto, Jamie; Fullagar, Jessica L.; Maddali, Kasthuraiah; Rostami, Shahrzad; Richman, Douglas D.; Pommier, Yves; Cohen, Seth M.

    2012-01-01

    A series of HIV integrase (HIV-1 IN) inhibitors were synthesized to evaluate the role of the metal-binding group (MBG) in this class of metalloenzyme inhibitors. A total of 21 different raltegravir-chelator derivative (RCD) compounds were prepared that differed only in the nature of the MBG. These IN strand-transfer inhibitors (INSTIs) were evaluated in vitro in cell-free enzyme activity assays, and the in vitro results were further validated in cell culture experiments. All of the active compounds showed selective inhibition of the strand-transfer reaction over 3′-processing, suggesting a common mode of action with raltegravir. The results of the in vitro activity suggest that the nature of the MBG donor atoms, the overall MBG structure, and the specific arrangement of the MBG donor atom triad are essential for obtaining maximal HIV-1 IN inhibition. At least two compounds (RCD-4, RCD-5) containing a hydroxypyrone MBG were found to display superior strand-transfer inhibition when compared to an abbreviated analogue of raltegravir (RCD-1). By isolating and examining the role of the MBG in a series of INSTIs, we have identified a scaffold (hydroxypyrones) that may provide access to a unique class of HIV-1 IN inhibitors, and may help overcome rising raltegravir resistance. PMID:22308350

  18. Catalytically-active complex of HIV-1 integrase with a viral DNA substrate binds anti-integrase drugs

    PubMed Central

    Alian, Akram; Griner, Sarah L.; Chiang, Vicki; Tsiang, Manuel; Jones, Gregg; Birkus, Gabriel; Geleziunas, Romas; Leavitt, Andrew D.; Stroud, Robert M.

    2009-01-01

    HIV-1 integration into the host cell genome is a multistep process catalyzed by the virally-encoded integrase (IN) protein. In view of the difficulty of obtaining a stable DNA-bound IN at high concentration as required for structure determination, we selected IN–DNA complexes that form disulfide linkages between 5′-thiolated DNA and several single mutations to cysteine around the catalytic site of IN. Mild reducing conditions allowed for selection of the most thermodynamically-stable disulfide-linked species. The most stable complexes induce tetramer formation of IN, as happens during the physiological integration reaction, and are able to catalyze the strand transfer step of retroviral integration. One of these complexes also binds strand-transfer inhibitors of HIV antiviral drugs, making it uniquely valuable among the mutants of this set for understanding portions of the integration reaction. This novel complex may help define substrate interactions and delineate the mechanism of action of known integration inhibitors. PMID:19416821

  19. Proteomic analysis in Daphnia magna exposed to As(III), As(V) and Cd heavy metals and their binary mixtures for screening potential biomarkers.

    PubMed

    Le, Thai-Hoang; Lim, Eun-Suk; Hong, Nam-Hui; Lee, Sung-Kyu; Shim, Yon Sik; Hwang, Jin Rae; Kim, Yang-Hoon; Min, Jiho

    2013-11-01

    In this study, the effects of three widespread heavy metals, As(III), As(V) and Cd, and their binary mixtures on the proteomic profile in D. magna were examined to screen novel protein biomarkers using the two-dimensional gel electrophoresis method (2DE). Ten 20d daphnia were exposed to the LC20 concentrations for each of a total of 8 treatments, including the control, As(III), As(V), Cd, [As(III)+As(V)], [As(III)+Cd], [As(V)+Cd], and [As(III), As(V), Cd], for 24h before protein isolation. Three replicates were performed for each treatment. These protein samples were employed for 2DE experiments with a pH gradient gel strip from pH 3 to pH 10. The protein spots were detected by a silver staining process and their intensities were analyzed by Progenesis software to discover the differentially expressed proteins (DEPs) in response to each heavy metal. A total of 117 differentially expressed proteins (DEPs) were found in daphnia responding to the 8 treatments and mapped onto a 2D proteome map, which provides some information of the molecular weight (MW) and pI value for each protein. All of these DEPs are considered as potential candidates for protein biomarkers in D. magna for detecting heavy metals in the aquatic ecosystem. Comparing the proteomic results among these treatments suggested that exposing D. magna to binary mixtures of heavy metals may result in some complex interactive molecular responses within them, rather than just the simple sum of the proteomic profiles of the individual chemicals, (As(III), As(V), and Cd).

  20. Crystal structure of the HIV-1 integrase core domain in complex with sucrose reveals details of an allosteric inhibitory binding site

    SciTech Connect

    Wielens, Jerome; Headey, Stephen J.; Jeevarajah, Dharshini; Rhodes, David I.; Deadman, John; Chalmers, David K.; Scanlon, Martin J.; Parker, Michael W.

    2010-04-19

    HIV integrase (IN) is an essential enzyme in HIV replication and an important target for drug design. IN has been shown to interact with a number of cellular and viral proteins during the integration process. Disruption of these important interactions could provide a mechanism for allosteric inhibition of IN. We present the highest resolution crystal structure of the IN core domain to date. We also present a crystal structure of the IN core domain in complex with sucrose which is bound at the dimer interface in a region that has previously been reported to bind integrase inhibitors.

  1. Styrylquinolines, Integrase Inhibitors Acting Prior to Integration: a New Mechanism of Action for Anti-Integrase Agents

    PubMed Central

    Bonnenfant, Sabine; Thomas, Claire Marie; Vita, Claudio; Subra, Frédéric; Deprez, Eric; Zouhiri, Fatima; Desmaële, Didier; d'Angelo, Jean; Mouscadet, Jean François; Leh, Hervé

    2004-01-01

    We have previously shown that styrylquinolines (SQLs) are integrase inhibitors in vitro. They compete with the long terminal repeat substrate for integrase. Here, we describe the cellular mode of action of these molecules. We show that SQLs do not interfere with virus entry. In fact, concentrations of up to 20 times the 50% inhibitory concentration did not inhibit cell-to-cell fusion or affect the interaction between GP120 and CD4 in vitro. Moreover, the pseudotype of the retrovirus envelope did not affect drug activity. Quantitative reverse transcription PCR experiments showed that SQLs do not inhibit the entry of the genomic RNA. In contrast, the treatment of human immunodeficiency virus type 1-infected cells with SQLs reduced the amount of the late cDNA, suggesting for the first time that integrase targeting molecules may affect the accumulation of DNA during reverse transcription. The cellular target of SQLs was confirmed by the appearance of mutations in the integrase gene when viruses were grown in the presence of increasing concentrations of SQLs. Finally, these mutations led to SQL-resistant viruses when introduced into the wild-type sequence. In contrast, SQLs were fully active against reverse transcriptase inhibitor- and diketo acid-resistant viruses, positioning SQLs as a second group of anti-integrase compounds. PMID:15140970

  2. Preparation and certification of arsenate [As(V)] reference material, NMIJ CRM 7912-a.

    PubMed

    Narukawa, Tomohiro; Kuroiwa, Takayoshi; Narushima, Izumi; Jimbo, Yasujiro; Suzuki, Toshihiro; Chiba, Koichi

    2010-05-01

    Arsenate [As(V)] solution reference material, National Metrology Institute of Japan (NMIJ) certified reference material (CRM) 7912-a, for speciation of arsenic species was developed and certified by NMIJ, the National Institute of Advanced Industrial Science and Technology. High-purity As(2)O(3) reagent powder was dissolved in 0.8 M HNO(3) solution and As(III) was oxidized to As(V) with HNO(3) to prepare 100 mg kg(-1) of As(V) candidate CRM solution. The solution was bottled in 400 bottles (50 mL each). The concentration of As(V) was determined by four independent analytical techniques-inductively coupled plasma mass spectrometry, inductively coupled plasma optical emission spectrometry, graphite furnace atomic absorption spectrometry, and liquid chromatography inductively coupled plasma mass spectrometry-according to As(V) calibration solutions, which were prepared from the arsenic standard of the Japan Calibration Service system and whose species was guaranteed to be As(V) by NMIJ. The uncertainties of all the measurements and preparation procedures were evaluated. The certified value of As(V) in the CRM is (99.53 +/- 1.67) mg kg(-1) (k = 2).

  3. Speciation of arsenic in Euglena gracilis cells exposed to As(V).

    PubMed

    Miot, Jennyfer; Morin, Guillaume; Skouri-Panet, Fériel; Férard, Céline; Poitevin, Antonine; Aubry, Emmanuel; Ona-Nguema, Georges; Juillot, Farid; Guyot, François; Brown, Gordon E

    2009-05-01

    Euglena gracilis is a photosynthetic eukaryote ubiquitous in arsenic-polluted acid mine drainages and is locally exposed to As(III) and As(V) concentrations up to 250 and 100 mg L(-1), respectively. Here, arsenic speciation in E. graciliswas determined by X-ray absorption spectroscopy and selected (bio)chemical methods on cells grown at nonlimiting phosphate concentrations. Our results suggest the following detoxification scheme: (1) uptake of As(V) from solution in competition with phosphate, (2) intracellular reduction to As(III), (3) complexation by cytoplasmic proteic thiol ligands of low molecular weight, and (4) As(III) export from the cell. However, at As(V) concentrations >100 mg L(-1), growth rate is markedly lowered and As(V) remains mostly unreduced during the extended lag period. Intracellular As(V) is found to be exclusively concentrated in the membrane + nucleus fraction, suggesting that arsenate could substitute for phosphate groups in membranes or in phosphate-containing macromolecules. Thus, arsenic species are partitioned, with As(III)-thiol compounds concentrated in the cytoplasmic proteic pool and As(V)-compounds associated with the membrane + nucleus fraction. The increasing growth delay observed with increasing initial As(V) concentration in the culture medium is proposed to result from the combination of a higher As(V) uptake and limiting intracellular As(V) reduction rate and As(III) export rate. Under high As(V) exposure conditions (200 mg L(-1)) the reduction step is found to be the most limiting step for detoxification.

  4. The role of lysine 186 in HIV-1 integrase multimerization

    SciTech Connect

    Berthoux, Lionel; Sebastian, Sarah; Muesing, Mark A.; Luban, Jeremy . E-mail: luban@irb.unisi.ch

    2007-07-20

    HIV-1 integrase (IN) catalyzes biochemical reactions required for viral cDNA insertion into host cell chromosomal DNA, an essential step in the HIV-1 replication cycle. In one of these reactions, the two ends of the linear viral cDNA are believed to be simultaneously ligated to chromosomal DNA by a tetrameric form of IN. The structure of the full-length IN tetramer is not known but a model consisting of the N-terminal domain and the catalytic core revealed basic residues 186 to 188 at the interface between the two IN dimers. We found that alteration of these residues, in particular changing IN lysine residue 186 to glutamate (K186Q), impairs IN oligomerization in the yeast two-hybrid system and decreases oligomeric forms of IN within virions. When expressed independently of other viral proteins in human cells, IN-K186Q did not concentrate in the nucleus as did wild-type IN. Co-expression of wild-type IN restored the multimerization defects of IN-K186Q, in both the two-hybrid system and in virions, and also rescued the nuclear targeting defects. Virions bearing IN-K186Q were not infectious in a single cycle of replication but when mixed virions containing two different IN mutants were produced, IN-K186Q was capable of complementing the catalytically inactive mutant IN-D116A. Our biochemical and functional data support the crystallographic model in which IN residue K186 lies at the interface between IN dimers and suggest that tetramerization is important, not only for concerted integration, but also for IN nuclear targeting.

  5. HIV-1 Integrase Strand Transfer Inhibitors with Reduced Susceptibility to Drug Resistant Mutant Integrases | Center for Cancer Research

    Cancer.gov

    Mutant forms of HIV-1 IN reduce the therapeutic effectiveness of integrase strand transfer inhibitors (INSTIs). The cover figure shows the IN of prototype foamy virus complexed to a novel INSTI (gold) that retains potency against resistant mutants of HIV-1 IN. Overlain are the host and viral DNA substrates (blue and green, respectively), showing substrate mimicry by the inhibitor.

  6. Adsorption of As(V) and As(III) by nanocrystalline titanium dioxide.

    PubMed

    Pena, Maria E; Korfiatis, George P; Patel, Manish; Lippincott, Lee; Meng, Xiaoguang

    2005-06-01

    This study evaluated the effectiveness of nanocrystalline titanium dioxide (TiO(2)) in removing arsenate [As(V)] and arsenite [As(III)] and in photocatalytic oxidation of As(III). Batch adsorption and oxidation experiments were conducted with TiO(2) suspensions prepared in a 0.04 M NaCl solution and in a challenge water containing the competing anions phosphate, silicate, and carbonate. The removal of As(V) and As(III) reached equilibrium within 4h and the adsorption kinetics were described by a pseudo-second-order equation. The TiO(2) was effective for As(V) removal at pH<8 and showed a maximum removal for As(III) at pH of about 7.5 in the challenge water. The adsorption capacity of the TiO(2) for As(V) and As(III) was much higher than fumed TiO(2) (Degussa P25) and granular ferric oxide. More than 0.5 mmol/g of As(V) and As(III) was adsorbed by the TiO(2) at an equilibrium arsenic concentration of 0.6mM. The presence of the competing anions had a moderate effect on the adsorption capacities of the TiO(2) for As(III) and As(V) in a neutral pH range. In the presence of sunlight and dissolved oxygen, As(III) (26.7 microM or 2mg/L) was completely converted to As(V) in a 0.2g/L TiO(2) suspension through photocatalytic oxidation within 25 min. The nanocrystalline TiO(2) is an effective adsorbent for As(V) and As(III) and an efficient photocatalyst.

  7. Single-molecule analysis of ϕC31 integrase-mediated site-specific recombination by tethered particle motion

    PubMed Central

    Fan, Hsiu-Fang; Hsieh, Tao-shih; Ma, Chien-Hui; Jayaram, Makkuni

    2016-01-01

    Serine and tyrosine site-specific recombinases (SRs and YRs, respectively) provide templates for understanding the chemical mechanisms and conformational dynamics of strand cleavage/exchange between DNA partners. Current evidence suggests a rather intriguing mechanism for serine recombination, in which one half of the cleaved synaptic complex undergoes a 180° rotation relative to the other. The ‘small’ and ‘large’ SRs contain a compact amino-terminal catalytic domain, but differ conspicuously in their carboxyl-terminal domains. So far, only one serine recombinase has been analyzed using single substrate molecules. We now utilized single-molecule tethered particle motion (TPM) to follow step-by-step recombination catalyzed by a large SR, phage ϕC31 integrase. The integrase promotes unidirectional DNA exchange between attB and attP sites to integrate the phage genome into the host chromosome. The recombination directionality factor (RDF; ϕC31 gp3) activates the excision reaction (attL × attR). From integrase-induced changes in TPM in the presence or absence of gp3, we delineated the individual steps of recombination and their kinetic features. The gp3 protein appears to regulate recombination directionality by selectively promoting or excluding active conformations of the synapse formed by specific att site partners. Our results support a ‘gated rotation’ of the synaptic complex between DNA cleavage and joining. PMID:27986956

  8. Simian immunodeficiency virus-Vpx for improving integrase defective lentiviral vector-based vaccines

    PubMed Central

    2012-01-01

    Background Integrase defective lentiviral vectors (IDLV) represent a promising delivery system for immunization purposes. Human dendritic cells (DC) are the main cell types mediating the immune response and are readily transduced by IDLV, allowing effective triggering of in vitro expansion of antigen-specific primed CD8+ T cells. However, IDLV expression in transduced DC is at lower levels than those of the integrase (IN) competent counterpart, thus requiring further improvement of IDLV for future use in the clinic. Results In this paper we show that the addition of simian immunodeficiency (SIV)-Vpx protein in the vector preparation greatly improves transduction of human and simian DC, but not of murine DC, thus increasing the ability of transduced DC to act as functional antigen presenting cells, in the absence of integrated vector sequences. Importantly, the presence of SIV-Vpx allows for using lower dose of input IDLV during in vitro transduction, thus further improving the IDLV safety profile. Conclusions These results have significant implications for the development of IDLV-based vaccines. PMID:22913641

  9. A novel family of integrases associated with prophages and genomic islands integrated within the tRNA-dihydrouridine synthase A (dusA) gene

    PubMed Central

    Farrugia, Daniel N.; Elbourne, Liam D. H.; Mabbutt, Bridget C.; Paulsen, Ian T.

    2015-01-01

    Genomic islands play a key role in prokaryotic genome plasticity. Genomic islands integrate into chromosomal loci such as transfer RNA genes and protein coding genes, whilst retaining various cargo genes that potentially bestow novel functions on the host organism. A gene encoding a putative integrase was identified at a single site within the 5′ end of the dusA gene in the genomes of over 200 bacteria. This integrase was discovered to be a component of numerous genomic islands, which appear to share a target site within the dusA gene. dusA encodes the tRNA-dihydrouridine synthase A enzyme, which catalyses the post-transcriptional reduction of uridine to dihydrouridine in tRNA. Genomic islands encoding homologous dusA-associated integrases were found at a much lower frequency within the related dusB and dusC genes, and non-dus genes. Excision of these dusA-associated islands from the chromosome as circularized intermediates was confirmed by polymerase chain reaction. Analysis of the dusA-associated islands indicated that they were highly diverse, with the integrase gene representing the only universal common feature. PMID:25883135

  10. Synthesis and HIV-1 integrase inhibition activity of some N-arylindoles.

    PubMed

    Xu, Hui; Liu, Wu-Qing; Fan, Ling-Ling; Chen, Yang; Yang, Liu-Meng; Lv, Lei; Zheng, Yong-Tang

    2008-05-01

    Eight simple N-arylindoles were designed, synthesized and evaluated as human immunodeficiency virus (HIV)-1 integrase inhibitors in vitro for the first time. Among these compounds, 3b, 3e and 3g demonstrated significant anti-HIV-1 integrase activity. Especially 3b showed the highest anti-HIV-1 integrase activity with EC50 value of 7.88 microg/ml and TI value of 24.61. Meantime, some structure-activity relationships were also observed and will provide a new lead for design and discovery of more potent N-arylindoles as HIV-1 integrase inhibitors.

  11. Prophage Integrase Typing Is a Useful Indicator of Genomic Diversity in Salmonella enterica.

    PubMed

    Colavecchio, Anna; D'Souza, Yasmin; Tompkins, Elizabeth; Jeukens, Julie; Freschi, Luca; Emond-Rheault, Jean-Guillaume; Kukavica-Ibrulj, Irena; Boyle, Brian; Bekal, Sadjia; Tamber, Sandeep; Levesque, Roger C; Goodridge, Lawrence D

    2017-01-01

    Salmonella enterica is a bacterial species that is a major cause of illness in humans and food-producing animals. S. enterica exhibits considerable inter-serovar diversity, as evidenced by the large number of host adapted serovars that have been identified. The development of methods to assess genome diversity in S. enterica will help to further define the limits of diversity in this foodborne pathogen. Thus, we evaluated a PCR assay, which targets prophage integrase genes, as a rapid method to investigate S. enterica genome diversity. To evaluate the PCR prophage integrase assay, 49 isolates of S. enterica were selected, including 19 clinical isolates from clonal serovars (Enteritidis and Heidelberg) that commonly cause human illness, and 30 isolates from food-associated Salmonella serovars that rarely cause human illness. The number of integrase genes identified by the PCR assay was compared to the number of integrase genes within intact prophages identified by whole genome sequencing and phage finding program PHASTER. The PCR assay identified a total of 147 prophage integrase genes within the 49 S. enterica genomes (79 integrase genes in the food-associated Salmonella isolates, 50 integrase genes in S. Enteritidis, and 18 integrase genes in S. Heidelberg). In comparison, whole genome sequencing and PHASTER identified a total of 75 prophage integrase genes within 102 intact prophages in the 49 S. enterica genomes (44 integrase genes in the food-associated Salmonella isolates, 21 integrase genes in S. Enteritidis, and 9 integrase genes in S. Heidelberg). Collectively, both the PCR assay and PHASTER identified the presence of a large diversity of prophage integrase genes in the food-associated isolates compared to the clinical isolates, thus indicating a high degree of diversity in the food-associated isolates, and confirming the clonal nature of S. Enteritidis and S. Heidelberg. Moreover, PHASTER revealed a diversity of 29 different types of prophages and 23

  12. Specific anti-integrase abzymes from HIV-infected patients: a comparison of the cleavage sites of intact globular HIV integrase and two 20-mer oligopeptides corresponding to its antigenic determinants.

    PubMed

    Odintsova, Elena S; Dmitrenok, Pavel S; Buneva, Valentina N; Nevinsky, Georgy A

    2013-03-01

    HIV-infected patients possess anti-integrase (IN) IgGs and IgMs that, after isolation by chromatography on IN-Sepharose, unlike canonical proteases, specifically hydrolyze only IN but not many other tested proteins. Hydrolysis of intact globular IN first leads to formation of many long fragments of protein, while its long incubation with anti-IN antibodies, especially in the case of abzymes (Abzs) with a high proteolytic activity, results in the formation of short and very short oligopeptides (OPs). To identify all sites of IgG-mediated proteolysis corresponding to known AGDs of integrase, we have used a combination of reverse-phase chromatography, matrix-assisted laser desorption/ionization spectrometry, and thin-layer chromatography to analyze the cleavage products of two 20-mer OPs corresponding to these AGDs. Both OPs contained 9-10 mainly clustered major, medium, and minor sites of cleavage. The main superficial cleavage sites of the AGDs in the intact IN and sites of partial or deep hydrolysis of the peptides analyzed do not coincide. The active sites of anti-IN Abzs are localized on their light chains, whereas the heavy chains are responsible for the affinity of protein substrates. Interactions of intact globular proteins with both light and heavy chains of Abzs provide high specificity of IN hydrolysis. The affinity of anti-IN Abzs for intact integrase was ~1000-fold higher than for the OPs. The data suggest that both OPs interact mainly with the light chains of different monoclonal Abzs of the total pool of IgGs, which possesses lower affinity for substrates; and therefore, depending on the oligopeptide sequences, their hydrolysis may be less specific and remarkably different in comparison with the cleavage of intact globular IN.

  13. Adsorption, desorption and fractionation of As(V) on untreated and mussel shell-treated granitic material

    NASA Astrophysics Data System (ADS)

    Seco-Reigosa, N.; Cutillas-Barreiro, L.; Nóvoa-Muñoz, J. C.; Arias-Estévez, M.; Álvarez-Rodríguez, E.; Fernández-Sanjurjo, M. J.; Núñez-Delgado, A.

    2015-03-01

    As(V) adsorption and desorption were studied on granitic material, coarse and fine mussel shell and granitic material amended with 12 and 24 t ha-1 fine shell, investigating the effect of different As(V) concentrations and different pH as well as the fractions where the adsorbed As(V) was retained. As(V) adsorption was higher on fine than on coarse shell. Mussel shell amendment increased As(V) adsorption on granitic material. Adsorption data corresponding to the unamended and shell-amended granitic material were satisfactory fitted to the Langmuir and Freundlich models. Desorption was always <19% when the highest As(V) concentration (100 mg L-1) was added. Regarding the effect of pH, the granitic material showed its highest adsorption (66%) at pH <6, and it was lower as pH increased. Fine shell presented notable adsorption in the whole pH range between 6 and 12, with a maximum of 83%. The shell-amended granitic material showed high As(V) adsorption, with a maximum (99%) at pH near 8, but decreased as pH increased. Desorption varying pH was always <26%. In the granitic material, desorption increased progressively when pH increased from 4 to 6, contrary to what happened to mussel shell. Regarding the fractionation of the adsorbed As(V), most of it was in the soluble fraction (weakly bound). The granitic material did not show high As(V) retention capacity, which could facilitate As(V) transfer to water courses and to the food chain in case of As(V) compounds being applied on this material; however, the mussel shell amendment increased As(V) retention, making this practice recommendable.

  14. As(V) Sorption/Desorption on Different Waste Materials and Soil Samples

    PubMed Central

    Quintáns-Fondo, Ana; Fernández-Sanjurjo, María J.; Álvarez-Rodríguez, Esperanza

    2017-01-01

    Aiming to investigate the efficacy of different materials as bio-sorbents for the purification of As-polluted waters, batch-type experiments were employed to study As(V) sorption and desorption on oak ash, pine bark, hemp waste, mussel shell, pyritic material, and soil samples, as a function of the As(V) concentration added. Pyritic material and oak ash showed high sorption (90% and >87%) and low desorption (<2% and <7%). Alternatively, hemp waste showed low retention (16% sorption and 100% desorption of the amount previously sorbed), fine shell and pine bark sorbed <3% and desorbed 100%, the vineyard soil sample sorbed 8% and released 85%, and the forest soil sample sorbed 32% and desorbed 38%. Sorption data fitted well to the Langmuir and Freundlich models in the case of both soil samples and the pyritic material, but only to the Freundlich equation in the case of the various by-products. These results indicate that the pyritic material and oak ash can be considered efficient As(V) sorbents (thus, useful in remediation of contaminated sites and removal of that pollutant), even when As(V) concentrations up to 6 mmol L−1 are added, while the other materials that were tested cannot retain or remove As(V) from polluted media. PMID:28753919

  15. Correlation between timing of ASV administration and complications in snake bites.

    PubMed

    Narvencar, K

    2006-09-01

    The present study was undertaken to study the relationship between the time of anti-snake venom (ASV) administration due to late arrival of patient at hospital and subsequent development of complications. All patients of snake bite that presented to our institution over a period of 1 1/2 years were included in the present study. A detailed clinical history, clinical examination and investigations were carried out. The patients were administered ASV within 10 minutes of presentation. The bite to needle time (time between the bite and start of ASV) was noted. The patients were then followed up to note any subsequent development of complications. The end-point of the study was normalization of haematological and neurological parameters. Fifty patients became eligible for the study. Twenty patients (40%) had complications while remaining 30 patients (60%) were uncomplicated. An attempt was made to study relationship between bite to needle time and subsequent development of complications. It was found to be significant at 5% level of significance (p<0.05) by chi square test. Incidence of complications was directly proportional to the duration of venom in the blood prior to neutralization by ASV due to late arrival of patient at hospital. The early institution of ASV is beneficial in preventing complications however severe is the systemic envenomation.

  16. An experimental design approach for modeling As(V) adsorption from aqueous solution by activated carbon.

    PubMed

    Bakkal Gula, C; Bilgin Simsek, E; Duranoglu, D; Beker, U

    2015-01-01

    The present paper discusses response surface methodology as an efficient approach for predictive model building and optimization of As(V) adsorption on activated carbon derived from a food industry waste: peach stones. The objectives of the study are application of a three-factor 2³ full factorial and central composite design technique for maximizing As(V) removal by produced activated carbon, and examination of the interactive effects of three independent variables (i.e., solution pH, temperature, and initial concentration) on As(V) adsorption capacity. Adsorption equilibrium was investigated by using Langmuir, Freundlich, and Dubinin-Radushkevich isotherm models. First-order and second-order kinetic equations were used for modeling of adsorption kinetics. Thermodynamic parameters (ΔG °, ΔH °, and ΔS °) were calculated and used to explain the As(V) adsorption mechanism. The negative value of ΔH (-7.778 kJ mol⁻¹) supported the exothermic nature of the sorption process and the Gibbs free energy values (ΔG°) were found to be negative, which indicates that the As(V) adsorption is feasible and spontaneous.

  17. Arsenic response of AtPCS1- and CePCS-expressing plants - effects of external As(V) concentration on As-accumulation pattern and NPT metabolism.

    PubMed

    Wojas, Sylwia; Clemens, Stephan; Skłodowska, Aleksandra; Maria Antosiewicz, Danuta

    2010-02-15

    Phytochelatins (PCs) are small, cysteine-rich peptides, known to play a major role in detoxification of both cadmium and arsenic. The aim of this study was to determine whether overexpression of either of two PC synthase (PCS) genes, AtPCS1 and CePCS in Nicotiana tabacum (previously shown to cause decrease and increase, respectively, of cadmium tolerance of tobacco - Wojas et al., 2008) also contributes to such contrasting phenotypes with respect to arsenic (As) tolerance and accumulation, and how observed responses relate to non-protein thiol (NPT) metabolism. The expression of both genes resulted in an increase of As-tolerance, with CePCS plants most tolerant. We showed for the first time that the response of PCS overexpressing plants to As qualitatively depends on the external As(V) concentration. At the less toxic 50muM As(V), AtPCS1 and CePCS transformants accumulated more As in roots and leaves than WT. An increase in PC production and the level of PC2 species was detected in leaves of AtPCS1 and CePCS plants, which might explain their enhanced As-accumulation and tolerance. In contrast, at the highly toxic 200muM As(V), several disturbances in thiol metabolism of PCS overexpressing plants were found, surprisingly, including decrease of PC levels both in roots and leaves of transgenic plants relative to WT. The increase in As-tolerance and accumulation due to AtPCS1 and CePCS overexpression, observed at the As(V) concentrations similar to those found in As-contaminated soils, makes these genes promising candidates for plant engineering for phytoremediation. Copyright 2009 Elsevier GmbH. All rights reserved.

  18. Novel and diverse integron integrase genes and integron-like gene cassettes are prevalent in deep-sea hydrothermal vents.

    PubMed

    Elsaied, Hosam; Stokes, H W; Nakamura, Takamichi; Kitamura, Keiko; Fuse, Hiroyuki; Maruyama, Akihiko

    2007-09-01

    The lack of information about mobile DNA in deep-sea hydrothermal vents limits our understanding of the phylogenetic diversity of the mobile genome of bacteria in these environments. We used culture-independent techniques to explore the diversity of the integron/mobile gene cassette system in a variety of hydrothermal vent communities. Three samples, which included two different hydrothermal vent fluids and a mussel species that contained essentially monophyletic sulfur-oxidizing bacterial endosymbionts, were collected from Suiyo Seamount, Izu-Bonin, Japan, and Pika site, Mariana arc. First, using degenerate polymerase chain reaction (PCR) primers, we amplified integron integrase genes from metagenomic DNA from each sample. From vent fluids, we discovered 74 new integrase genes that were classified into 11 previously undescribed integron classes. One integrase gene was recorded in the mussel symbiont and was phylogenetically distant from those recovered from vent fluids. Second, using PCR primers targeting the gene cassette recombination site (59-be), we amplified and subsequently identified 60 diverse gene cassettes. In multicassette amplicons, a total of 13 59-be sites were identified. Most of these sites displayed features that were atypical of the features previously well conserved in this family. The Suiyo vent fluid was characterized by gene cassette open reading frames (ORFs) that had significant homologies with transferases, DNA-binding proteins and metal transporter proteins, while the majority of Pika vent fluid gene cassettes contained novel ORFs with no identifiable homologues in databases. The symbiont gene cassette ORFs were found to be matched with DNA repair proteins, methionine aminopeptidase, aminopeptidase N, O-sialoglycoprotein endopeptidase and glutamate synthase, which are proteins expected to play a role in animal/symbiont metabolism. The success of this study indicates that the integron/gene cassette system is common in deep-sea hydrothermal

  19. HIV‑1 Integrase Strand Transfer Inhibitors with Reduced Susceptibility to Drug Resistant Mutant Integrases | Center for Cancer Research

    Cancer.gov

    On the cover: Mutant forms of HIV-1 IN reduce the therapeutic effectiveness of integrase strand transfer inhibitors (INSTIs). The cover figure shows the IN of prototype foamy virus complexed to a novel INSTI (gold) that retains potency against resistant mutants of HIV-1 IN. Overlain are the host and viral DNA substrates (blue and green, respectively), showing substrate mimicry by the inhibitor. Cover design by Joseph Myer

  20. Next-generation site-directed transgenesis in the malaria vector mosquito Anopheles gambiae: self-docking strains expressing germline-specific phiC31 integrase.

    PubMed

    Meredith, Janet M; Underhill, Ann; McArthur, Clare C; Eggleston, Paul

    2013-01-01

    Diseases transmitted by mosquitoes have a devastating impact on global health and the situation is complicated due to difficulties with both existing control measures and the impact of climate change. Genetically modified mosquitoes that are refractory to disease transmission are seen as having great potential in the delivery of novel control strategies. The Streptomyces phage phiC31 integrase system has been successfully adapted for site-directed transgene integration in a range of insects, thus overcoming many limitations due to size constraints and random integration associated with transposon-mediated transformation. Using this technology, we previously published the first site-directed transformation of Anopheles gambiae, the principal vector of human malaria. Mosquitoes were initially engineered to incorporate the phiC31 docking site at a defined genomic location. A second phase of genetic modification then achieved site-directed integration of an anti-malarial effector gene. In the current publication we report improved efficiency and utility of the phiC31 integrase system following the generation of Anopheles gambiae self-docking strains. Four independent strains, with docking sites at known locations on three different chromosome arms, were engineered to express integrase under control of the regulatory regions of the nanos gene from Anopheles gambiae. The resulting protein accumulates in the posterior oocyte to provide integrase activity at the site of germline development. Two self-docking strains, exhibiting significantly different levels of integrase expression, were assessed for site-directed transgene integration and found to demonstrate greatly improved survival and efficiency of transformation. In the fight against malaria, it is imperative to establish a broad repertoire of both anti-malarial effector genes and tissue-specific promoters to regulate their expression, enabling those offering maximum effect with minimum fitness cost to be identified

  1. Dialysis purification of integrase-DNA complexes provides high-resolution atomic force microscopy images: dimeric recombinant HIV-1 integrase binding and specific looping on DNA.

    PubMed

    Tsuruyama, Tatsuaki; Nakai, Tonau; Ohmori, Rei; Ozeki, Munetaka; Tamaki, Keiji; Yoshikawa, Kenichi

    2013-01-01

    It remains difficult to obtain high-resolution atomic force microscopy images of HIV-1 integrase bound to DNA in a dimeric or tetrameric fashion. We therefore constructed specific target DNAs to assess HIV-1 integrase binding and purified the complex by dialysis prior to analysis. Our resulting atomic force microscopy analyses indicated precise size of binding human immunodeficiency virus type 1 (HIV-1) recombinant integrase in a tetrameric manner, inducing formation of a loop-like or figure-eight-like secondary structure in the target DNA. Our findings regarding the target DNA secondary structure provide new insights into the intermediate states of retroviral integration.

  2. Development of a phenotypic susceptibility assay for HIV-1 integrase inhibitors.

    PubMed

    Heger, Eva; Theis, Alexandra Andrée; Remmel, Klaus; Walter, Hauke; Pironti, Alejandro; Knops, Elena; Di Cristanziano, Veronica; Jensen, Björn; Esser, Stefan; Kaiser, Rolf; Lübke, Nadine

    2016-12-01

    Phenotypic resistance analysis is an indispensable method for determination of HIV-1 resistance and cross-resistance to novel drug compounds. Since integrase inhibitors are essential components of recent antiretroviral combination therapies, phenotypic resistance data, in conjunction with the corresponding genotypes, are needed for improving rules-based and data-driven tools for resistance prediction, such as HIV-Grade and geno2pheno[integrase]. For generation of phenotypic resistance data to recent integrase inhibitors, a recombinant phenotypic integrase susceptibility assay was established. For validation purposes, the phenotypic resistance to raltegravir, elvitegravir and dolutegravir of nine subtype-B virus strains, isolated from integrase inhibitor-naïve and raltegravir-treated patients was determined. Genotypic resistance analysis identified four virus strains harbouring RAL resistance-associated mutations. Phenotypic resistance analysis was performed as follows. The HIV-1 integrase genes were cloned into a modified pNL4-3 vector and transfected into 293T cells for the generation of recombinant virus. The integrase-inhibitor susceptibility of the recombinant viruses was determined via an indicator cell line. While raltegravir resistance profiles presented a high cross-resistance to elvitegravir, dolutegravir maintained in-vitro activity in spite of the Y143R and N155H mutations, confirming the strong activity of dolutegravir against raltegravir-resistant viruses. Solely a Q148H+G140S variant presented reduced susceptibility to dolutegravir. In conclusion, our phenotypic susceptibility assay permits resistance analysis of the integrase gene of patient-derived viruses for integrase inhibitors by replication-competent recombinants. Thus, this assay can be used to analyze phenotypic drug resistance of integrase inhibitors in vitro. It provides the possibility to determine the impact of newly appearing mutational patterns to drug resistance of recent integrase

  3. Adsorption of As(V) inside the pores of porous hematite in water.

    PubMed

    Dai, Min; Xia, Ling; Song, Shaoxian; Peng, Changsheng; Lopez-Valdivieso, Alejandro

    2016-04-15

    As(V) adsorption inside the pores of porous hematite in water has been studied in this work. This study was performed on nonporous hematite and porous hematite prepared from the thermal decomposition of goethite and siderite through the measurements of adsorption isotherm, SEM-EDX, XRD and BET. The results demonstrated that the As(V) adsorption was difficult to be realized inside pores if they were too small. This observation might be due to that the pore entrances were blocked by the adsorbed ions and thus the inside surfaces became invalid for the adsorption. Only if the pore size is large enough, the effective surface area inside pores would be close to that on non-porous hematite for As(V) adsorption. In addition, it was found that siderite is better than goethite for preparing porous hematite with thermal decomposition as adsorbent for arsenic removal. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Magnetite nanoparticles coated glass wool for As(V) removal from drinking water

    NASA Astrophysics Data System (ADS)

    Kango, Sarita; Kumar, Rajesh

    2015-08-01

    Arsenic (As) removal from contaminated groundwater is a key environmental concern worldwide. In this study, glass wool was coated with magnetite nanoparticles under argon gas flow and magnetite coated glass wool have been investigated for application as an adsorbent for As(V) removal from water. The adsorbent was characterized by using Scanning Electron Microscopy (SEM) and arsenic contaminated water treated with adsorbent was analyzed by Inductively Coupled Plasma Mass Spectroscopy (ICP-MS). The ICP-MS results showed that 10 g/L of adsorbent removed 99.4% of As(V) within 5 hours at pH-7 and initial arsenic concentration of 360µg/L. Adsorption kinetics data fitted well in pseudo-first-order kinetics model with high correlation coefficient (R2 = 0.995). As magnetite nanoparticles coated glass wool showed favorable adsorption behavior for As(V), it can be a promising tool for water purification.

  5. Magnetite nanoparticles coated glass wool for As(V) removal from drinking water

    SciTech Connect

    Kango, Sarita; Kumar, Rajesh

    2015-08-28

    Arsenic (As) removal from contaminated groundwater is a key environmental concern worldwide. In this study, glass wool was coated with magnetite nanoparticles under argon gas flow and magnetite coated glass wool have been investigated for application as an adsorbent for As(V) removal from water. The adsorbent was characterized by using Scanning Electron Microscopy (SEM) and arsenic contaminated water treated with adsorbent was analyzed by Inductively Coupled Plasma Mass Spectroscopy (ICP-MS). The ICP-MS results showed that 10 g/L of adsorbent removed 99.4% of As(V) within 5 hours at pH-7 and initial arsenic concentration of 360µg/L. Adsorption kinetics data fitted well in pseudo-first-order kinetics model with high correlation coefficient (R{sup 2} = 0.995). As magnetite nanoparticles coated glass wool showed favorable adsorption behavior for As(V), it can be a promising tool for water purification.

  6. As(V) and P Competitive Sorption on Soils, By-Products and Waste Materials

    PubMed Central

    Rivas-Pérez, Ivana María; Paradelo-Núñez, Remigio; Nóvoa-Muñoz, Juan Carlos; Arias-Estévez, Manuel; Fernández-Sanjurjo, María José; Álvarez-Rodríguez, Esperanza; Núñez-Delgado, Avelino

    2015-01-01

    Batch-type experiments were used to study competitive As(V) and P sorption on various soils and sorbent materials. The materials assayed were a forest soil, a vineyard soil, pyritic material, granitic material, coarsely and finely ground mussel shell, calcinated mussel shell ash, pine sawdust and slate processing fines. Competition between As(V) and P was pronounced in the case of both soils, granitic material, slate fines, both shells and pine sawdust, showing more affinity for P. Contrary, the pyritic material and mussel shell ash showed high and similar affinity for As(V) and P. These results could be useful to make a correct use of the soils and materials assayed when focusing on As and P removal in solid or liquid media, in circumstances where both pollutants may compete for sorption sites. PMID:26690456

  7. Raltegravir, elvitegravir, and metoogravir: the birth of "me-too" HIV-1 integrase inhibitors

    PubMed Central

    Serrao, Erik; Odde, Srinivas; Ramkumar, Kavya; Neamati, Nouri

    2009-01-01

    Merck's MK-0518, known as raltegravir, has recently become the first FDA-approved HIV-1 integrase (IN) inhibitor and has since risen to blockbuster drug status. Much research has in turn been conducted over the last few years aimed at recreating but optimizing the compound's interactions with the protein. Resulting me-too drugs have shown favorable pharmacokinetic properties and appear drug-like but, as expected, most have a highly similar interaction with IN to that of raltegravir. We propose that, based upon conclusions drawn from our docking studies illustrated herein, most of these me-too MK-0518 analogues may experience a low success rate against raltegravir-resistant HIV strains. As HIV has a very high mutational competence, the development of drugs with new mechanisms of inhibitory action and/or new active substituents may be a more successful route to take in the development of second- and third-generation IN inhibitors. PMID:19265512

  8. Design of cell-permeable stapled peptides as HIV-1 integrase inhibitors.

    PubMed

    Long, Ya-Qiu; Huang, Shao-Xu; Zawahir, Zahrah; Xu, Zhong-Liang; Li, Huiyuan; Sanchez, Tino W; Zhi, Ying; De Houwer, Stephanie; Christ, Frauke; Debyser, Zeger; Neamati, Nouri

    2013-07-11

    HIV-1 integrase (IN) catalyzes the integration of viral DNA into the host genome, involving several interactions with the viral and cellular proteins. We have previously identified peptide IN inhibitors derived from the α-helical regions along the dimeric interface of HIV-1 IN. Herein, we show that appropriate hydrocarbon stapling of these peptides to stabilize their helical structure remarkably improves the cell permeability, thus allowing inhibition of the HIV-1 replication in cell culture. Furthermore, the stabilized peptides inhibit the interaction of IN with the cellular cofactor LEDGF/p75. Cellular uptake of the stapled peptide was confirmed in four different cell lines using a fluorescein-labeled analogue. Given their enhanced potency and cell permeability, these stapled peptides can serve as not only lead IN inhibitors but also prototypical biochemical probes or "nanoneedles" for the elucidation of HIV-1 IN dimerization and host cofactor interactions within their native cellular environment.

  9. Impairment of Human Immunodeficiency Virus Type-1 Integrase SUMOylation Correlates with an Early Replication Defect*

    PubMed Central

    Zamborlini, Alessia; Coiffic, Audrey; Beauclair, Guillaume; Delelis, Olivier; Paris, Joris; Koh, Yashuiro; Magne, Fabian; Giron, Marie-Lou; Tobaly-Tapiero, Joelle; Deprez, Eric; Emiliani, Stephane; Engelman, Alan; de Thé, Hugues; Saïb, Ali

    2011-01-01

    HIV-1 integrase (IN) orchestrates the integration of the reverse transcribed viral cDNA into the host cell genome and participates also in other steps of HIV-1 replication. Cellular and viral factors assist IN in performing its multiple functions, and post-translational modifications contribute to modulate its activities. Here, we show that HIV-1 IN is modified by SUMO proteins and that phylogenetically conserved SUMOylation consensus motifs represent major SUMO acceptor sites. Viruses harboring SUMOylation site IN mutants displayed a replication defect that was mapped during the early stages of infection, before integration but after reverse transcription. Because SUMOylation-defective IN mutants retained WT catalytic activity, we hypothesize that SUMOylation might regulate the affinity of IN for co-factors, contributing to efficient HIV-1 replication. PMID:21454548

  10. Biological responses of duckweed (Lemna minor L.) exposed to the inorganic arsenic species As(III) and As(V): effects of concentration and duration of exposure.

    PubMed

    Duman, Fatih; Ozturk, Fatma; Aydin, Zeki

    2010-06-01

    The accumulation of arsenic (As) and physiological responses of Lemna minor L. under different concentration (0, 1, 4, 16 and 64 microM) and duration (1, 2, 4 and 6 days) of two species As, NaAsO(2) and Na(2)HAsO(4).7H(2)O, were studied in hydroponics. The accumulation of both As species depended on As concentration and exposure duration. The highest accumulation of As was found as 17408 and 8674 microg g(-1), for plants exposed to 64 microM of As(III) and As(V), respectively, after 6 days. Two-way ANOVA analyses indicated that, for plants exposed to arsenite (As(III)), exposure duration had a greater effect than concentration on As accumulation. Conversely, exposure concentration had a greater effect on As accumulation in plants exposed to arsenate (As(V)). Arsenic exposure levels, approaching 16 microM for As(III) and 64 microM for As(V), did not significantly affect EC values. Beyond these exposure concentrations, EC values increased in a manner that depended on duration. Significant effect of As(III) on lipid peroxidation was observed at 1 microM application whereas, this effect started to be significant after an exposure to 16 microM As(V). For both As(III) and As(V), photosynthetic pigment levels slightly increased for the first day with respect to the control, followed by a gradual decline at higher concentrations and durations. An increase in protein content and enzyme activity was observed at moderate exposure conditions, followed by a decrease. Significant positive correlations were determined between accumulated As and ion leakage and lipid peroxidation. Negative correlations were found between accumulated As and total chlorophyll and protein content. Our results suggested that exposure duration and concentration had a strong synergetic effect on antioxidant enzyme activity. The findings of the present study may be useful when this plant is used as a phytoremediator in arsenic-polluted water.

  11. A metal-induced conformational change and activation of HIV-1 integrase.

    PubMed

    Asante-Appiah, E; Skalka, A M

    1997-06-27

    Retroviral integrases are composed of three independently folding domains whose organization relevant to one another is largely unknown. As an approach to understanding its structure, we have investigated the effect of the required metal cofactor(s), Mn2+ or Mg2+, on the conformation of human immunodeficiency virus type 1 (HIV-1) integrase (IN) using monoclonal antibodies (mAbs) that are specific for each of these three domains. Upon the addition of increasing concentrations of the divalent cations to immobilized HIV-1 IN in ELISA assays, binding of mAbs specific for either the C-terminal domain or for an epitope in the catalytic core domain was lost, whereas binding of an N terminus-specific mAb was unaffected. Size exclusion chromatography of a nonaggregating derivative of HIV-1 IN showed that the oligomeric state of the protein did not change under conditions in which recognition of the core and C terminus-specific mAbs was lost. Preincubation with Mn2+ increased the resistance of HIV-1 IN to proteolytic digestion and produced a digestion pattern that was significantly different from that observed with the apoprotein. A derivative that lacked the N-terminal domain, IN(50-288), exhibited the same metal-dependent changes observed with the full-length protein, whereas the isolated catalytic core domain IN(50-212) did not. From this we conclude that the metal-induced conformational change comprises a reorganization of the core and C-terminal domains. Preincubation with Mn2+ increased the specific activity of HIV-1 IN 5-fold. Enzymatic activity was inhibited by the conformation-sensitive C terminus-specific mAb, but this inhibition was reduced greatly if the enzyme was first preincubated with metal ions. Thus, it appears that apo-HIV-1 IN exists predominantly in an inactive conformation that is converted into a catalytically competent form upon the addition of metal ions.

  12. Functional Characteristics of a Highly Specific Integrase Encoded by an LTR-Retrotransposon

    PubMed Central

    Peyretaillade, Eric; Brasset, Emilie; Dastugue, Bernard; Vaury, Chantal

    2008-01-01

    Background The retroviral Integrase protein catalyzes the insertion of linear viral DNA into host cell DNA. Although different retroviruses have been shown to target distinctive chromosomal regions, few of them display a site-specific integration. ZAM, a retroelement from Drosophila melanogaster very similar in structure and replication cycle to mammalian retroviruses is highly site-specific. Indeed, ZAM copies target the genomic 5′-CGCGCg-3′ consensus-sequences. To enlighten the determinants of this high integration specificity, we investigated the functional properties of its integrase protein denoted ZAM-IN. Principal Findings Here we show that ZAM-IN displays the property to nick DNA molecules in vitro. This endonuclease activity targets specific sequences that are present in a 388 bp fragment taken from the white locus and known to be a genomic ZAM integration site in vivo. Furthermore, ZAM-IN displays the unusual property to directly bind specific genomic DNA sequences. Two specific and independent sites are recognized within the 388 bp fragment of the white locus: the CGCGCg sequence and a closely apposed site different in sequence. Conclusion This study strongly argues that the intrinsic properties of ZAM-IN, ie its binding properties and its endonuclease activity, play an important part in ZAM integration specificity. Its ability to select two binding sites and to nick the DNA molecule reminds the strategy used by some site-specific recombination enzymes and forms the basis for site-specific integration strategies potentially useful in a broad range of genetic engineering applications. PMID:18784842

  13. HIV-1 Protease, Reverse Transcriptase, and Integrase Variation

    PubMed Central

    Sankaran, Kris; Varghese, Vici; Winters, Mark A.; Hurt, Christopher B.; Eron, Joseph J.; Parkin, Neil; Holmes, Susan P.; Holodniy, Mark; Shafer, Robert W.

    2016-01-01

    ABSTRACT HIV-1 protease (PR), reverse transcriptase (RT), and integrase (IN) variability presents a challenge to laboratories performing genotypic resistance testing. This challenge will grow with increased sequencing of samples enriched for proviral DNA such as dried blood spots and increased use of next-generation sequencing (NGS) to detect low-abundance HIV-1 variants. We analyzed PR and RT sequences from >100,000 individuals and IN sequences from >10,000 individuals to characterize variation at each amino acid position, identify mutations indicating APOBEC-mediated G-to-A editing, and identify mutations resulting from selective drug pressure. Forty-seven percent of PR, 37% of RT, and 34% of IN positions had one or more amino acid variants with a prevalence of ≥1%. Seventy percent of PR, 60% of RT, and 60% of IN positions had one or more variants with a prevalence of ≥0.1%. Overall 201 PR, 636 RT, and 346 IN variants had a prevalence of ≥0.1%. The median intersubtype prevalence ratios were 2.9-, 2.1-, and 1.9-fold for these PR, RT, and IN variants, respectively. Only 5.0% of PR, 3.7% of RT, and 2.0% of IN variants had a median intersubtype prevalence ratio of ≥10-fold. Variants at lower prevalences were more likely to differ biochemically and to be part of an electrophoretic mixture compared to high-prevalence variants. There were 209 mutations indicative of APOBEC-mediated G-to-A editing and 326 mutations nonpolymorphic treatment selected. Identification of viruses with a high number of APOBEC-associated mutations will facilitate the quality control of dried blood spot sequencing. Identifying sequences with a high proportion of rare mutations will facilitate the quality control of NGS. IMPORTANCE Most antiretroviral drugs target three HIV-1 proteins: PR, RT, and IN. These proteins are highly variable: many different amino acids can be present at the same position in viruses from different individuals. Some of the amino acid variants cause drug

  14. HIV-1 Protease, Reverse Transcriptase, and Integrase Variation.

    PubMed

    Rhee, Soo-Yon; Sankaran, Kris; Varghese, Vici; Winters, Mark A; Hurt, Christopher B; Eron, Joseph J; Parkin, Neil; Holmes, Susan P; Holodniy, Mark; Shafer, Robert W

    2016-07-01

    HIV-1 protease (PR), reverse transcriptase (RT), and integrase (IN) variability presents a challenge to laboratories performing genotypic resistance testing. This challenge will grow with increased sequencing of samples enriched for proviral DNA such as dried blood spots and increased use of next-generation sequencing (NGS) to detect low-abundance HIV-1 variants. We analyzed PR and RT sequences from >100,000 individuals and IN sequences from >10,000 individuals to characterize variation at each amino acid position, identify mutations indicating APOBEC-mediated G-to-A editing, and identify mutations resulting from selective drug pressure. Forty-seven percent of PR, 37% of RT, and 34% of IN positions had one or more amino acid variants with a prevalence of ≥1%. Seventy percent of PR, 60% of RT, and 60% of IN positions had one or more variants with a prevalence of ≥0.1%. Overall 201 PR, 636 RT, and 346 IN variants had a prevalence of ≥0.1%. The median intersubtype prevalence ratios were 2.9-, 2.1-, and 1.9-fold for these PR, RT, and IN variants, respectively. Only 5.0% of PR, 3.7% of RT, and 2.0% of IN variants had a median intersubtype prevalence ratio of ≥10-fold. Variants at lower prevalences were more likely to differ biochemically and to be part of an electrophoretic mixture compared to high-prevalence variants. There were 209 mutations indicative of APOBEC-mediated G-to-A editing and 326 mutations nonpolymorphic treatment selected. Identification of viruses with a high number of APOBEC-associated mutations will facilitate the quality control of dried blood spot sequencing. Identifying sequences with a high proportion of rare mutations will facilitate the quality control of NGS. Most antiretroviral drugs target three HIV-1 proteins: PR, RT, and IN. These proteins are highly variable: many different amino acids can be present at the same position in viruses from different individuals. Some of the amino acid variants cause drug resistance and occur mainly

  15. X-ray and molecular modelling in fragment-based design of three small quinoline scaffolds for HIV integrase inhibitors.

    PubMed

    Majerz-Maniecka, Katarzyna; Musiol, Robert; Skórska-Stania, Agnieszka; Tabak, Dominik; Mazur, Pawel; Oleksyn, Barbara J; Polanski, Jaroslaw

    2011-03-01

    Crystal structures of three small molecular scaffolds based on quinoline, 2-methylquinoline-5,8-dione, 5-hydroxy-quinaldine-6-carboxylic acid and 8-hydroxy-quinaldine-7-carboxylic acid, were characterised. 5-Hydroxy-quinaldine-6-carboxylic acid was co-crystallized with cobalt(II) chloride to form a model of divalent metal cation-ligand interactions for potential HIV integrase inhibitors. Molecular docking into active site of HIV IN was also performed on 1WKN PDB file. Selected ligand-protein interactions have been found specific for active compounds. Studied structures can be used as scaffolds in fragment-based design of new potent drugs.

  16. Identification of key structural determinants of the IntI1 integron integrase that influence attC × attI1 recombination efficiency

    PubMed Central

    Demarre, Gaëlle; Frumerie, Clara; Gopaul, Deshmukh N.; Mazel, Didier

    2007-01-01

    The integron platform codes for an integrase (IntI) from the tyrosine family of recombinases that mediates recombination between a proximal double-strand recombination site, attI and a single-strand target recombination site, attC. The attI site is only recognized by its cognate integrase, while the various tested attCs sites are recombined by several different IntI integrases. We have developed a genetic system to enrich and select mutants of IntI1 that provide a higher yield of recombination in order to identify key protein structural elements important for attC × attI1 recombination. We isolated mutants with higher activity on wild type and mutant attC sites. Interestingly, three out of four characterized IntI1 mutants selected on different substrates are mutants of the conserved aspartic acid in position 161. The IntI1 model we made based on the VchIntIA 3D structure suggests that substitution at this position, which plays a central role in multimer assembly, can increase or decrease the stability of the complex and accordingly influence the rate of attI × attC recombination versus attC × attC. These results suggest that there is a balance between the specificity of the protein and the protein/protein interactions in the recombination synapse. PMID:17884913

  17. Alternative nucleophilic substrates for the endonuclease activities of human immunodeficiency virus type 1 integrase

    SciTech Connect

    Ealy, Julie B.; Sudol, Malgorzata; Krzeminski, Jacek; Amin, Shantu; Katzman, Michael

    2012-11-10

    Retroviral integrase can use water or some small alcohols as the attacking nucleophile to nick DNA. To characterize the range of compounds that human immunodeficiency virus type 1 integrase can accommodate for its endonuclease activities, we tested 45 potential electron donors (having varied size and number or spacing of nucleophilic groups) as substrates during site-specific nicking at viral DNA ends and during nonspecific nicking reactions. We found that integrase used 22 of the 45 compounds to nick DNA, but not all active compounds were used for both activities. In particular, 13 compounds were used for site-specific and nonspecific nicking, 5 only for site-specific nicking, and 4 only for nonspecific nicking; 23 other compounds were not used for either activity. Thus, integrase can accommodate a large number of nucleophilic substrates but has selective requirements for its different activities, underscoring its dynamic properties and providing new information for modeling and understanding integrase.

  18. Design, synthesis and biological activity of aromatic diketone derivatives as HIV-1 integrase inhibitors.

    PubMed

    Hu, Liming; Li, Zhipeng; Wang, Zhanyang; Liu, Gengxin; He, Xianzhuo; Wang, Xiaoli; Zeng, Chengchu

    2015-01-01

    A series of aromatic diketone derivatives were designed and synthesized as potential HIV-1 integrase (IN) inhibitors and evaluated to determine their ability to inhibit the strand transfer process of HIV-1 integrase. The results indicate that (Z)-1-(3-acetyl-2-hydroxy-4,6-dimethoxyphenyl)-3-hydroxy-3-(substituted)phenylprop-2-en-1-one (5a-5d) can moderately inhibit HIV-1 integrase. The cyclization and condensation products (6a-6c and 7e-7f) of compounds 5a-5d show poor inhibitory activity against HIV-1 integrase. The molecular docking results indicate that the different types of compounds act on HIV-1 integrase in different ways, and these results can explain the differences in the inhibitory activities.

  19. Diversity of phage integrases in Enterobacteriaceae: development of markers for environmental analysis of temperate phages.

    PubMed

    Balding, Claire; Bromley, Stephen A; Pickup, Roger W; Saunders, Jon R

    2005-10-01

    Viruses are the most abundant biological entities in aquatic systems. Temperate bacteriophages have enormous influences on microbial diversity, genetic exchange and bacterial population dynamics. However, development of molecular tools for their detection in the environment has been problematic. The integrase gene is used here as a molecular marker to analyse the diversity of temperate bacteriophages in a population of freshwater bacteria. Interrogation of the GenBank database revealed 32 non-cryptic enteric phage integrase sequences, leading to the development of a suite of 11 degenerate primer sets specific to the extant sequences elucidated. Application of these primer sets to enterobacterial isolates recovered from a freshwater pond and the temperate phages induced from them revealed a number of diverse integrase genes, including novel integrase-like sequences not represented in the databases. This highlights the potential of utilizing the integrase gene family as a marker for phage diversity.

  20. As(V) and As(III) reactions on pristine pyrite and on surface-oxidized pyrite.

    PubMed

    Sun, Fenglong; Dempsey, Brian A; Osseo-Asare, Kwadwo A

    2012-12-15

    Reactions of As(III) and As(V) with pyrite were investigated using pristine pyrite (produced and reacted in a rigorously anoxic environment with P(O2)<10(-8)atm) and using surface-oxidized pyrite (produced under anoxic conditions, exposed to air, then stored and reacted under rigorously anoxic conditions). Results with surface-oxidized pyrite were similar to previously reported arsenic-pyrite results. However As(III) adsorbed over a broader pH range on pristine pyrite than on surface-oxidized pyrite, As(V) adsorbed over a narrower pH range on pristine pyrite than on surface-oxidized pyrite, and adsorbed As(V) on pristine pyrite was reduced to As(III) but adsorbed As(V) was not reduced with surface-oxidized pyrite. Reduction of As(V) with pristine pyrite was first-order in total As(V), Fe(II) was released, and sulfur was oxidized. The proposed mechanism for pyrite oxidation by As(V) was similar to the published mechanism for oxidation by O(2) and rates were compared. The results can be used to predict the removals of As(V) and As(III) on pyrite in continuously anoxic environments or on pyrite in intermittently oxic/anoxic environments. Rigorous cleanup and continuous maintenance of strictly anoxic conditions are required if commercial or produced pyrites are to be used as surrogates for pristine pyrite.

  1. Chicoric acid analogues as HIV-1 integrase inhibitors.

    PubMed

    Lin, Z; Neamati, N; Zhao, H; Kiryu, Y; Turpin, J A; Aberham, C; Strebel, K; Kohn, K; Witvrouw, M; Pannecouque, C; Debyser, Z; De Clercq, E; Rice, W G; Pommier, Y; Burke, T R

    1999-04-22

    The present study was undertaken to examine structural features of L-chicoric acid (3) which are important for potency against purified HIV-1 integrase and for reported cytoprotective effects in cell-based systems. Through a progressive series of analogues, it was shown that enantiomeric D-chicoric acid (4) retains inhibitory potency against purified integrase equal to its L-counterpart and further that removal of either one or both carboxylic functionalities results in essentially no loss of inhibitory potency. Additionally, while two caffeoyl moieties are required, attachment of caffeoyl groups to the central linking structure can be achieved via amide or mixed amide/ester linkages. More remarkable is the finding that blockage of the catechol functionality through conversion to tetraacetate esters results in almost no loss of potency, contingent on the presence of at least one carboxyl group on the central linker. Taken as a whole, the work has resulted in the identification of new integrase inhibitors which may be regarded as bis-caffeoyl derivatives of glycidic acid and amino acids such as serine and beta-aminoalanine. The present study also examined the reported ability of chicoric acid to exert cytoprotective effects in HIV-infected cells. It was demonstrated in target and cell-based assays that the chicoric acids do not significantly inhibit other targets associated with HIV-1 replication, including reverse transcription, protease function, NCp7 zinc finger function, or replication of virus from latently infected cells. In CEM cells, for both the parent chicoric acid and selected analogues, antiviral activity was observable under specific assay conditions and with high dependence on the multiplicity of viral infection. However, against HIV-1- and HIV-2-infected MT-4 cells, the chicoric acids and their tetraacetylated esters exhibited antiviral activity (50% effective concentration (EC50) ranging from 1.7 to 20 microM and 50% inhibitory concentration (IC50

  2. Tolerability of integrase inhibitors in a real-life setting.

    PubMed

    Peñafiel, Judit; de Lazzari, Elisa; Padilla, Mireia; Rojas, Jhon; Gonzalez-Cordon, Ana; Blanco, Jose L; Blanch, Jordi; Marcos, Maria A; Lonca, Montserrat; Martinez-Rebollar, Maria; Laguno, Montserrat; Tricas, Amparo; Rodriguez, Ana; Mallolas, Josep; Gatell, Jose M; Martinez, Esteban

    2017-06-01

    Integrase inhibitors have shown better tolerability than other drugs in clinical trials, but some post-marketing data have suggested potential differences among them. We compared rates and reasons for discontinuation of raltegravir-, elvitegravir- and dolutegravir-based regimens in a large cohort of HIV-infected patients. Retrospective analysis of a prospectively followed cohort including all antiretroviral-naive and all virologically suppressed antiretroviral-experienced patients prescribed a first regimen containing raltegravir, elvitegravir or dolutegravir with at least one follow-up visit. Major outcomes were early discontinuation (≤1 year) due to any reason and more specifically due to toxicity. Incidence was calculated as number of episodes per 1000 person-years. Risk factors for discontinuation were assessed by multivariate Cox models. Early discontinuations due to any reason were 271 (raltegravir), 168 (elvitegravir) and 264 (dolutegravir) per 1000 patient-years ( P  =   0.0821). Early discontinuations due to toxicity were 76 (raltegravir), 103 (elvitegravir) and 81 (dolutegravir) per 1000 patient-years ( P  =   0.6792). Overall, the most common toxicities leading to discontinuation were neuropsychiatric, osteomuscular or digestive. Most frequent neuropsychiatric manifestations reported at discontinuation were insomnia, dizziness, headache and anxiety irrespective of the integrase inhibitor. Among discontinuations due to toxicity, neuropsychiatric effects were more common with dolutegravir than with raltegravir or elvitegravir ( P  =   0.0046). Age (HR 1.04, 95% CI 1.02-1.07, P  =   0.0007) was the only independent risk factor for early discontinuation due to toxicity. Discontinuations due to any reason tended to be less common with elvitegravir, but discontinuations due to toxicity did not differ among integrase inhibitors. Neuropsychiatric toxicity leading to drug discontinuation was more frequent with dolutegravir.

  3. Examining structural analogs of elvitegravir as potential inhibitors of HIV-1 integrase.

    PubMed

    Shah, Kavita; Gupta, Saumya; Mishra, Hirdyesh; Sharma, Prashant K; Jayaswal, Amit

    2014-08-01

    Acquired immunodeficiency syndrome (AIDS) is a major health problem in many parts of the world. The human immunodeficiency virus-1 integrase (HIV-1 IN) enzyme has been targeted in HIV patients for therapy. Several integrase inhibitors have been reported, but only elvitegravir (EVG), a new-generation drug, is clinically approved for HIV treatment. In the present work, we investigated two structural analogs of EVG as potential inhibitors of the target molecule, HIV-1 IN. The ligand binding site on HIV-1 IN was identified using Q-SiteFinder, and the HIV-1 IN protein was docked with ligand (EVG and/or analogs) using AutoDock 4. The results suggest that Lys173, Thr125, and His171 are involved in enzyme-substrate binding through hydrogen bonds. Single mutations carried out at Lys173, viz. Lys173Leu (polar > nonpolar) and Lys173Gln (polar > polar), in chain B using PyMOL showed the mutants to have lower binding energy when docked with analog 2, suggesting it to be more stable than analog 1. In conclusion, the mutant HIV-1 IN can bind EVG and its analogs. The physicochemical and pharmacokinetic parameters also show analog 2 to be a promising molecule that can be developed as an alternative to EVG to help overcome the problem of drug resistance by HIV to this inhibitor. Analog 2 may be used as an HIV-1 IN inhibitor with similar potential to that of EVG. Further validation through wet-lab studies, however, is required for future applications.

  4. ON-SITE MERCURY ANALYSIS OF SOIL AT HAZARDOUS WASTE SITES BY IMMUNOASSAY AND ASV

    EPA Science Inventory

    Two field methods for Hg, immunoassay and anodic stripping voltammetry (ASV), that can provide onsite results for quick decisions at hazardous waste sites were evaluated. Each method was applied to samples from two Superfund sites that contain high levels of Hg; Sulphur Bank Me...

  5. ON-SITE MERCURY ANALYSIS OF SOIL AT HAZARDOUS WASTE SITES BY IMMUNOASSAY AND ASV

    EPA Science Inventory

    Two field methods for Hg, immunoassay and anodic stripping voltammetry (ASV), that can provide onsite results for quick decisions at hazardous waste sites were evaluated. Each method was applied to samples from two Superfund sites that contain high levels of Hg; Sulphur Bank Me...

  6. Biosorption of As(V) onto dried alligator weed root: role of metal (hydro) oxides.

    PubMed

    Chen, Jian; Tao, Weihua; Sun, Cheng

    2016-01-01

    The present work investigates the adsorption of As(V) onto the dried powder of alligator weed root as bio-sorbent, using acid pre-treated alligator weed root powder as the reference. The isotherm study suggested there is a favorable As(V) adsorption happened on the AWR surface. The batch adsorption experimental results indicated that the ionic strength has little impact on the adsorption, while the solution pH has a significant effect on the adsorption with apparent inhibition appearing in both extreme acidic and alkaline pH region. In addition, the properties of the biosorbent were characterized by various techniques including SEM-EDS, FT-IR, and ICP detection. The analysis results suggested that the metals including Mn, Fe, and Al enrich over the alligator weed root surface in the morphology of metal (hydro) oxide. Based on the nature of the biosorbent and As(V) besides the adsorption performance, the metal (hydro) oxides over biosorbent surface is suggested as the essential role to drive the adsorption. With the metal (hydro) oxides denuded in the pre-treatment, the biosorbent loses its adsorption capability for As(V) totally.

  7. Effects of Mn(II) on the sorption and mobilization of As(V) in the presence of hematite.

    PubMed

    Ren, Hai-Tao; Jia, Shao-Yi; Liu, Yong; Wu, Song-Hai; Han, Xu

    2012-05-30

    In this study, the effects of Mn(II) on the sorption and mobilization of As(V) by synthetic hematite were investigated. Our results showed that As(V) removal by hematite was evidently dependent on pH, and simultaneous addition of Mn(II) and As(V) into hematite suspension resulted in more removal of As(V) via electrostatic attraction at pH 4.0, 7.0 and 8.3. However, in Mn(II) pre-loaded system, the removal percentages of As(V) at pH 8.3 decreased by 17.0%, 20.7% and 26.7% after 24h at the aging time of 2, 12 and 36 h, respectively. The concentrations of the released As(V) after the addition of 1mM Mn(II) were 23.6, 12.9 and 7.0 μM at pH 8.5 in 2, 3 and 4 g L(-1) hematite suspension, respectively. But Ca(2+) did not show such an effect under similar experimental conditions. Abiotic oxidation of Mn(II) on hematite played an important role in As(V) mobilization. The growing thin layer of Mn(III, IV) (hydr)oxides (MnO(x)) formed on hematite would take up the sorption sites pre-occupied by As(V) and resulted in the release of the adsorbed As(V) back into solution. This study enriched our understanding on As(V) fate in the coexistence of iron oxides and Mn(II). Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.

  8. Adsorption, desorption and fractionation of As(V) on untreated and mussel shell-treated granitic material

    NASA Astrophysics Data System (ADS)

    Seco-Reigosa, N.; Cutillas-Barreiro, L.; Nóvoa-Muñoz, J. C.; Arias-Estévez, M.; Álvarez-Rodríguez, E.; Fernández-Sanjurjo, M. J.; Núñez-Delgado, A.

    2014-12-01

    As(V) adsorption and desorption were studied on granitic material, coarse and fine mussel shell, and granitic material amended with 12 and 24 t ha-1 fine shell, investigating the effect of different As(V) concentrations and different pH, as well as the fractions where the adsorbed As(V) was retained. As(V) adsorption was higher on fine than on coarse shell. Mussel shell amendment increased As(V) adsorption on granitic material. Adsorption data corresponding to the un-amended and shell-amended granitic material were satisfactory fitted to the Langmuir and Freundlich models. Desorption was always < 19% when the highest As(V) concentration (100 mg L-1) was added. Regarding the effect of pH, the granitic material showed its highest adsorption (66%) at pH < 6, and it was lower as pH increased. Fine shell presented notable adsorption in the whole pH range between 6 and 12, with a maximum of 83%. The shell-amended granitic material showed high As(V) adsorption, with a maximum (99%) at pH near 8, but decreasing as pH increased. Desorption varying pH was always < 26%. In the granitic material, desorption increased progressively when pH increased from 4 to 6, contrary to what happened to mussel shell. Regarding the fractionation of the adsorbed As(V), most of it was in the soluble fraction (weakly bound). Globally, the granitic material did not show high As(V) retention capacity, which implies risks of water pollution and transfer to the food chain; however, the mussel shell amendment increased As(V) retention, making this practice recommendable.

  9. Treatment of As(V) and As(III) by electrocoagulation using Al and Fe electrode.

    PubMed

    Kuan, W H; Hu, C Y; Chiang, M C

    2009-01-01

    A batch electrocoagulation (EC) process with bipolar electrode and potentiodynamic polarization tests with monopolar systems were investigated as methods to explore the effects of electrode materials and initial solution pH on the As(V) and As(III) removal. The results displayed that the system with Al electrode has higher reaction rate during the initial period from 0 to 25 minutes than that of Fe electrode for alkaline condition. The pH increased with the EC time because the As(V) and As(III) removal by either co-precipitation or adsorption resulted in that the OH positions in Al-hydroxide or Fe-hydroxide were substituted by As(V) and As(III). The pH in Fe electrode system elevate higher than that in Al electrode because the As(V) removal substitutes more OH position in Fe-hydroxide than that in Al-hydroxide. EC system with Fe electrode can successfully remove the As(III) but system with Al electrode cannot because As(III) can strongly bind to the surface of Fe-hydroxide with forming inner-sphere species but weakly adsorb to the Al-hydroxide surface with forming outer-sphere species. The acidic solution can destroy the deposited hydroxide passive film then allow the metallic ions liberate into the solution, therefore, the acidic initial solution can enhance the As(V) and As(III) removal. The over potential calculation and potentiodynamic polarization tests reveal that the Fe electrode systems possess higher over potential and pitting potential than that of Al electrode system due to the fast hydrolysis of and the occurrence of Fe-hydroxide passive film.

  10. As(V) adsorption onto nanoporous titania adsorbents (NTAs): effects of solution composition.

    PubMed

    Han, Dong Suk; Batchelor, Bill; Park, Sung Hyuk; Abdel-Wahab, Ahmed

    2012-08-30

    This study has focused on developing two nanoporous titania adsorbents (NTA) to enhance removal efficiency of adsorption process for As(V) by characterizing the effects of pH and phosphate concentration on their sorption capacities and behaviors. One type of adsorbent is a mesoporous titania (MT) solid phase and the other is group of a highly ordered mesoporous silica solids (SBA-15) that can incorporate different levels of reactive titania sorption sites. Microscopic analysis showed that Ti((25))-SBA-15 (Ti/SBA=0.25 g/g) had titania nanostructured mesopores that do not rupture the highly ordered hexagonal silica framework. However, MT has disordered, wormhole-like mesopores that are caused by interparticle porosity. Adsorption experiments showed that Ti((25))-SBA-15 had a greater sorption capacity for As(V) than did Ti((15))-SBA-15 or Ti((35))-SBA-15 and the amount of As(V) adsorbed generally decreased as pH increased. Higher removal of As(V) was observed with Ti((25))-SBA-15 than with MT at pH 4, but MT had higher removals at higher pH (7, 9.5), even though MT has a lower specific surface area. However, in the presence of phosphate, MT showed higher removal of As(V) at low pH rather than did Ti((25))-SBA-15. As expected, the NTAs showed very fast sorption kinetics, but they followed a bi-phasic sorption pattern. Copyright © 2012 Elsevier B.V. All rights reserved.

  11. Influence of Drug Resistance Mutations on the Activity of HIV-1 Subtypes A and B Integrases: a Comparative Study

    PubMed Central

    Shadrina, O. A.; Zatsepin, T. S.; Agapkina, Yu. Yu.; Isaguliants, M. G.; Gottikh, M. B.

    2015-01-01

    Integration of human immunodeficiency virus (HIV-1) DNA into the genome of an infected cell is one of the key steps in the viral replication cycle. The viral enzyme integrase (IN), which catalyzes the integration, is an attractive target for the development of new antiviral drugs. However, the HIV-1 therapy often results in the IN gene mutations inducing viral resistance to integration inhibitors. To assess the impact of drug resistance mutations on the activity of IN of HIV-1 subtype A strain FSU-A, which is dominant in Russia, variants of the consensus IN of this subtype containing the primary resistance mutations G118R and Q148K and secondary compensatory substitutions E138K and G140S were prepared and characterized. Comparative study of these enzymes with the corresponding mutants of IN of HIV-1 subtype B strains HXB-2 was performed. The mutation Q148K almost equally reduced the activity of integrases of both subtypes. Its negative effect was partially compensated by the secondary mutations E138K and G140S. Primary substitution G118R had different influence on the activity of proteins of the subtypes A and B, and the compensatory effect of the secondary substitution E138K also depended on the viral subtype. Comparison of the mutants resistance to the known strand transfer inhibitors raltegravir and elvitegravir, and a new inhibitor XZ-259 (a dihydro-1H-isoindol derivative), showed that integrases of both subtypes with the Q148K mutation were insensitive to raltegravir and elvitegravir but were effectively inhibited by XZ-259. The substitution G118R slightly reduced the efficiency of IN inhibition by raltegravir and elvitegravir and caused no resistance to XZ_259. PMID:25927004

  12. Structure-Guided Optimization of HIV Integrase Strand Transfer Inhibitors.

    PubMed

    Zhao, Xue Zhi; Smith, Steven J; Maskell, Daniel P; Métifiot, Mathieu; Pye, Valerie E; Fesen, Katherine; Marchand, Christophe; Pommier, Yves; Cherepanov, Peter; Hughes, Stephen H; Burke, Terrence R

    2017-09-14

    Integrase mutations can reduce the effectiveness of the first-generation FDA-approved integrase strand transfer inhibitors (INSTIs), raltegravir (RAL) and elvitegravir (EVG). The second-generation agent, dolutegravir (DTG), has enjoyed considerable clinical success; however, resistance-causing mutations that diminish the efficacy of DTG have appeared. Our current findings support and extend the substrate envelope concept that broadly effective INSTIs can be designed by filling the envelope defined by the DNA substrates. Previously, we explored 1-hydroxy-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamides as an INSTI scaffold, making a limited set of derivatives, and concluded that broadly effective INSTIs can be developed using this scaffold. Herein, we report an extended investigation of 6-substituents as well the first examples of 7-substituted analogues of this scaffold. While 7-substituents are not well-tolerated, we have identified novel substituents at the 6-position that are highly effective, with the best compound (6p) retaining better efficacy against a broad panel of known INSTI resistant mutants than any analogues we have previously described.

  13. Use of a Novel Integrase-Deficient Lentivirus for Targeted Anti-Cancer Therapy With Survivin Promoter-Driven Diphtheria Toxin A

    PubMed Central

    Lin, Baoshun; Gao, Anding; Zhang, Rui; Ma, Hongyu; Shen, Haifeng; Hu, Qiong; Zhang, Hua; Zhao, Meng; Lan, Xiaopeng; Liu, Kuancan

    2015-01-01

    Abstract As an immunotoxin, diphtheria toxin has been widely used in gene therapy and gene function assays for its roles in protein synthesis inhibition, and the aim of our study is to set up a nonintegrating lentiviral system for specific expression of diphtheria toxin A (DTA) used in cancer gene therapy. Here, we established a lentiviral system that could coordinately express fluorescent protein and DTA driven by the cytomegalovirus (CMV) promoter, which is convenient for us to precisely trace the expression of DTA and monitor the process of lentivirus packaging. To achieve safer cancer therapy, we replaced the CMV promoter with the Survivin promoter, a specific promoter that is dramatically activated in cancer tissues and cells, but not in normal tissues and cells, and that will impose greater therapeutic potential because a significant expression difference occurred between these 2 groups. Meanwhile, we obtained integrase-deficient lentivirus (IDLV) after packaging with the integrase mutant, which expresses defective integrase RRK262263264AAH, to minimize the side effects that derived from the insertional mutagenesis of the host genome. Our results suggest that the IDLV system that we generated possesses therapeutic potential in cancers in vitro and in vivo. PMID:26252309

  14. Absence of primary integrase resistance mutations in HIV type 1-infected patients in Venezuela.

    PubMed

    Rangel, Héctor R; Garzaro, Domingo; Fabbro, Rona; Martinez, Nahir; Ossenkop, John; Torres, Jaime R; Gutiérrez, Cristina R; Pujol, Flor H

    2010-08-01

    The preexistence of mutations to integrase inhibitors in HIV-1-infected Venezuelan patients was evaluated. The integrase region of the HIV-1 genome was amplified by nested-PCR and sequenced in 57 isolates from both naive (n = 24) and treated patients who received protease and/or reverse transcriptase inhibitors (PI and RTI, n = 33), but were never exposed to integrase inhibitors. Only one primary integrase resistance mutation, not conferring drug resistance by itself, was found among these patients, although several minor viral mutations, equally distributed among naive and PI- and RTI-treated patients, were also found. In the limited number of samples, no relation was found among the presence of resistance mutations to PI or RTI and the presence of minor mutations to integrase. The absence of resistance to integrase inhibitors may be related to the recent introduction of these drugs in our country. The availability of in-house assays allows for a more comprehensive surveillance of drug resistance to integrase inhibitors in Venezuela.

  15. Bovine Lactoferrampin, Human Lactoferricin, and Lactoferrin 1-11 Inhibit Nuclear Translocation of HIV Integrase.

    PubMed

    Wang, Winston Yan; Wong, Jack Ho; Ip, Denis Tsz Ming; Wan, David Chi Cheong; Cheung, Randy Chifai; Ng, Tzi Bun

    2016-08-01

    This study aimed to investigate fragments derived from human and bovine lactoferrins for ability to inhibit nuclear translocation of HIV-1 integrase. It was shown that human lactoferricin, human lactoferrin 1-11, and bovine lactoferrampin reduced nuclear distribution of HIV-1 integrase. Bovine lactoferrampin could inhibit both the activity and nuclear translocation of HIV-1 integrase. Human lactoferrampin, bovine lactoferricin, and bovine lactoferrin 1-11 had no effect on HIV-1 integrase nuclear translocation. Human lactoferrampin which inhibited the activity of integrase did not prevent its nuclear translocation. Human lactoferricin and lactoferrin 1-11 did not inhibit HIV-1 integrase nuclear translocation despite their ability to attenuate the enzyme activity. The discrepancy between the findings on reduction of HIV-1 activity and inhibition of nuclear translocation of HIV-1 integrase was due to the different mechanisms involved. A similar reasoning can also be applied to the different inhibitory potencies of the milk peptides on different HIV enzymes, i.e., nuclear translocation.

  16. Effect of HIV-1 Subtype C integrase mutations implied using molecular modeling and docking data.

    PubMed

    Sachithanandham, Jaiprasath; Konda Reddy, Karnati; Solomon, King; David, Shoba; Kumar Singh, Sanjeev; Vadhini Ramalingam, Veena; Alexander Pulimood, Susanne; Cherian Abraham, Ooriyapadickal; Rupali, Pricilla; Sridharan, Gopalan; Kannangai, Rajesh

    2016-01-01

    The degree of sequence variation in HIV-1 integrase genes among infected patients and their impact on clinical response to Anti retroviral therapy (ART) is of interest. Therefore, we collected plasma samples from 161 HIV-1 infected individuals for subsequent integrase gene amplification (1087 bp). Thus, 102 complete integrase gene sequences identified as HIV-1 subtype-C was assembled. This sequence data was further used for sequence analysis and multiple sequence alignment (MSA) to assess position specific frequency of mutations within pol gene among infected individuals. We also used biophysical geometric optimization technique based molecular modeling and docking (Schrodinger suite) methods to infer differential function caused by position specific sequence mutations towards improved inhibitor selection. We thus identified accessory mutations (usually reduce susceptibility) leading to the resistance of some known integrase inhibitors in 14% of sequences in this data set. The Stanford HIV-1 drug resistance database provided complementary information on integrase resistance mutations to deduce molecular basis for such observation. Modeling and docking analysis show reduced binding by mutants for known compounds. The predicted binding values further reduced for models with combination of mutations among subtype C clinical strains. Thus, the molecular basis implied for the consequence of mutations in different variants of integrase genes of HIV-1 subtype C clinical strains from South India is reported. This data finds utility in the design, modification and development of a representative yet an improved inhibitor for HIV-1 integrase.

  17. Architecture of a Full-length Retroviral Integrase Monomer and Dimer, Revealed by Small Angle X-ray Scattering and Chemical Cross-linking

    SciTech Connect

    Bojja, Ravi S.; Andrake, Mark D.; Weigand, Steven; Merkel, George; Yarychkivska, Olya; Henderson, Adam; Kummerling, Marissa; Skalka, Anna Marie

    2012-02-07

    We determined the size and shape of full-length avian sarcoma virus (ASV) integrase (IN) monomers and dimers in solution using small angle x-ray scattering. The low resolution data obtained establish constraints for the relative arrangements of the three component domains in both forms. Domain organization within the small angle x-ray envelopes was determined by combining available atomic resolution data for individual domains with results from cross-linking coupled with mass spectrometry. The full-length dimer architecture so revealed is unequivocally different from that proposed from x-ray crystallographic analyses of two-domain fragments, in which interactions between the catalytic core domains play a prominent role. Core-core interactions are detected only in cross-linked IN tetramers and are required for concerted integration. The solution dimer is stabilized by C-terminal domain (CTD-CTD) interactions and by interactions of the N-terminal domain in one subunit with the core and CTD in the second subunit. These results suggest a pathway for formation of functional IN-DNA complexes that has not previously been considered and possible strategies for preventing such assembly.

  18. Architecture of a Full-length Retroviral Integrase Monomer and Dimer, Revealed by Small Angle X-ray Scattering and Chemical Cross-linking*

    PubMed Central

    Bojja, Ravi S.; Andrake, Mark D.; Weigand, Steven; Merkel, George; Yarychkivska, Olya; Henderson, Adam; Kummerling, Marissa; Skalka, Anna Marie

    2011-01-01

    We determined the size and shape of full-length avian sarcoma virus (ASV) integrase (IN) monomers and dimers in solution using small angle x-ray scattering. The low resolution data obtained establish constraints for the relative arrangements of the three component domains in both forms. Domain organization within the small angle x-ray envelopes was determined by combining available atomic resolution data for individual domains with results from cross-linking coupled with mass spectrometry. The full-length dimer architecture so revealed is unequivocally different from that proposed from x-ray crystallographic analyses of two-domain fragments, in which interactions between the catalytic core domains play a prominent role. Core-core interactions are detected only in cross-linked IN tetramers and are required for concerted integration. The solution dimer is stabilized by C-terminal domain (CTD-CTD) interactions and by interactions of the N-terminal domain in one subunit with the core and CTD in the second subunit. These results suggest a pathway for formation of functional IN-DNA complexes that has not previously been considered and possible strategies for preventing such assembly. PMID:21454648

  19. Removal of arsenic from water using manganese (III) oxide: Adsorption of As(III) and As(V).

    PubMed

    Babaeivelni, Kamel; Khodadoust, Amid P

    2016-01-01

    Removal of arsenic from water was evaluated with manganese (III) oxide (Mn2O3) as adsorbent. Adsorption of As(III) and As(V) onto Mn2O3 was favorable according to the Langmuir and Freundlich adsorption equilibrium equations, while chemisorption of arsenic occurred according to the Dubinin-Radushkevich equation. Adsorption parameters from the Langmuir, Freundlich, and Temkin equations showed a greater adsorption and removal of As(III) than As(V) by Mn2O3. Maximum removal of As(III) and As(V) occurred at pH 3-9 and at pH 2, respectively, while removal of As(V) in the pH range of 6-9 was 93% (pH 6) to 61% (pH 9) of the maximum removal. Zeta potential measurements for Mn2O3 in As(III) was likely converted to As(V) solutions indicated that As(III) was likely converted to As(V) on the Mn2O3 surface at pH 3-9. Overall, the effective Mn2O3 sorbent rapidly removed As(III) and As(V) from water in the pH range of 6-9 for natural waters.

  20. HIV Integrase Inhibitors with Nucleobase Scaffolds: Discovery of a Highly Potent anti-HIV Agent

    PubMed Central

    Nair, Vasu; Chi, Guochen; Ptak, Roger; Neamati, Nouri

    2008-01-01

    HIV integrase is essential for HIV replication. However, there are currently no integrase inhibitors in clinical use for AIDS. We have discovered a conceptually new β-diketo acid that is a powerful inhibitor of both the 3′-processing and strand transfer steps of HIV-1 integrase. The in vitro anti-HIV data of this inhibitor were remarkable as exemplified by its highly potent antiviral therapeutic efficacy against HIVTEKI and HIV-1NL4-3 replication in PBMC (TI >4,000 and >10,000, respectively). PMID:16420027

  1. Structural Basis for the Recognition Between HIV-1 Integrase and Transcriptional Coactivator p75

    SciTech Connect

    Cherepanov,P.; Ambrosio, A.; Rahman, S.; Ellenberger, T.; Engelman, A.

    2005-01-01

    Integrase (IN) is an essential retroviral enzyme, and human transcriptional coactivator p75, which is also referred to as lens epithelium-derived growth factor (LEDGF), is the dominant cellular binding partner of HIV-1 IN. Here, we report the crystal structure of the dimeric catalytic core domain of HIV-1 IN complexed to the IN-binding domain of LEDGF. Previously identified LEDGF hotspot residues anchor the protein to both monomers at the IN dimer interface. The principal structural features of IN that are recognized by the host factor are the backbone conformation of residues 168-171 from one monomer and a hydrophobic patch that is primarily comprised of {alpha}-helices 1 and 3 of the second IN monomer. Inspection of diverse retroviral primary and secondary sequence elements helps to explain the apparent lentiviral tropism of the LEDGF-IN interaction. Because the lethal phenotypes of HIV-1 mutant viruses unable to interact with LEDGF indicate that IN function is highly sensitive to perturbations of the structure around the LEDGF-binding site, we propose that small molecule inhibitors of the protein-protein interaction might similarly disrupt HIV-1 replication.

  2. D77, one benzoic acid derivative, functions as a novel anti-HIV-1 inhibitor targeting the interaction between integrase and cellular LEDGF/p75

    SciTech Connect

    Du Li; Zhao Yaxue; Chen, Jing; Yang Liumeng; Zheng Yongtang; Tang Yun Shen Xu Jiang Hualiang

    2008-10-10

    Integration of viral-DNA into host chromosome mediated by the viral protein HIV-1 integrase (IN) is an essential step in the HIV-1 life cycle. In this process, Lens epithelium-derived growth factor (LEDGF/p75) is discovered to function as a cellular co-factor for integration. Since LEDGF/p75 plays an important role in HIV integration, disruption of the LEDGF/p75 interaction with IN has provided a special interest for anti-HIV agent discovery. In this work, we reported that a benzoic acid derivative, 4-[(5-bromo-4-{l_brace}[2,4-dioxo-3-(2-oxo-2-phenylethyl) -1,3-thiazolidin-5-ylidene]methyl{r_brace}-2-ethoxyphenoxy)methyl]benzoic acid (D77) could potently inhibit the IN-LEDGF/p75 interaction and affect the HIV-1 IN nuclear distribution thus exhibiting antiretroviral activity. Molecular docking with site-directed mutagenesis analysis and surface plasmon resonance (SPR) binding assays has clarified possible binding mode of D77 against HIV-1 integrase. As the firstly discovered small molecular compound targeting HIV-1 integrase interaction with LEDGF/p75, D77 might supply useful structural information for further anti-HIV agent discovery.

  3. Incorporation of a fluorescent guanosine analog into oligonucleotides and its application to a real time assay for the HIV-1 integrase 3'-processing reaction.

    PubMed Central

    Hawkins, M E; Pfleiderer, W; Mazumder, A; Pommier, Y G; Balis, F M

    1995-01-01

    We have synthesized a highly fluorescent (quantum yield 0.88) guanosine analog, (3-methyl-8-(2-deoxy-beta-D-ribofuranosyl) isoxanthopterin (3-Mi) in a dimethoxytrityl, phosphoramidite protected form, which can be site-specifically inserted into oligonucleotides through a 3',5'-phosphodiester linkage using an automated DNA synthesizer. Fluorescence is partially quenched within an oligonucleotide and the degree of quench is a function of the fluorophore's proximity to purines and its position in the oligonucleotide. As an example of the potential utility of this class of fluorophores, we developed a continuous assay for HIV-1 integrase 3'-processing reaction by incorporating 3-MI at the cleavage site in a double-stranded oligonucleotide identical to the U5 terminal sequence of the HIV genome. Integrase cleaves the 3'-terminal dinucleotide containing the fluorophore, resulting in an increase in fluorescence which can be monitored on a spectrofluorometer. Substitution of the fluorophore for guanosine at the cleavage site does not inhibit integrase activity. This assay is specific for the 3'-processing reaction. The change in fluorescence intensity is linear over time and proportional to the rate of the reaction. This assay demonstrates the potential utility of this new class of fluorophore for continuous monitoring of protein/DNA interactions. PMID:7659509

  4. CCD Observations of ERS with the 60 cm Telescope at ASV

    NASA Astrophysics Data System (ADS)

    Damljanovic, G.; Milic, I. S.

    2013-05-01

    We present the observations of extragalactic radio sources (ERS) which are possible in the optical domain and can be used to establish the link between the ICRF2 and the future Gaia Celestial Reference Frame (GCRF). Our telescope of small aperture size (< 1 m) is located in the south of Serbia, near the town of Prokuplje, at the Astronomical Station Vidojevica (ASV) which belongs to the Astronomical Observatory of Belgrade (AOB). It is a Cassegrain-type optical system (D=60 cm, F=600 cm) of equatorial mount. About 40 ERS, from ICRF2 list, were observed at ASV during 2011 and 2012. These observations are of importance to compare the ERS optical and radio positions (VLBI ones), and to investigate the relation between optical and radio reference frames. Also, they are useful to check the possibilities of the instrument. We observed ERS with the CCD Apogeee Alta U42. The observations, reduction and preliminary results of some ERS are presented here.

  5. Plasmid integration in a wide range of bacteria mediated by the integrase of Lactobacillus delbrueckii bacteriophage mv4.

    PubMed Central

    Auvray, F; Coddeville, M; Ritzenthaler, P; Dupont, L

    1997-01-01

    Bacteriophage mv4 is a temperate phage infecting Lactobacillus delbrueckii subsp. bulgaricus. During lysogenization, the phage integrates its genome into the host chromosome at the 3' end of a tRNA(Ser) gene through a site-specific recombination process (L. Dupont et al., J. Bacteriol., 177:586-595, 1995). A nonreplicative vector (pMC1) based on the mv4 integrative elements (attP site and integrase-coding int gene) is able to integrate into the chromosome of a wide range of bacterial hosts, including Lactobacillus plantarum, Lactobacillus casei (two strains), Lactococcus lactis subsp. cremoris, Enterococcus faecalis, and Streptococcus pneumoniae. Integrative recombination of pMC1 into the chromosomes of all of these species is dependent on the int gene product and occurs specifically at the pMC1 attP site. The isolation and sequencing of pMC1 integration sites from these bacteria showed that in lactobacilli, pMC1 integrated into the conserved tRNA(Ser) gene. In the other bacterial species where this tRNA gene is less or not conserved; secondary integration sites either in potential protein-coding regions or in intergenic DNA were used. A consensus sequence was deduced from the analysis of the different integration sites. The comparison of these sequences demonstrated the flexibility of the integrase for the bacterial integration site and suggested the importance of the trinucleotide CCT at the 5' end of the core in the strand exchange reaction. PMID:9068626

  6. Interrogating HIV integrase for compounds that bind- a SAMPL challenge

    PubMed Central

    Peat, Thomas S; Dolezal, Olan; Newman, Janet; Mobley, David; Deadman, John J

    2015-01-01

    Tremendous gains and novel methods are often developed when people are challenged to do something new or difficult. This process is enhanced when people compete against each other-this can be seen in sport as well as in science and technology (e.g. the space race). The SAMPL challenges, like the CASP challenges, aim to challenge modellers and software developers to develop new ways of looking at molecular interactions so the community as a whole can progress in the accurate prediction of these interactions. In order for this challenge to occur, data must be supplied so the prospective test can be done. We have supplied unpublished data related to a drug discovery program run several years ago on HIV integrase for the SAMPL4 challenge. This paper describes the methods used to obtain these data and the chemistry involved. PMID:24532034

  7. Interrogating HIV integrase for compounds that bind- a SAMPL challenge

    NASA Astrophysics Data System (ADS)

    Peat, Thomas S.; Dolezal, Olan; Newman, Janet; Mobley, David; Deadman, John J.

    2014-04-01

    Tremendous gains and novel methods are often developed when people are challenged to do something new or difficult. This process is enhanced when people compete against each other-this can be seen in sport as well as in science and technology (e.g. the space race). The SAMPL challenges, like the CASP challenges, aim to challenge modellers and software developers to develop new ways of looking at molecular interactions so the community as a whole can progress in the accurate prediction of these interactions. In order for this challenge to occur, data must be supplied so the prospective test can be done. We have supplied unpublished data related to a drug discovery program run several years ago on HIV integrase for the SAMPL4 challenge. This paper describes the methods used to obtain these data and the chemistry involved.

  8. Design of second generation HIV-1 integrase inhibitors.

    PubMed

    Deng, Jinxia; Dayam, Raveendra; Al-Mawsawi, Laith Q; Neamati, Nouri

    2007-01-01

    The prospect of HIV-1 integrase (IN) as a therapeutically viable retroviral drug target is on the verge of realization. The observed preclinical and clinical performance of beta-diketo containing and naphthyridine carboxamide compounds provides direct proof for the clinical application of IN inhibition. These validated lead compounds are useful in the design and development of second generation IN inhibitors. The results from preclinical and clinical studies on the first generation IN inhibitors reiterate a demand for novel second generation inhibitors with improved pharmacokinetic and metabolic properties. Pharmacophore-based drug design techniques facilitate the discovery of novel compounds on the basis of validated lead compounds specific for a drug target. In this article we have comprehensively reviewed the application of pharmacophore-based drug design methods in the field of IN inhibitor discovery.

  9. Computer tools in the discovery of HIV-I integrase inhibitors

    PubMed Central

    Liao, Chenzhong; Nicklaus, Marc C

    2010-01-01

    Computer-aided drug design (CADD) methodologies have made great advances and contributed significantly to the discovery and/or optimization of many clinically used drugs in recent years. CADD tools have likewise been applied to the discovery of inhibitors of HIV-I integrase, a difficult and worthwhile target for the development of efficient anti-HIV drugs. This article reviews the application of CADD tools, including pharmacophore search, quantitative structure–activity relationships, model building of integrase complexed with viral DNA and quantum-chemical studies in the discovery of HIV-I integrase inhibitors. Different structurally diverse integrase inhibitors have been identified by, or with significant help from, various CADD tools. PMID:21426160

  10. Unmasking the ancestral activity of integron integrases reveals a smooth evolutionary transition during functional innovation

    PubMed Central

    Escudero, Jose Antonio; Loot, Celine; Parissi, Vincent; Nivina, Aleksandra; Bouchier, Christiane; Mazel, Didier

    2016-01-01

    Tyrosine (Y)-recombinases have evolved to deliver mechanistically different reactions on a variety of substrates, but these evolutionary transitions are poorly understood. Among them, integron integrases are hybrid systems recombining single- and double-stranded DNA partners. These reactions are asymmetric and need a replicative resolution pathway, an exception to the canonical second strand exchange model of Y-recombinases. Integron integrases possess a specific domain for this specialized pathway. Here we show that despite this, integrases are still capable of efficiently operating the ancestral second strand exchange in symmetrical reactions between double-stranded substrates. During these reactions, both strands are reactive and Holliday junction resolution can follow either pathway. A novel deep-sequencing approach allows mapping of the crossover point for the second strand exchange. The persistence of the ancestral activity in integrases illustrates their robustness and shows that innovation towards new recombination substrates and resolution pathways was a smooth evolutionary process. PMID:26961432

  11. Oxidation of As(III) to As(V) using ozone microbubbles.

    PubMed

    Khuntia, Snigdha; Majumder, Subrata Kumar; Ghosh, Pallab

    2014-02-01

    The use of ozone in the treatment of water and wastewater is rapidly increasing due to its high oxidizing power. Arsenic is one the most toxic elements found in water. As(III) and As(V) are the major sources of arsenic poisoning. It is known that As(V) can be more easily removed from water by adsorptive methods than As(III). In this work, oxidation of more toxic As(III) to less toxic As(V) was studied in a pilot-plant by using ozone microbubbles. The microbubbles were effective in dissolving ozone in water. The oxidation was fast over a wide range of pH (e.g., 4-9). The role of hydroxyl radical in the oxidation of As(III) under acidic conditions was investigated by using 2-propanol as the hydroxyl radical scavenger. Under acidic conditions, the addition of 2-propanol slowed down the oxidation, which proves that hydroxyl radicals were involved in the oxidation process. The effect of carbonate ions on the rate of oxidation was investigated. It was found that the generation of carbonate ion radical from the carbonate ion accelerated the oxidation of As(III). The kinetics of oxidation of As(III) by ozone was studied. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. Electromembrane extraction and spectrophotometric determination of As(V) in water samples.

    PubMed

    Kamyabi, Mohammad Ali; Aghaei, Ali

    2016-12-01

    In this study, for the first time electromembrane extraction (EME) was used as a highly efficient sample pre-treatment method for the UV-VIS spectrophotometric determination of As(V) in water samples. The influences of experimental parameters during EME were investigated and optimized using one-variable-at-a-time methodology as follows: organic solvent: 1-octanol+2.5% (V/V) di-(2-ethylhexyl) phosphate, applied voltage: 70V, extraction time: 15min, pH of acceptor: 13, stirring rate: 750rpm. The method allowed the determination of As(V) in the range of 5-300ngmL(-1). The relative standard deviation was found to be within the range of 3.4-7.6%. The limit of detection, corresponding to a signal to noise ratio of three, was 1.5ngmL(-1). The proposed method was finally applied to the determination of As(V) in water samples and relative recoveries ranging from 95 to 102% were obtained.

  13. Modeling and fixed bed column adsorption of As(V) on laterite soil.

    PubMed

    Maji, Sanjoy K; Pal, Anjali; Pal, Tarasankar; Adak, Asok

    2007-09-01

    Laterite soil, an abundant locally available natural adsorbent, has been evaluated for As(V) removal from aqueous solutions in column mode operation. The column studies were conducted using columns of 10, 20, 30 cm bed depth with 2 cm internal diameter. Initial As(V) concentration was 0.5 mg/L and flow rate was 7.75 mL/min. Bohart and Adams sorption model was employed for the determination of different parameters like height of exchange zone, adsorption rate, time required for exchange zone to move, and the adsorption capacity. Effect of flow rate and initial concentration was studied. The adsorption capacity of the laterite soil for 0.5 mg/L of As(V) was found to be 62.32 mg/L, and the adsorption rate constant was 1.0911 L/mg h for the minimum bed depth of 8.47 cm. The column was designed by the BDST model. Freundlich isotherm model was used to compare the theoretical and experimental breakthrough profile in the dynamic process. The bed saturation obtained was 36-80%. Regeneration of the exhausted column was possible with 1M NaOH.

  14. HIV-1 group O integrase displays lower susceptibility to raltegravir and has a different mutational pathway for resistance than HIV-1 group M.

    PubMed

    Depatureaux, Agnès; Mesplède, Thibault; Quashie, Peter; Oliveira, Maureen; Moisi, Daniela; Brenner, Bluma; Wainberg, Mark

    2014-01-01

    HIV-1 group O (HIV-O) is a rare HIV-1 variant characterized by a high number of polymorphisms, especially in the integrase gene, e.g. positions L74I, S153A, G163Q and T206S. As HIV-O integrase enzymes have not previously been studied, our aim was to assess the impact of HIV-O integrase polymorphisms on susceptibility to integrase inhibitors and emergence of resistance associated mutations. Viruses and Methods: We cloned and purified integrase proteins from each of HIV-1 Group O clades A (HIV-O/A) and B (HIV-O/B), a HIV-O divergent strain (HIV-O/Div), and HIV-1 group M (subtype B, HIV-M/B) and characterized these enzymes for susceptibility to integrase strand transfer inhibitors (INSTIs) in cell-free assays and in tissue culture, in the absence or presence of varying concentrations of several INSTIs. The inhibition constant (Ki) and IC50 were calculated and compared for HIV-M and HIV-O integrases. Selections for resistance-related mutations were performed using cord blood mononuclear cells and increasing concentration of INSTIs. HIV-O integrase and viruses were more susceptible to raltegravir (RAL) in competitive inhibition assays and in tissue culture than were HIV-M enzymes and viruses, respectively. During selection, we observed different pathways of resistance depending on the drug and clade. Mutations selected in HIV-O can be classified as follows: (1) mutations described for HIV-M such as T97A, Q148R, V151A/I (RAL), T66I, E92Q, E157Q (EVG) and M50I, R263K (DTG) and (2) signature mutations for HIV-O (i.e. not described in HIV-M) F121C (HIV-O/B for RAL), V75I (HIV-O/A for RAL) and S153V (HIV-O/A for DTG). Only the HIV-O/Div selected the Q148R mutation for RAL and R263K+M50I for DTG, as previously described for HIV-M. None of the HIV-O viruses selected either N155H or Y143C. The selection of the specific S153V mutation could be explained at the nucleotide level: HIV-O at this position contains an alanine and substitution of alanine to valine (153AGGC→153VGTC

  15. Design of a series of bicyclic HIV-1 integrase inhibitors. Part 2: azoles: effective metal chelators.

    PubMed

    Le, Giang; Vandegraaff, Nick; Rhodes, David I; Jones, Eric D; Coates, Jonathan A V; Thienthong, Neeranat; Winfield, Lisa J; Lu, Long; Li, Xinming; Yu, Changjiang; Feng, Xiao; Deadman, John J

    2010-10-01

    Synthesis of a diverse set of azoles and their utilizations as an amide isostere in the design of HIV integrase inhibitors is described. The Letter identified thiazole, oxazole, and imidazole as the most promising heterocycles. Initial SAR studies indicated that these novel series of integrase inhibitors are amenable to lead optimization. Several compounds with low nanomolar inhibitory potency are reported. Copyright © 2010 Elsevier Ltd. All rights reserved.

  16. Structure-Guided Optimization of HIV Integrase Strand Transfer Inhibitors | Center for Cancer Research

    Cancer.gov

    Integrase mutations can reduce the effectiveness of the first-generation FDA-approved integrase strand transfer inhibitors (INSTIs), raltegravir (RAL) and elvitegravir (EVG). The second-generation agent, dolutegravir (DTG), has enjoyed considerable clinical success; however, resistance-causing mutations that diminish the efficacy of DTG have appeared. Our current findings support and extend the substrate envelope concept that broadly effective INSTIs can be designed by filling the envelope defined by the DNA substrates.

  17. Development of a receptor model for efficient in silico screening of HIV-1 integrase inhibitors.

    PubMed

    Quevedo, Mario A; Ribone, Sergio R; Briñón, Margarita C; Dehaen, Wim

    2014-07-01

    Integrase (IN) is a key viral enzyme for the replication of the type-1 human immunodeficiency virus (HIV-1), and as such constitutes a relevant therapeutic target for the development of anti-HIV agents. However, the lack of crystallographic data of HIV IN complexed with the corresponding viral DNA has historically hindered the application of modern structure-based drug design techniques to the discovery of new potent IN inhibitors (INIs). Consequently, the development and validation of reliable HIV IN structural models that may be useful for the screening of large databases of chemical compounds is of particular interest. In this study, four HIV-1 IN homology models were evaluated respect to their capability to predict the inhibition potency of a training set comprising 36 previously reported INIs with IC50 values in the low nanomolar to the high micromolar range. Also, 9 inactive structurally related compounds were included in this training set. In addition, a crystallographic structure of the IN-DNA complex corresponding to the prototype foamy virus (PFV) was also evaluated as structural model for the screening of inhibitors. The applicability of high throughput screening techniques, such as blind and ligand-guided exhaustive rigid docking was assessed. The receptor models were also refined by molecular dynamics and clustering techniques to assess protein sidechain flexibility and solvent effect on inhibitor binding. Among the studied models, we conclude that the one derived from the X-ray structure of the PFV integrase exhibited the best performance to rank the potencies of the compounds in the training set, with the predictive power being further improved by explicitly modeling five water molecules within the catalytic side of IN. Also, accounting for protein sidechain flexibility enhanced the prediction of inhibition potencies among the studied compounds. Finally, an interaction fingerprint pattern was established for the fast identification of potent IN

  18. Virtual screening of integrase inhibitors by large scale binding free energy calculations: the SAMPL4 challenge

    PubMed Central

    Gallicchio, Emilio; Deng, Nanjie; He, Peng; Wickstrom, Lauren; Perryman, Alexander L.; Santiago, Daniel N.; Forli, Stefano; Olson, Arthur J.; Levy, Ronald M.

    2014-01-01

    As part of the SAMPL4 blind challenge, filtered AutoDock Vina ligand docking predictions and large scale binding energy distribution analysis method binding free energy calculations have been applied to the virtual screening of a focused library of candidate binders to the LEDGF site of the HIV integrase protein. The computational protocol leveraged docking and high level atomistic models to improve enrichment. The enrichment factor of our blind predictions ranked best among all of the computational submissions, and second best overall. This work represents to our knowledge the first example of the application of an all-atom physics-based binding free energy model to large scale virtual screening. A total of 285 parallel Hamiltonian replica exchange molecular dynamics absolute protein-ligand binding free energy simulations were conducted starting from docked poses. The setup of the simulations was fully automated, calculations were distributed on multiple computing resources and were completed in a 6-weeks period. The accuracy of the docked poses and the inclusion of intramolecular strain and entropic losses in the binding free energy estimates were the major factors behind the success of the method. Lack of sufficient time and computing resources to investigate additional protonation states of the ligands was a major cause of mispredictions. The experiment demonstrated the applicability of binding free energy modeling to improve hit rates in challenging virtual screening of focused ligand libraries during lead optimization. PMID:24504704

  19. Virtual screening of integrase inhibitors by large scale binding free energy calculations: the SAMPL4 challenge

    NASA Astrophysics Data System (ADS)

    Gallicchio, Emilio; Deng, Nanjie; He, Peng; Wickstrom, Lauren; Perryman, Alexander L.; Santiago, Daniel N.; Forli, Stefano; Olson, Arthur J.; Levy, Ronald M.

    2014-04-01

    As part of the SAMPL4 blind challenge, filtered AutoDock Vina ligand docking predictions and large scale binding energy distribution analysis method binding free energy calculations have been applied to the virtual screening of a focused library of candidate binders to the LEDGF site of the HIV integrase protein. The computational protocol leveraged docking and high level atomistic models to improve enrichment. The enrichment factor of our blind predictions ranked best among all of the computational submissions, and second best overall. This work represents to our knowledge the first example of the application of an all-atom physics-based binding free energy model to large scale virtual screening. A total of 285 parallel Hamiltonian replica exchange molecular dynamics absolute protein-ligand binding free energy simulations were conducted starting from docked poses. The setup of the simulations was fully automated, calculations were distributed on multiple computing resources and were completed in a 6-weeks period. The accuracy of the docked poses and the inclusion of intramolecular strain and entropic losses in the binding free energy estimates were the major factors behind the success of the method. Lack of sufficient time and computing resources to investigate additional protonation states of the ligands was a major cause of mispredictions. The experiment demonstrated the applicability of binding free energy modeling to improve hit rates in challenging virtual screening of focused ligand libraries during lead optimization.

  20. Discovery of a small-molecule HIV-1 integrase inhibitor-binding site

    PubMed Central

    Al-Mawsawi, Laith Q.; Fikkert, Valery; Dayam, Raveendra; Witvrouw, Myriam; Burke, Terrence R.; Borchers, Christoph H.; Neamati, Nouri

    2006-01-01

    Herein, we report the identification of a unique HIV-1 integrase (IN) inhibitor-binding site using photoaffinity labeling and mass spectrometric analysis. We chemically incorporated a photo-activatable benzophenone moiety into a series of coumarin-containing IN inhibitors. A representative of this series was covalently photo-crosslinked with the IN core domain and subjected to HPLC purification. Fractions were subsequently analyzed by using MALDI-MS and electrospray ionization (ESI)-MS to identify photo-crosslinked products. In this fashion, a single binding site for an inhibitor located within the tryptic peptide 128AACWWAGIK136 was identified. Site-directed mutagenesis followed by in vitro inhibition assays resulted in the identification of two specific amino acid residues, C130 and W132, in which substitutions resulted in a marked resistance to the IN inhibitors. Docking studies suggested a specific disruption in functional oligomeric IN complex formation. The combined approach of photo-affinity labeling/MS analysis with site-directed mutagenesis/molecular modeling is a powerful approach for elucidating inhibitor-binding sites of proteins at the atomic level. This approach is especially important for the study of proteins that are not amenable to traditional x-ray crystallography and NMR techniques. This type of structural information can help illuminate processes of inhibitor resistance and thereby facilitate the design of more potent second-generation inhibitors. PMID:16785440

  1. Biologically-validated HIV integrase inhibitors with nucleobase scaffolds: structure, synthesis, chemical biology, molecular modeling, and antiviral activity.

    PubMed

    Nair, Vasu; Uchil, Vinod; Chi, Guochen; Dams, Iwona; Cox, Arthur; Seo, Byung

    2007-01-01

    Integrase, an enzyme of the pol gene of HIV, is a significant viral target for the discovery of anti-HIV agents. In this presentation, we report on the continuation of our work on the discovery of diketo acids, constructed on nucleobase scaffolds, that are inhibitors of HIV integrase. An example of our synthetic approach to inhibitors with purine nucleobase scaffolds is given. Comparison is made between integrase inhibition data arising from compounds with pyrimidine versus purine nucleobase scaffold. Antiviral results are cited.

  2. Secondary Integrase Resistance Mutations Found in HIV-1 Minority Quasispecies in Integrase Therapy-Naive Patients Have Little or No Effect on Susceptibility to Integrase Inhibitors▿

    PubMed Central

    Ceccherini-Silberstein, Francesca; Van Baelen, Kurt; Armenia, Daniele; Trignetti, Maria; Rondelez, Evelien; Fabeni, Lavinia; Scopelliti, Fernanda; Pollicita, Michela; Van Wesenbeeck, Liesbeth; Van Eygen, Veerle; Dori, Luca; Sarmati, Loredana; Aquaro, Stefano; Palamara, Guido; Andreoni, Massimo; Stuyver, Lieven J.; Perno, Carlo Federico

    2010-01-01

    The goal of this study was to explore the presence of integrase strand transfer inhibitor (InSTI) resistance mutations in HIV-1 quasispecies present in InSTI-naïve patients and to evaluate their in vitro effects on phenotypic susceptibility to InSTIs and their replication capacities. The RT-RNase H-IN region was PCR amplified from plasma viral RNA obtained from 49 HIV-1 subtype B-infected patients (21 drug naïve and 28 failing highly active antiretroviral therapy [HAART] not containing InSTIs) and recombined with an HXB2-based backbone with RT and IN deleted. Recombinant viruses were tested against raltegravir and elvitegravir and for replication capacity. Three-hundred forty-four recombinant viruses from 49 patients were successfully analyzed both phenotypically and genotypically. The majority of clones were not phenotypically resistant to InSTIs: 0/344 clones showed raltegravir resistance, and only 3 (0.87%) showed low-level elvitegravir resistance. No primary resistance mutations for raltegravir and elvitegravir were found as major or minor species. The majority of secondary mutations were also absent or rarely present. Secondary mutations, such as T97A and G140S, found rarely and only as minority quasispecies, were present in the elvitegravir-resistant clones. A novel mutation, E92G, although rarely found in minority quasispecies, showed elvitegravir resistance. Preexisting genotypic and phenotypic raltegravir resistance was extremely rare in InSTI-naïve patients and confined to only a restricted minority of secondary variants. Overall, these results, together with others based on population and ultradeep sequencing, suggest that at this point IN genotyping in all patients before raltegravir treatment may not be cost-effective and should not be recommended until evidence of transmitted drug resistance to InSTIs or the clinical relevance of IN minor variants/polymorphisms is determined. PMID:20479206

  3. The HIV-1 integrase E157Q polymorphism per se does not alter susceptibility to raltegravir and dolutegravir in vitro.

    PubMed

    Saladini, Francesco; Giannini, Alessia; Boccuto, Adele; Tiezzi, Diletta; Vicenti, Ilaria; Zazzi, Maurizio

    2017-08-21

    : The HIV-1 integrase E157Q natural polymorphism has been reported to cause one case of raltegravir (RAL) and dolutegravir (DTG) failure. We constructed six recombinant viruses containing integrase from HIV-1 isolates found to harbour E157Q as the only integrase strand inhibitor (INSTI) resistance-related mutation. Phenotypic analysis showed that E157Q results in minimal changes in RAL and DTG susceptibility. Together with data retrieved from the Stanford HIV database, our results indicate that E157Q is not a relevant INSTI resistance mutation per se. The previously reported case of E157Q based resistance must have resulted from combined as yet unidentified integrase polymorphisms.

  4. Dolutegravir Interactions with HIV-1 Integrase-DNA: Structural Rationale for Drug Resistance and Dissociation Kinetics

    PubMed Central

    DeAnda, Felix; Hightower, Kendra E.; Nolte, Robert T.; Hattori, Kazunari; Yoshinaga, Tomokazu; Kawasuji, Takashi; Underwood, Mark R.

    2013-01-01

    Signature HIV-1 integrase mutations associated with clinical raltegravir resistance involve 1 of 3 primary genetic pathways, Y143C/R, Q148H/K/R and N155H, the latter 2 of which confer cross-resistance to elvitegravir. In accord with clinical findings, in vitro drug resistance profiling studies with wild-type and site-directed integrase mutant viruses have shown significant fold increases in raltegravir and elvitegravir resistance for the specified viral mutants relative to wild-type HIV-1. Dolutegravir, in contrast, has demonstrated clinical efficacy in subjects failing raltegravir therapy due to integrase mutations at Y143, Q148 or N155, which is consistent with its distinct in vitro resistance profile as dolutegravir’s antiviral activity against these viral mutants is equivalent to its activity against wild-type HIV-1. Kinetic studies of inhibitor dissociation from wild-type and mutant integrase-viral DNA complexes have shown that dolutegravir also has a distinct off-rate profile with dissociative half-lives substantially longer than those of raltegravir and elvitegravir, suggesting that dolutegravir’s prolonged binding may be an important contributing factor to its distinct resistance profile. To provide a structural rationale for these observations, we constructed several molecular models of wild-type and clinically relevant mutant HIV-1 integrase enzymes in complex with viral DNA and dolutegravir, raltegravir or elvitegravir. Here, we discuss our structural models and the posited effects that the integrase mutations and the structural and electronic properties of the integrase inhibitors may have on the catalytic pocket and inhibitor binding and, consequently, on antiviral potency in vitro and in the clinic. PMID:24146996

  5. Blind prediction of HIV integrase binding from the SAMPL4 challenge

    PubMed Central

    Mobley, David L.; Liu, Shuai; Lim, Nathan M.; Wymer, Karisa L.; Perryman, Alexander L.; Forli, Stefano; Deng, Nanjie; Su, Justin; Branson, Kim; Olson, Arthur J.

    2015-01-01

    Here, we give an overview of the protein-ligand binding portion of the SAMPL4 challenge, which focused on predicting binding of HIV integrase inhibitors in the catalytic core domain. The challenge encompassed three components – a small “virtual screening” challenge, a binding mode prediction component, and a small affinity prediction component. Here, we give summary results and statistics concerning the performance of all submissions at each of these challenges. Virtual screening was particularly challenging here in part because, in contrast to more typical virtual screening test sets, the inactive compounds were tested because they were thought to be likely binders, so only the very top predictions performed significantly better than random. Pose prediction was also quite challenging, in part because inhibitors in the set bind to three different sites, so even identifying the correct binding site was challenging. Still, the best methods managed low RMSD predictions in many cases. Here, we give an overview of results, highlight some features of methods which worked particularly well, and refer the interested reader to papers in this issue which describe specific submissions for additional details. PMID:24595873

  6. Design of inhibitors of the HIV-1 integrase core domain using virtual screening

    PubMed Central

    Regon, Preetom; Gogoi, Dhrubajyoti; Rai, Ashok Kumar; Bordoloi, Manabjyoti; Bezbaruah, Rajib Lochan

    2014-01-01

    Acquired immunodeficiency syndrome (AIDS) is a disease of the human immune system caused by the human immunodeficiency virus (HIV). The integrase (IN) enzyme of HIV interacts with several cellular and viral proteins during the integration process. Thus, it represents an appropriate target for antiretroviral drugs (ARVs). We performed virtual screening of database compounds and designed analogues using Elvitegravir (EVG) as a standard compound. The 378 screened compounds were retrieved from ZINC, ChemSpider, PubChem, and ChemBank Chemical Databases based on chemical similarity and literature searches related to the structure of EVG. The Physiochemical properties, Bioactivity, Toxicity and Absorption, Distribution, Metabolism and Excretion of Molecules (ADME) of these compounds were predicted and docking Experiments were conducted using Molegro Virtual Docker software. The docking and ADME suggested very significant results in regard to EVG. The MolDock and Rerank scores were used to analyze the results. The compounds ZINC26507991 (-84.22), Analogue 9 (-68.49), ZINC20731658 (-66.79), ZINC00210363 (-43.44) showed better binding orientation with IN receptor model with respect to EVG (182.52). The ZINC26507991 has showed significant ADME result. PMID:24616558

  7. The long-acting integrase inhibitor GSK744 protects macaques from repeated intravaginal SHIV challenge.

    PubMed

    Radzio, Jessica; Spreen, William; Yueh, Yun Lan; Mitchell, James; Jenkins, Leecresia; García-Lerma, J Gerardo; Heneine, Walid

    2015-01-14

    Daily preexposure prophylaxis (PrEP) with Truvada is a proven HIV prevention strategy; however, its effectiveness is limited by low adherence. Antiretroviral drug formulations that require infrequent dosing may increase adherence and thus PrEP effectiveness. We investigated whether monthly injections of a long-acting formulation of the HIV integrase inhibitor GSK1265744 (GSK744 LA) prevented simian/human immunodeficiency virus (SHIV) infection by vaginal challenge in macaques. Female pigtail macaques (n = 12) were exposed to intravaginal inoculations of SHIV twice a week for up to 11 weeks. Half of the animals received a GSK744 LA injection every 4 weeks, and half received placebo. GSK744 LA, at plasma concentrations achievable with quarterly injections in humans, protected all six macaques from infection. Placebo controls were all infected after a median of 4 (range, 2 to 20) vaginal challenges with SHIV. Efficacy was related to high and sustained vaginal and plasma drug concentrations that remained above the protein-adjusted 90% inhibitory concentration during the dosing cycles. These data support advancement of GSK744 LA as a potential PrEP candidate for women.

  8. Development and use of SIV-based Integrase defective lentiviral vector for immunization

    PubMed Central

    Michelini, Zuleika; Negri, Donatella RM; Baroncelli, Silvia; Spada, Massimo; Leone, Pasqualina; Bona, Roberta; Klotman, Mary E.; Cara, Andrea

    2009-01-01

    Integrase (IN) defective lentiviral vectors have a high safety profile and might prove useful as immunizing agents especially against HIV-1. However, IN defective SIV-based vectors must be developed in order to test their potential in the non human primate models (NHP) of AIDS. To this aim we tested a novel SIV-based IN defective lentiviral vector for its ability to induce sustained immune responses in mice. BALB/c mice were immunized once intramuscularly with a SIV-based IN defective lentiviral vector expressing the model antigen enhanced green fluorescence protein (eGFP). Immune responses were evaluated 90 days after the injection and compared with those elicited with the IN competent counterpart. The IN defective vector was able to efficiently elicit specific and long-lasting polyfunctional immune responses as evaluated by enzyme-linked immunospot (ELISPOT) assays for interferon-γ (IFN-γ) in spleens, bone marrow (BM) and draining lymph nodes, and by intracellular staining (ICS) for IFN-γ, Interleukin-2 (IL-2) and tumor necrosis factor (TNF-α) in both splenocytes and BM cells without integration of the vector into the host genome. This is the first demonstration that an IN defective SIV-based lentiviral vector provides effective immunization, thus paving the way for the construction of IN defective vectors expressing SIV antigen(s) and test their efficacy against a SIV virus challenge in the NHP model of AIDS. PMID:19523909

  9. As(V), Cr(III) and Cr(VI) sorption on biochars and soil

    NASA Astrophysics Data System (ADS)

    Diamadopoulos, Evan; Agrafioti, Evita; Kalderis, Dimitrios

    2014-05-01

    The use of biochar, as a cost effective sorbent for heavy metal removal from contaminated water and soils is becoming a very promising practice. In this study, rice husk, the organic fraction of solid wastes, as well as sewage sludge were used as precursors for biochar production. The first was chosen as one of the most abundant types of biomass worldwide and the other two in order to find alternative innovative uses of these wastes. A series of batch kinetic and equilibrium (sorption and desorption) experiments was conducted using As(V), Cr(III) and Cr(VI) as adsorbates. The specific heavy metals were chosen in order to assess biochars removal capacity towards both anionic and cationic metals. Apart from biochars, a sandy loam soil was also used as adsorbent for metal removal. Knowing the separate behavior of biochars and soil towards metal sorption, it could be the first step in explaining the fate of heavy metals in a biochar amended soil. The kinetic study showed that, for all adsorbents and metals examined, sorption can be well described by the pseudo-second order kinetic model. What is more, simulation of sorption isotherms gave a better fit for the Freundlich model, possibly due to the heterogeneous surface of the initial biomasses and the fine aggregates that soil consists of. Based on the equilibrium study, the materials examined removed more than 95% of the initial Cr(III). This is possibly related to the electrostatic interactions between adsorbents negative surface charge and Cr(III) cations. However, removal rates for As(V) and Cr(VI) anions were significantly lower. Biochar derived from sewage sludge was efficient in removing 89% of Cr(VI) and 53% of As(V). Its ash high Fe2O3 content may have enhanced metal adsorption via precipitation. Soil was the most effective material for the removal of As(V), yet it could not strongly retain metal anions compared to biochars, as a significant amount of the adsorbed metal was released during desorption

  10. Nucleic acid amplification of HIV-1 integrase sequence subtypes CRF01_AE and B for development of HIV anti-integrase drug resistance genotyping assay

    NASA Astrophysics Data System (ADS)

    Adlar, F. R.; Bela, B.

    2017-08-01

    To anticipate the potential use of anti-integrase drugs in Indonesia for treatment of HIV-1 infection, the development of a drug resistance genotyping assay for anti-integrase is crucial in identifying the genetic drug resistance profile of Indonesian HIV-1 strains. This experiment aimed to amplify a target region in the integrase gene of Indonesian HIV-1 subtypes CRF01_AE and B that contain genetic mutations known to confer resistance to anti-integrase drug. Eleven archived plasma samples from individuals living with HIV-1 were obtained from the Virology and Cancer Pathobiology Research Center for Health Service (VCPRC FKUI-RSCM) laboratory. One of the plasma samples contained HIV-1 subtype B, and the remaining plasma samples contained subtype CRF01_AE. The target regions for all samples were amplified through RT-PCR, with an annealing temperature of 55 °C, using the primer pair AE_POL 4086F and AE_POL 5232R that were designed by VCPRC FKUI-RSCM. The results of this experiment show that 18.2% (2/11) of the samples were successfully amplified using the one-step RT-PCR. While the primer pair was effective in amplifying the target region in the integrase gene sequence for subtype B (100%; 1/1), it had a low efficacy (10%, 1/10) for subtype CRF01_AE. In conclusion, the primer pair can be used to amplify the target region in Indonesian HIV-1 strain subtypes CRF01_AE and B. However, optimization of the PCR condition and an increased number of samples would help to determine an accurate representation of the efficacy of the primer pair.

  11. Dynamic Oligomerization of Integrase Orchestrates HIV Nuclear Entry

    PubMed Central

    Borrenberghs, Doortje; Dirix, Lieve; De Wit, Flore; Rocha, Susana; Blokken, Jolien; De Houwer, Stéphanie; Gijsbers, Rik; Christ, Frauke; Hofkens, Johan; Hendrix, Jelle; Debyser, Zeger

    2016-01-01

    Nuclear entry is a selective, dynamic process granting the HIV-1 pre-integration complex (PIC) access to the chromatin. Classical analysis of nuclear entry of heterogeneous viral particles only yields averaged information. We now have employed single-virus fluorescence methods to follow the fate of single viral pre-integration complexes (PICs) during infection by visualizing HIV-1 integrase (IN). Nuclear entry is associated with a reduction in the number of IN molecules in the complexes while the interaction with LEDGF/p75 enhances IN oligomerization in the nucleus. Addition of LEDGINs, small molecule inhibitors of the IN-LEDGF/p75 interaction, during virus production, prematurely stabilizes a higher-order IN multimeric state, resulting in stable IN multimers resistant to a reduction in IN content and defective for nuclear entry. This suggests that a stringent size restriction determines nuclear pore entry. Taken together, this work demonstrates the power of single-virus imaging providing crucial insights in HIV replication and enabling mechanism-of-action studies. PMID:27830755

  12. Interactions between Integrase Inhibitors and human Arginase 1.

    PubMed

    Lisi, Lucia; Pizzoferrato, Michela; Miscioscia, Fabiola Teresa; Topai, Alessandra; Navarra, Pierluigi

    2017-04-11

    The neuro-pathogenic mechanism(s) underlying HIV-associated neurocognitive disorders are mostly unknown. HIV-infected macrophages and microglial cells play a crucial role and the metabolic fate of L-arginine may be highly relevant for microglia activation. In this context Arginase (ARG), which uses L-arginine as substrate, can be on the same time a target and source of oxidative stress and inflammation. In the present study, we investigated whether Integrase Strand Transfer Inhibitors (INSTIs) share with the other antiretroviral drugs the ability to inhibit ARG activity. We used the previously validated cell model, namely the human microglia cell line (CHME-5), as well as the computational chemistry approach. Furthermore, here we characterized the activity of purified human ARG in a cell-free in vitro system, and investigated the effects of INSTIs in this newly validated model. Overall evidence shows that Dolutegravir, Raltegravir and Elvitegravir inhibit ARG activity. This article is protected by copyright. All rights reserved.

  13. Structural dynamics of native and V260E mutant C-terminal domain of HIV-1 integrase

    NASA Astrophysics Data System (ADS)

    Sangeetha, Balasubramanian; Muthukumaran, Rajagopalan; Amutha, Ramaswamy

    2015-04-01

    The C-terminal domain (CTD) of HIV-1 integrase is a five stranded β-barrel resembling an SH3 fold. Mutational studies on isolated CTD and full-length IN have reported V260E mutant as either homo-dimerization defective or affecting the stability and folding of CTD. In this study, molecular dynamics simulation techniques were used to unveil the effect of V260E mutation on isolated CTD monomer and dimer. Both monomeric and dimeric forms of wild type and V260E mutant are highly stable during the simulated period. However, the stabilizing π-stacking interaction between Trp243 and Trp243' at the dimer interface is highly disturbed in CTD-V260E (>6 Å apart). The loss in entropy for dimerization is -30 and -25 kcal/mol for CTD-wt and CTD-V260E respectively signifying a weak hydrophobic interaction and its perturbation in CTD-V260E. The mutant Glu260 exhibits strong attraction/repulsion with all the basic/acidic residues of CTD. In addition to this, the dynamics of CTD-wild type and V260E monomers at 498 K was analyzed to elucidate the effect of V260E mutation on CTD folding. Increase in SASA and reduction in the number of contacts in CTD-V260E during simulation highlights the instability caused by the mutation. In general, V260E mutation affects both multimerization and protein folding with a pronounced effect on protein folding rather than multimerization. This study emphasizes the importance of the hydrophobic nature and SH3 fold of CTD in proper functioning of HIV integrase and perturbing this nature would be a rational approach toward designing more selective and potent allosteric anti-HIV inhibitors.

  14. Removal of As(III) and As(V) from water by copper oxide incorporated mesoporous alumina.

    PubMed

    Pillewan, Pradnya; Mukherjee, Shrabanti; Roychowdhury, Tarit; Das, Sera; Bansiwal, Amit; Rayalu, Sadhana

    2011-02-15

    In the present manuscript a new adsorbent namely copper oxide incorporated mesoporous alumina (COIMA) for removal of arsenic from water is reported. The COIMA was prepared by treating mesoporous alumina with copper sulphate solution followed by calcination at 450°C in the presence of air. Various adsorption isotherm and kinetic parameters were computed using batch adsorption studies to determine the adsorption capacity for As(III) and As(V) and to understand the mechanism of adsorption. It was observed that incorporation of copper oxide improves the adsorption capacity of unmodified alumina from 0.92 to 2.16 mg g(-1) for As(III) and from 0.84 to 2.02 mg g(-1) for As(V). The results revealed that the adsorption follows Langmuir isotherm and pseudo-second-order kinetic models for both As(III) and As(V). The material is capable of simultaneously removing As(III) and As(V) with removal efficiencies of more than 95% for both As(III) and As(V). Assessment of the water quality before and after treatment with COIMA also confirmed that the there is no leaching of copper and other parameters were also within permissible limits of Indian drinking water standard indicating that the COIMA can be used for treatment of arsenic contaminated drinking water.

  15. Radiation induced emulsion graft polymerization of 4-vinylpyridine onto PE/PP nonwoven fabric for As(V) adsorption

    NASA Astrophysics Data System (ADS)

    Akkaş Kavaklı, Pınar; Kavaklı, Cengiz; Seko, Noriaki; Tamada, Masao; Güven, Olgun

    2016-10-01

    A novel nonwoven fabric adsorbent having 4-vinylpyridine functional groups was prepared by using radiation-induced emulsion graft polymerization method and grafting 4-vinylpyridine monomer onto a polyethylene-coated polypropylene nonwoven fabric (NWF) in aqueous emulsion solution. The grafting conditions of the 4-vinylpyridine monomer onto the NWF were optimised and 150% Dg VP-g-NWF was prepared using 30 kGy pre-irradiation dose, 5% VP monomer concentration and 0.5% (w/w) Tween 20 in aqueous emulsion. Grafted 4-vinylpyridine chains on the NWF were then quaternized for the preparation of QVP-g-NWF adsorbent. All fabric structures were characterized by using Fourier-transform infrared spectrometer, x-ray photoelectron spectrometer and scanning electron microscope. QVP-g-NWF adsorbent was used in batch adsorption experiments for As(V) ions by studying the pH, contact time, and initial As(V) ion concentration parameters. Results showed that QVP-g-NWF adsorbent has significant As(V) adsorption and experimental As(V) adsorption capacity was 98.04 mg As(V)/g polymer from 500 mg/L initial As(V) concentration at pH 7.00.

  16. Discovery of a small-molecule HIV-1 integrase inhibitor-binding site | Center for Cancer Research

    Cancer.gov

    The lowest energy-binding conformation of an inhibitor bound to the dimeric interface of HIV-1 integrase core domain. The yellow region represents a unique allosteric binding site identified by affinity labeling and mass spectrometry and validated through mutagenesis. This site can provide a potential platform for the rational design of inhibitors selective for disruption of integrase multimerization.

  17. Intramolecular integration assay validates integrase phi C31 and R4 potential in a variety of insect cells.

    PubMed

    Chompoosri, Jakkrawarn; Fraser, Tresa; Rongsriyam, Yupha; Komalamisra, Narumon; Siriyasatien, Padet; Thavara, Usavadee; Tawatsin, Apiwat; Fraser, Malcolm J

    2009-11-01

    Phage phi C31 and R4 integrases are site-specific and unidirectional serine recombinases. We have analyzed the ability of these integrases to mediate intramolecular integration between their attB and attP sites in 7 important insect cell lines as a means of predicting their relative mobility in the corresponding insect species. Both integrases exhibit significantly higher frequencies in Drosophila S2 cells than in the other insect cell lines examined, but do work well in all of the species tested. Our results, coupled with previous results of the activity of phi C31 integrase in D. melanogaster and Aedes aegypti, suggest the family of serine catalyzed integrases will be useful site-specific integration tools for functional genome analysis and genetic engineering in a wide range of insect species.

  18. Dolutegravir (S/GSK1349572) Exhibits Significantly Slower Dissociation than Raltegravir and Elvitegravir from Wild-Type and Integrase Inhibitor-Resistant HIV-1 Integrase-DNA Complexes▿†

    PubMed Central

    Hightower, Kendra E.; Wang, Ruolan; DeAnda, Felix; Johns, Brian A.; Weaver, Kurt; Shen, Yingnian; Tomberlin, Ginger H.; Carter, H. Luke; Broderick, Timothy; Sigethy, Scott; Seki, Takahiro; Kobayashi, Masanori; Underwood, Mark R.

    2011-01-01

    The integrase inhibitor (INI) dolutegravir (DTG; S/GSK1349572) has significant activity against HIV-1 isolates with raltegravir (RAL)- and elvitegravir (ELV)-associated resistance mutations. As an initial step in characterizing the different resistance profiles of DTG, RAL, and ELV, we determined the dissociation rates of these INIs with integrase (IN)-DNA complexes containing a broad panel of IN proteins, including IN substitutions corresponding to signature RAL and ELV resistance mutations. DTG dissociates slowly from a wild-type IN-DNA complex at 37°C with an off-rate of 2.7 × 10−6 s−1 and a dissociative half-life (t1/2) of 71 h, significantly longer than the half-lives for RAL (8.8 h) and ELV (2.7 h). Prolonged binding (t1/2, at least 5 h) was observed for DTG with IN-DNA complexes containing E92, Y143, Q148, and N155 substitutions. The addition of a second substitution to either Q148 or N155 typically resulted in an increase in the off-rate compared to that with the single substitution. For all of the IN substitutions tested, the off-rate of DTG from IN-DNA complexes was significantly slower (from 5 to 40 times slower) than the off-rate of RAL or ELV. These data are consistent with the potential for DTG to have a higher genetic barrier to resistance, provide evidence that the INI off-rate may be an important component of the mechanism of INI resistance, and suggest that the slow dissociation of DTG may contribute to its distinctive resistance profile. PMID:21807982

  19. Use of a hexasubstituted benzene scaffold in the development of multivalent HIV-1 integrase inhibitors.

    PubMed

    Tupchiangmai, Wipa; Choksakulporn, Saowanaporn; Tewtrakul, Supinya; Pianwanit, Somsak; Sritana-anant, Yongsak

    2014-01-01

    The highly directional hexasubstituted benzene moiety was used as the central scaffold to create new human immunodeficiency virus (HIV)-1 integrase inhibitors through the attachment of multiple active groups. A series of potential inhibitors having substituted polyhydroxylated mono, bis and tris-cinnamoyl derivatives connected on the scaffold were prepared through Claisen-Schmidt condensations with substituted benzaldehydes, followed by partial demethylation to uncover the active phenolic groups required for the interactions with the integrase enzyme active sites. Using a multiplate integration assay method, four compounds carrying at least two sets of interacting moieties were found to be relatively potent integrase inhibitors with IC50 values in the low micromolar range. The results confirmed that multiple polyhydroxylated groups were required on the platform in order to effectively interact with the enzyme. The results from molecular docking studies consistently complemented the experimental results and revealed the nature of the potential key binding interactions responsible for the apparent activity of the active compounds.

  20. Preliminary study on the DNA-binding properties of phage ΦC31 integrase.

    PubMed

    Li, Zhihui; Fang, Yuxiang; Wang, Rencheng; Xue, Jinglun; Chen, Jinzhong

    2011-09-15

    ΦC31 integrase is a member of the large serine subfamily and is required for the recombination of the phage genome into the host chromosome, either to establish or exit from the lysogenic state. This enzyme can also mediate site-specific integration in mammalian cells in a cofactor-independent manner and has been considered as a potentially powerful tool for gene therapy. It has previously been reported that DAXX interacts with ΦC31 integrase and markedly inhibits its integration efficiency, and the 451RFGK454 tetramer of ΦC31 integrase has been identified as the interacting motif. Here, we report that both the deletion of the tetramer or the replacement of Arg with His greatly reduced the recombination activity of the ΦC31 integrase. Electrophoretic mobility shift assays further demonstrated that the DNA-binding ability and binding specificity of the two mutants were dramatically reduced. Bioinformatic analysis indicated a probable helix-turn-helix-like DNA-binding motif between residues 415-525, a region that contains the tetramer motif. However, neither truncated Int(415-525) nor Int(△415-525) alone could bind to the attB target sequence. Results of a circular dichroism spectroscopy assay indicated that Int(415-525) did not fold correctly into a helix-turn-helix-like structure, which may be one of the reasons for its lack of DNA-binding ability. Thus, the identification and confirmation of four key amino acids in the DNA-binding specificity and recombination activity of ΦC31 integrase provide information about the domain structure and function of the large C-terminal region and suggest important implications for the more efficient use of integrase in gene transfer and gene therapy. Copyright © 2011 Elsevier B.V. All rights reserved.

  1. Dicaffeoylquinic and Dicaffeoyltartaric Acids Are Selective Inhibitors of Human Immunodeficiency Virus Type 1 Integrase

    PubMed Central

    McDougall, Brenda; King, Peter J.; Wu, Bor Wen; Hostomsky, Zdenek; Reinecke, Manfred G.; Robinson, W. Edward

    1998-01-01

    Current pharmacological agents for human immunodeficiency virus (HIV) infection include drugs targeted against HIV reverse transcriptase and HIV protease. An understudied therapeutic target is HIV integrase, an essential enzyme that mediates integration of the HIV genome into the host chromosome. The dicaffeoylquinic acids (DCQAs) and the dicaffeoyltartaric acids (DCTAs) have potent activity against HIV integrase in vitro and prevent HIV replication in tissue culture. However, their specificity against HIV integrase in cell culture has been questioned. Thus, the ability of the DCQAs and DCTAs to inhibit binding of HIV type 1 (HIV-1) gp120 to CD4 and their activities against HIV-1 reverse transcriptase and HIV RNase H were studied. The DCQAs and DCTAs inhibited HIV-1 integrase at concentrations between 150 and 840 nM. They inhibited HIV replication at concentrations between 2 and 12 μM. Their activity against reverse transcriptase ranged from 7 μM to greater than 100 μM. Concentrations that inhibited gp120 binding to CD4 exceeded 80 μM. None of the compounds blocked HIV-1 RNase H by 50% at concentrations exceeding 80 μM. Furthermore, when the effects of the DCTAs on reverse transcription in acutely infected cells were measured, they were found to have no activity. Therefore, the DCQAs and DCTAs exhibit >10- to >100-fold specificity for HIV integrase, and their activity against integrase in biochemical assays is consistent with their observed anti-HIV activity in tissue culture. Thus, the DCQAs and DCTAs are a potentially important class of HIV inhibitors that act at a site distinct from that of current HIV therapeutic agents. PMID:9449274

  2. TG1 integrase-based system for site-specific gene integration into bacterial genomes.

    PubMed

    Muroi, Tetsurou; Kokuzawa, Takaaki; Kihara, Yoshihiko; Kobayashi, Ryuichi; Hirano, Nobutaka; Takahashi, Hideo; Haruki, Mitsuru

    2013-05-01

    Serine-type phage integrases catalyze unidirectional site-specific recombination between the attachment sites, attP and attB, in the phage and host bacterial genomes, respectively; these integrases and DNA target sites function efficiently when transferred into heterologous cells. We previously developed an in vivo site-specific genomic integration system based on actinophage TG1 integrase that introduces ∼2-kbp DNA into an att site inserted into a heterologous Escherichia coli genome. Here, we analyzed the TG1 integrase-mediated integrations of att site-containing ∼10-kbp DNA into the corresponding att site pre-inserted into various genomic locations; moreover, we developed a system that introduces ∼10-kbp DNA into the genome with an efficiency of ∼10(4) transformants/μg DNA. Integrations of attB-containing DNA into an attP-containing genome were more efficient than integrations of attP-containing DNA into an attB-containing genome, and integrations targeting attP inserted near the replication origin, oriC, and the E. coli "centromere" analogue, migS, were more efficient than those targeting attP within other regions of the genome. Because the genomic region proximal to the oriC and migS sites is located at the extreme poles of the cell during chromosomal segregation, the oriC-migS region may be more exposed to the cytosol than are other regions of the E. coli chromosome. Thus, accessibility of pre-inserted attP to attB-containing incoming DNA may be crucial for the integration efficiency by serine-type integrases in heterologous cells. These results may be beneficial to the development of serine-type integrases-based genomic integration systems for various bacterial species.

  3. Diketo acids derivatives as integrase inhibitors: the war against the acquired immunodeficiency syndrome.

    PubMed

    Henao-Mejia, Jorge; Góez, Yenny; Patiño, Pablo; Rugeles, Maria T

    2006-06-01

    Since the human immunodeficiency virus was identified as etiological agent of the acquired immunodeficiency syndrome, great advances have been accomplished in the therapeutic field leading to reduced morbidity and mortality among infected patients. However, the high mutation rate of the viral genome generates strains resistant to multiple drugs, pointing to the importance of finding new therapeutic targets. Among the HIV structural genes, the POL gene codes for three essential enzymes: reverse transcriptase, protease, and integrase; nineteen of the twenty drugs currently approved by the Food and Drug Administration to treat this viral infection, inhibit the reverse transcriptase and the protease. Although intense research has been carried out in this area during the last 10 years, HIV integrase inhibitors are not yet approved for clinical use; however the fact that presence of this enzyme is a sine qua non for a productive HIV life cycle joined to its unique properties makes it a promissory target for anti-HIV therapy. Many compounds have been claimed to inhibit integrase in vitro; however, few of them have proven to have antiviral activity and low cytotoxicity in cell systems. Diketoacid derivatives are the most promising integrase inhibitors so far reported. Initially discovered independently by Shionogi & Co. and the Merck Research Laboratories, these compounds are highly specific for the integrase with potent antiviral activity in vitro and in vivo, and low cytotoxicity in cell cultures. Some of these compounds have recently entered clinical trials. Due to the high relevance of integrase inhibitors, and specifically of diketoacid derivatives, we review the latest findings and patents in this important field of research.

  4. A method for producing transgenic cells using a multi-integrase system on a human artificial chromosome vector.

    PubMed

    Yamaguchi, Shigeyuki; Kazuki, Yasuhiro; Nakayama, Yuji; Nanba, Eiji; Oshimura, Mitsuo; Ohbayashi, Tetsuya

    2011-02-24

    The production of cells capable of expressing gene(s) of interest is important for a variety of applications in biomedicine and biotechnology, including gene therapy and animal transgenesis. The ability to insert transgenes at a precise location in the genome, using site-specific recombinases such as Cre, FLP, and ΦC31, has major benefits for the efficiency of transgenesis. Recent work on integrases from ΦC31, R4, TP901-1 and Bxb1 phages demonstrated that these recombinases catalyze site-specific recombination in mammalian cells. In the present study, we examined the activities of integrases on site-specific recombination and gene expression in mammalian cells. We designed a human artificial chromosome (HAC) vector containing five recombination sites (ΦC31 attP, R4 attP, TP901-1 attP, Bxb1 attP and FRT; multi-integrase HAC vector) and de novo mammalian codon-optimized integrases. The multi-integrase HAC vector has several functions, including gene integration in a precise locus and avoiding genomic position effects; therefore, it was used as a platform to investigate integrase activities. Integrases carried out site-specific recombination at frequencies ranging from 39.3-96.8%. Additionally, we observed homogenous gene expression in 77.3-87.5% of colonies obtained using the multi-integrase HAC vector. This vector is also transferable to another cell line, and is capable of accepting genes of interest in this environment. These data suggest that integrases have high DNA recombination efficiencies in mammalian cells. The multi-integrase HAC vector enables us to produce transgene-expressing cells efficiently and create platform cell lines for gene expression.

  5. Site-specific bacterial chromosome engineering mediated by IntA integrase from Rhizobium etli.

    PubMed

    Hernández-Tamayo, Rogelio; Torres-Tejerizo, Gonzalo; Brom, Susana; Romero, David

    2016-06-29

    The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range. Integration into the chromosome circumvents issues such as plasmid replication, stability, incompatibility, and copy number variance. The site-specific integrase IntA from Rhizobium etli CFN42 catalyzes a direct recombination between two specific DNA sites: attA and attD (23 bp). This recombination is stable. The aim of this work was to develop a R. etli derivative that may be used as recipient for the integration of foreign DNA in the chromosome, adapting the IntA catalyzed site-specific recombination system. To fulfill our aim, we designed a Rhizobium etli CFN42 derivative, containing a "landing pad" (LP) integrated into the chromosome. The LP sector consists of a green fluorescent protein gene under the control of the lacZ promoter and a spectinomycin resistance gene. Between the lacZ promoter and the GFP gene we inserted an IntA attachment site, which does not affect transcription from the lac promoter. Also, a mobilizable donor vector was generated, containing an attA site and a kanamycin resistance gene; to facilitate insertion of foreign DNA, this vector also contains a multicloning site. There are no promoters flanking the multicloning site. A biparental mating protocol was used to transfer the donor vector into the landing pad strain; insertion of the donor vector into the landing pad sector via IntA-mediated attA X attA recombination thereby interrupted the expression of the green fluorescent protein, generating site-specific cointegrants. Cointegrants were easily recognized by screening for antibiotic sensitivity and lack of GFP expression, and were obtained with an efficiency of 6.18 %. Integration of foreign DNA in Rhizobium, lacking any similarity with the genome, can be easily achieved by IntA-mediated recombination. This protocol contains the mating and selection procedures for creating and isolating integrants.

  6. HIV type 1 integrase polymorphisms in treatment-naive and treatment-experienced HIV type 1-infected patients in Thailand where HIV type 1 subtype A/E predominates.

    PubMed

    Phuphuakrat, Angsana; Pasomsub, Ekawat; Kiertiburanakul, Sasisopin; Chantratita, Wasun; Sungkanuparph, Somnuek

    2012-08-01

    Integrase inhibitor (INI) is a novel antiretroviral drug recommended for both treatment-naive and treatment-experienced HIV-1-infected patients. Limited data are available on INI resistance in Thailand, where HIV-1 subtype A/E predominates. We aimed to investigate INI resistance-associated mutations (RAMs) among treatment-naive patients and patients who experienced treatment failure with NNRTI-based or PI-based antiretroviral therapy (ART) in Thailand. One hundred and eight plasma samples of 58 treatment-naive and 50 treatment-experienced HIV-1-infected individuals were collected. The HIV-1 integrase coding region was sequenced. Polymorphisms were compared between subtype A/E and B circulating in Thailand and between treatment-naive and treatment-experienced groups. Resulting amino acids were interpreted for drug resistance according to Stanford algorithms. Ninety-seven samples were HIV-1 subtype A/E, 10 were subtype B, and one was subtype C. Age, gender, and CD4 cell counts were similar between treatment-naive and treatment-experienced groups, while the treatment-failure group showed a statistically significant longer awareness time of HIV-1 infection and lower viral load than the treatment-naive group. Major INI-RAM was not found in this study, but some minor INI-RAMs, such asV54I, L68I, L74M, T97A, and S230N, were found. Comparing INI-RAMs between subtype A/E and B, the prevalence of V54I and V72I was higher in subtype B than subtype E, while V201I was found in all sequences of subtype A/E. In subtype A/E, integrase polymorphisms were not different between treatment-naive and treatment-experienced groups. However, the number of amino acid substitutions was significantly higher in the treatment-experienced group (p=0.009). One NNRTI-based ART-treated patient was found to have potential low-level INI-RAMs. INI-RAMs are rare in both treatment-naive and treatment-experienced patients in Thailand. This suggested that INI should be active in patients who are naive to

  7. Antiviral Characteristics of GSK1265744, an HIV Integrase Inhibitor Dosed Orally or by Long-Acting Injection

    PubMed Central

    Kobayashi, Masanori; Seki, Takahiro; Miki, Shigeru; Wakasa-Morimoto, Chiaki; Suyama-Kagitani, Akemi; Kawauchi-Miki, Shinobu; Taishi, Teruhiko; Kawasuji, Takashi; Johns, Brian A.; Underwood, Mark R.; Garvey, Edward P.; Sato, Akihiko; Fujiwara, Tamio

    2014-01-01

    GSK1265744 is a new HIV integrase strand transfer inhibitor (INSTI) engineered to deliver efficient antiviral activity with a once-daily, low-milligram dose that does not require a pharmacokinetic booster. The in vitro antiviral profile and mechanism of action of GSK1265744 were established through integrase enzyme assays, resistance passage experiments, and cellular assays with site-directed molecular (SDM) HIV clones resistant to other classes of anti-HIV-1 agents and earlier INSTIs. GSK1265744 inhibited HIV replication with low or subnanomolar efficacy and with a selectivity index of at least 22,000 under the same culture conditions. The protein-adjusted half-maximal inhibitory concentration (PA-EC50) extrapolated to 100% human serum was 102 nM. When the virus was passaged in the presence of GSK1265744, highly resistant mutants with more than a 10-fold change (FC) in EC50 relative to that of the wild-type were not observed for up to 112 days of culture. GSK1265744 demonstrated activity against SDM clones containing the raltegravir (RAL)-resistant Y143R, Q148K, N155H, and G140S/Q148H signature variants (FC less than 6.1), while these mutants had a high FC in the EC50 for RAL (11 to >130). Either additive or synergistic effects were observed when GSK1265744 was tested in combination with representative anti-HIV agents, and no antagonistic effects were seen. These findings demonstrate that, similar to dolutegravir, GSK1265744 is differentiated as a new INSTI, having a markedly distinct resistance profile compared with earlier INSTIs, RAL, and elvitegravir (EVG). The collective data set supports further clinical development of GSK1265744. PMID:25367908

  8. Microwave assisted organic synthesis (MAOS) of small molecules as potential HIV-1 integrase inhibitors.

    PubMed

    Ferro, Stefania; Grazia, Sara De; De Luca, Laura; Gitto, Rosaria; Faliti, Caterina Elisa; Debyzer, Zeger; Chimirri, Alba

    2011-08-11

    Integrase (IN) represents a clinically validated target for the development of antivirals against human immunodeficiency virus (HIV). In recent years our research group has been engaged in the stucture-function study of this enzyme and in the development of some three-dimensional pharmacophore models which have led to the identification of a large series of potent HIV-1 integrase strand-transfer inhibitors (INSTIs) bearing an indole core. To gain a better understanding of the structure-activity relationships (SARs), herein we report the design and microwave-assisted synthesis of a novel series of 1-H-benzylindole derivatives.

  9. Ethyl malonate amides: a diketo acid offspring fragment for HIV integrase inhibition.

    PubMed

    Serafin, Katarzyna; Mazur, Pawel; Bak, Andrzej; Laine, Elodie; Tchertanov, Luba; Mouscadet, Jean-François; Polanski, Jaroslaw

    2011-08-15

    While searching for new HIV integrase inhibitors we discovered that some ethyl malonate amides (EMA) are active against this enzyme. Surprisingly, the main function can only very rarely be found among the reported drug candidates. We synthesised a series of compounds in order to establish and analyse the structure-activity relationship. The similarity to the important classes of HIV integrase inhibitors as well as the synthetic availability of the different targets including this pharmacophore makes EMA compounds an interesting object of investigations. Copyright © 2011 Elsevier Ltd. All rights reserved.

  10. The mechanisms of detoxification of As(III), dimethylarsinic acid (DMA) and As(V) in the microalga Chlorella vulgaris.

    PubMed

    Pantoja Munoz, L; Purchase, D; Jones, H; Raab, A; Urgast, D; Feldmann, J; Garelick, H

    2016-06-01

    The response of Chlorella vulgaris when challenged by As(III), As(V) and dimethylarsinic acid (DMA) was assessed through experiments on adsorption, efflux and speciation of arsenic (reduction, oxidation, methylation and chelation with glutathione/phytochelatin [GSH/PC]). Our study indicates that at high concentrations of phosphate (1.62mM of HPO4(2-)), upon exposure to As(V), cells are able to shift towards methylation of As(V) rather than PC formation. Treatment with As(V) caused a moderate decrease in intracellular pH and a strong increase in the concentration of free thiols (GSH). Passive surface adsorption was found to be negligible for living cells exposed to DMA and As(V). However, adsorption of As(III) was observed to be an active process in C. vulgaris, because it did not show saturation at any of the exposure periods. Chelation of As(III) with GS/PC and to a lesser extent hGS/hPC is a major detoxification mechanism employed by C. vulgaris cells when exposed to As(III). The increase of bound As-GS/PC complexes was found to be strongly related to an increase in concentration of As(III) in media. C. vulgaris cells did not produce any As-GS/PC complex when exposed to As(V). This may indicate that a reduction step is needed for As(V) complexation with GSH/PC. C. vulgaris cells formed DMAS(V)-GS upon exposure to DMA independent of the exposure period. As(III) triggers the formation of arsenic complexes with PC and homophytochelatins (hPC) and their compartmentalisation to vacuoles. A conceptual model was devised to explain the mechanisms involving ABCC1/2 transport. The potential of C. vulgaris to bio-remediate arsenic from water appeared to be highly selective and effective without the potential hazard of reducing As(V) to As(III), which is more toxic to humans. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Bacterial Formation of As(V) and As(III) Ferric Oxyhydroxides in Acid Mine Drainage.

    NASA Astrophysics Data System (ADS)

    Morin, G.; Juillot, F.; Lebrun, S.; Casiot, C.; Elbaz-Poulichet, F.; Bruneel, O.; Personne, J.; Leblanc, M.; Ildefonse, P.; Calas, G.

    2002-12-01

    The oxidation of dissolved Fe(II) which is often promoted by acidophilic bacteria in acid mine drainage (AMD) and some hot springs, leads to the precipitation of Fe(III) oxy-hydroxides which incorporate toxic elements within their structure or adsorb them at their surface, thus limiting their mobility. In such complex natural systems, synchrotron-based techniques as X-ray absorption spectroscopy offer the opportunity to monitor surface/solution interactions as well as redox changes affecting the mobility and toxicity of trace elements as arsenic. Spatial and seasonal variations of the (bio-) oxidation of Fe(II) and As(III), and the subsequent precipitation of As-Fe gels, were followed by XANES, XRD, and SEM along the CarnoulŠs AMD (Gard, France). Chemical and mineralogical data collected on sediments, stromatolite, and bioassay samples showed that some indigenous bacteria living in the As-rich CarnoulŠs water ([As] = up to 350 mg.l-1) play an important role in the nature and composition of the solid phases that sequester arsenic at the site. The formation of nano-crystalline and amorphous As(III) ferric oxy-hydroxides has been related to the presence of bacteria able to oxidize Fe(II) but not As(III), which are only present in winter in the upstream area. A rare ferric arsenite sulfate oxy-hydroxide mineral was discovered in this context. Other types of bacteria, occurring in the downstream area whatever the season, are able to catalyze As(III) to As(V) oxidation and, provided that enough Fe(II) oxidizes, promote the formation of amorphous As(V) rich ferric oxy-hydroxides. These bacterially mediated reactions significantly reduce the concentration of dissolved As(III), which is more toxic and mobile than As(V), and might thus be helpful for designing As-removal processes. This work was supported by the French PEVS and ACI Ecologie Quantitative Programs and the PIRAMID EC program. ?Deceased, 26 October 1999 Juillot F., Ildefonse Ph., Morin G., Calas G., De

  12. Lack of impact of pre-existing T97A HIV-1 integrase mutation on integrase strand transfer inhibitor resistance and treatment outcome

    PubMed Central

    Ram, Renee R.; Margot, Nicolas A.; Barnes, Tiffany L.; White, Kirsten L.; Callebaut, Christian; Miller, Michael D.

    2017-01-01

    T97A is an HIV-1 integrase polymorphism associated with integrase strand transfer inhibitor (INSTI) resistance. Using pooled data from 16 clinical studies, we investigated the prevalence of T97A (pre-existing and emergent) and its impact on INSTI susceptibility and treatment response in INSTI-naive patients who enrolled on elvitegravir (EVG)- or raltegravir (RAL)-based regimens. Prior to INSTI-based therapy, primary INSTI resistance-associated mutations (RAMs) were absent and T97A pre-existed infrequently (1.4%; 47 of 3367 integrase sequences); most often among non-B (5.3%) than B (0.9%) HIV-1 subtypes. During INSTI-based therapy, few patients experienced virologic failure with emergent INSTI RAMs (3%; 122 of 3881 patients), among whom T97A emerged infrequently in the presence (n = 6) or absence (n = 8) of primary INSTI RAMs. A comparison between pre-existing and emergent T97A patient populations (i.e., in the absence of primary INSTI RAMs) showed no significant differences in EVG or RAL susceptibility in vitro. Furthermore, among all T97A-containing viruses tested, only 38–44% exhibited reduced susceptibility to EVG and/or RAL (all of low magnitude; <11-fold), while all maintained susceptibility to dolutegravir. Of the patients with pre-existing T97A, 17 had available clinical follow-up: 16 achieved virologic suppression and 1 maintained T97A and INSTI sensitivity without further resistance development. Overall, T97A is an infrequent integrase polymorphism that is enriched among non-B HIV-1 subtypes and can confer low-level reduced susceptibility to EVG and/or RAL. However, detection of T97A does not affect response to INSTI-based therapy with EVG or RAL. These results suggest a very low risk of initiating INSTI-based therapy in patients with pre-existing T97A. PMID:28212411

  13. Role of Human Immunodeficiency Virus Type 1 Integrase in Uncoating of the Viral Core▿

    PubMed Central

    Briones, Marisa S.; Dobard, Charles W.; Chow, Samson A.

    2010-01-01

    After membrane fusion with a target cell, the core of human immunodeficiency virus type 1 (HIV-1) enters into the cytoplasm, where uncoating occurs. The cone-shaped core is composed of the viral capsid protein (CA), which disassembles during uncoating. The underlying factors and mechanisms governing uncoating are poorly understood. Several CA mutations can cause changes in core stability and a block at reverse transcription, demonstrating the requirement for optimal core stability during viral replication. HIV-1 integrase (IN) catalyzes the insertion of the viral cDNA into the host genome, and certain IN mutations are pleiotropic. Similar to some CA mutants, two IN mutants, one with a complete deletion of IN (NL-ΔIN) and the other with a Cys-to-Ser substitution (NL-C130S), were noninfectious, with a replication block at reverse transcription. Compared to the wild type (WT), the cytoplasmic CA levels of the IN mutants in infected cells were reduced, suggesting accelerated uncoating. The role of IN during uncoating was examined by isolating and characterizing cores from NL-ΔIN and NL-C130S. Both IN mutants could form functional cores, but the core yield and stability were decreased. Also, virion incorporation of cyclophilin A (CypA), a cellular peptidyl-prolyl isomerase that binds specifically to CA, was decreased in the IN mutants. Cores isolated from WT virus depleted of CypA had an unstable-core phenotype, confirming a role of CypA in promoting optimal core stability. Taken together, our results indicate that IN is required during uncoating for maintaining CypA-CA interaction, which promotes optimal stability of the viral core. PMID:20219923

  14. TISSUE DISTRIBUTION OF INORGANIC ARSENIC (AS) AND ITS METHYLATED METABOLITES IN MICE FOLLOWING ORAL ADMINISTRATION OF ARSENATE (ASV)

    EPA Science Inventory

    TISSUE DISTRIBUTION OF INORGANIC ARSENIC (iAs) AND ITS METHYLATED METABOLITES IN MICE FOLLOWING ORAL ADMINISTRATION OF ARSENATE (AsV). E M Kenyon1, L M Del Razo2, and M F Hughes1. 1NHEERL, ORD, US EPA, RTP, NC, USA; 2CINVESTAV-IPN, Mexico City, Mexico.

    The relationship o...

  15. Toxicity and bioremediation of As(III) and As(V) in the green microalgae Botryococcus braunii: A laboratory study.

    PubMed

    Podder, M S; Majumder, C B

    2017-02-01

    Worldwide threats of fuel shortages in the near future and climate change because of greenhouse gas emissions are posing severe challenges and therefore it is vital to search for sustainable ways of preventing the consequences. The dual use of microalgae for phycoremediation and biomass production for sustainable biofuel production is a viable choice. Phycoremediation of As(III) and As(V) ions using microalgae was investigated in a two-staged batch reactor. Accumulation and toxicity of inorganic arsenic forms (As(III) and As(V)) to green microalgae Botryococcus braunii depend on environmental factors. Dissolved oxygen and pH cycles did not significantly differ due to the absence or presence of arsenic (either As(III) or As(V)) ions in the culture. Monod model was utilized for representing the growth kinetics of microalgae in pure media containing various concentrations of nitrate ions. Maximum specific growth rate and saturation constant were found to be 0.14788 d(-1) and 0.00105 g/L, respectively. With the increase in concentration of phosphate in growth medium, the growth of microalgae increased. Media with NaCl (1.0 g/L) and NaHCO3 (1 g/L) resulted in higher maximum biomass concentration. Effect of coexisting ions on phycoremediation of As(III) and As(V) ions using microalgae was studied.

  16. TISSUE DISTRIBUTION OF INORGANIC ARSENIC (AS) AND ITS METHYLATED METABOLITES IN MICE FOLLOWING ORAL ADMINISTRATION OF ARSENATE (ASV)

    EPA Science Inventory

    TISSUE DISTRIBUTION OF INORGANIC ARSENIC (iAs) AND ITS METHYLATED METABOLITES IN MICE FOLLOWING ORAL ADMINISTRATION OF ARSENATE (AsV). E M Kenyon1, L M Del Razo2, and M F Hughes1. 1NHEERL, ORD, US EPA, RTP, NC, USA; 2CINVESTAV-IPN, Mexico City, Mexico.

    The relationship o...

  17. Removal of Cr(VI) and As(V) ions from aqueous solutions by polyacrylate and polystyrene anion exchange resins

    NASA Astrophysics Data System (ADS)

    Jachuła, Justyna; Hubicki, Zbigniew

    2013-09-01

    The sorption of Cr(VI) and As(V) from the aqueous solutions with the polyacrylate anion exchangers of the strong base functional groups Amberlite IRA 458 and Amberlite IRA 958 was studied. The studies were carried out by the static-batch method. The concentration of Cr(VI) and As(V) ions in the aqueous solution was determined by the UV-VIS spectrophotometer. The influence of several parameters was studied with respect to sorption equilibrium. The phase contact time and the concentration affect the sorption process. The equilibrium state was established already after 15 min of phase contact time. Maximum uptake of Cr(VI) and As(V) occurred at pH 5 and 10, respectively. The determined kinetic parameters imply that the sorption process proceeds according to the equation type of pseudo second-order. Sorption equilibrium data were correlated with the Langmuir and Freundlich isotherms. Removal of As(V) ions on macroporous Amberlite IRA 900 decreased about 12 % in presence of other anions (Cl-, NO3 -, SO4 2-) in the solution. The sorption was temperature dependent.

  18. Adsorption Characteristics of Different Adsorbents and Iron(III) Salt for Removing As(V) from Water

    PubMed Central

    Ćurko, Josip; Matošić, Marin; Crnek, Vlado; Stulić, Višnja

    2016-01-01

    Summary The aim of this study is to determine the adsorption performance of three types of adsorbents for removal of As(V) from water: Bayoxide® E33 (granular iron(III) oxide), Titansorb® (granular titanium oxide) and a suspension of precipitated iron(III) hydroxide. Results of As(V) adsorption stoichiometry of two commercial adsorbents and precipitated iron(III) hydroxide in tap and demineralized water were fitted to Freundlich and Langmuir adsorption isotherm equations, from which adsorption constants and adsorption capacity were calculated. The separation factor RL for the three adsorbents ranged from 0.04 to 0.61, indicating effective adsorption. Precipitated iron(III) hydroxide had the greatest, while Titansorb had the lowest capacity to adsorb As(V). Comparison of adsorption from tap or demineralized water showed that Bayoxide and precipitated iron(III) hydroxide had higher adsorption capacity in demineralized water, whereas Titansorb showed a slightly higher capacity in tap water. These results provide mechanistic insights into how commonly used adsorbents remove As(V) from water. PMID:27904416

  19. ILG1 : a new integrase-like gene that is a marker of bacterial contamination by the laboratory Escherichia coli strain TOP10F'.

    PubMed

    Tian, Wenzhi; Chua, Kevin; Strober, Warren; Chu, Charles C

    2002-07-01

    Identification of differentially expressed genes between normal and diseased states is an area of intense current medical research that can lead to the discovery of new therapeutic targets. However, isolation of differentially expressed genes by subtraction often suffers from unreported contamination of the resulting subtraction library with clones containing DNA sequences not from the original RNA samples. Subtraction using cDNA representational difference analysis (RDA) was performed on human B cells from normal or common variable immunodeficiency patients. The material remaining after the subtraction was cloned and individual clones were sequenced. The sequence of one clone with similarity to integrases (ILG1, integrase-like gene-1) was used to obtain the full length cDNA sequence and as a probe for the presence of this sequence in RNA or genomic DNA samples. After five rounds of cDNA RDA, 23.3% of the clones from the resulting subtraction library contained Escherichia coli DNA. In addition, three clones contained the sequence of a new integrase, ILG1. The full length cDNA sequence of ILG1 exhibits prokaryotic, but not eukaryotic, features. At the DNA level, ILG1 is not similar to any known gene. At the protein level, ILG1 has 58% similarity to integrases from the cryptic P4 bacteriophage family (S clade). The catalytic domain of ILG1 contains the conserved features found in site-specific recombinases. The critical residues that form the catalytic active site pocket are conserved, including the highly conserved R-H-R-Y hallmark of these recombinases. Interestingly, ILG1 was not present in the original B cell populations. By probing genomic DNA, ILG1 could only be detected in the E. coli TOP10F' strain used in our laboratory for molecular cloning, but not in any of its precursor strains, including TOP10. Furthermore, bacteria cultured from the mouth of the laboratory worker who performed cDNA RDA were also positive for ILG1. In the course of our studies using c

  20. The Stringent Response Promotes Antibiotic Resistance Dissemination by Regulating Integron Integrase Expression in Biofilms

    PubMed Central

    Strugeon, Emilie; Tilloy, Valentin; Ploy, Marie-Cécile

    2016-01-01

    ABSTRACT Class 1 integrons are genetic systems that enable bacteria to capture and express gene cassettes. These integrons, when isolated in clinical contexts, most often carry antibiotic resistance gene cassettes. They play a major role in the dissemination of antibiotic resistance among Gram-negative bacteria. The key element of integrons is the integrase, which allows gene cassettes to be acquired and shuffled. Planktonic culture experiments have shown that integrase expression is regulated by the bacterial SOS response. In natural settings, however, bacteria generally live in biofilms, which are characterized by strong antibiotic resilience and by increased expression of stress-related genes. Here, we report that under biofilm conditions, the stringent response, which is induced upon starvation, (i) increases basal integrase and SOS regulon gene expression via induction of the SOS response and (ii) exerts biofilm-specific regulation of the integrase via the Lon protease. This indicates that biofilm environments favor integron-mediated acquisition of antibiotic resistance and other adaptive functions encoded by gene cassettes. PMID:27531906

  1. [Integrase inhibitors - new challenges for the treatment of HIV-1 infections].

    PubMed

    Stock, Ingo

    2013-12-01

    Integrase inhibitors are a promising new group of antiretroviral drugs that suppress the integrase yielded by human immunodeficiency viruses (HIV) via inhibiting the ,,integration" of the viral deoxyribonucleic acid (DNA) into the hosts' DNA genome. In 2007, raltegravir was the first integrase inhibitor that has been approved for the treatment of HIV-1 infections in antiretroviral-pretreated (-experienced) and antiretroviral-naive patients. Recently, elvitegravir, as a fixed coformulation with cobicistat, tenofovir und emtricitabine, has been approved for the treatment of HIV-1-infected antiretroviral-naive patients. InAugust of 2013, dolutegravir, a third integrase inhibitor, has been approved by the US Food and Drug Adiministation (FDA) for the treatment of HIV-1 infections in adults and children aged 12 years and older. Raltegravir has to be applied twice daily without a boosting agent. Elvitegravir and dolutegravir are applied once daily in the presence of a booster (elvitegravir) or unboosted (dolutegravir). In contrast to raltegravir and elvitegravir, dolutegravir shows a high genetic barrier to resistance, and is also applicable for the treatment of several HIV-1 infections with raltegravir and elvitegravir-resistant HIV variants. During the last years, raltegravir, elvitegravir and dolutegravir have been proven and established in the antiretroviral treatment of HIV-1 infections as effective, safe and well-tolerated agents. However, reliable statement forecasts of long-term toxicity of these substances can not yet be made.

  2. Integrase Inhibitor Prodrugs: Approaches to Enhancing the Anti-HIV Activity of β-Diketo Acids.

    PubMed

    Nair, Vasu; Okello, Maurice

    2015-07-13

    HIV integrase, encoded at the 3'-end of the HIV pol gene, is essential for HIV replication. This enzyme catalyzes the incorporation of HIV DNA into human DNA, which represents the point of "no-return" in HIV infection. Integrase is a significant target in anti-HIV drug discovery. This review article focuses largely on the design of integrase inhibitors that are β-diketo acids constructed on pyridinone scaffolds. Methodologies for synthesis of these compounds are discussed. Integrase inhibition data for the strand transfer (ST) step are compared with in vitro anti-HIV data. The review also examines the issue of the lack of correlation between the ST enzymology data and anti-HIV assay results. Because this disconnect appeared to be a problem associated with permeability, prodrugs of these inhibitors were designed and synthesized. Prodrugs dramatically improved the anti-HIV activity data. For example, for compound, 96, the anti-HIV activity (EC50) improved from 500 nM for this diketo acid to 9 nM for its prodrug 116. In addition, there was excellent correlation between the IC50 and IC90 ST enzymology data for 96 (6 nM and 97 nM, respectively) and the EC50 and EC90 anti-HIV data for its prodrug 116 (9 nM and 94 nM, respectively). Finally, it was confirmed that the prodrug 116 was rapidly hydrolyzed in cells to the active compound 96.

  3. Development of an integrase-based ELISA for specific diagnosis of individuals infected with HIV.

    PubMed

    Rikhtegaran Tehrani, Zahra; Azadmanesh, Kayhan; Mostafavi, Ehsan; Soori, Shahrzad; Azizi, Mohammad; Khabiri, Alireza

    2015-04-01

    Currently, enzyme immunoassays (EIAs) are the most common immunological diagnostic methods that are used as the screening tool in HIV detection. Among all three major genes of HIV, the products of gag and env are usually used in EIAs (ELISAs and rapid tests). Hence, the presence of cross reacting antibodies against these antigens leads to the appearance of repetitive false positive results in screening tests. Re-testing the primary reactive samples with EIAs using other HIV antigens can considerably reduce the rate of false positive results. The products of pol gene may act as an appropriate candidate in this context. Integrase is a conserved and immunogenic product of HIV, encoded by the pol gene. The aim of this research was to determine the sensitivity and specificity of an ELISA detecting integrase antibodies. Recombinant integrase was produced in Escherichia coli to develop the integrase-based ELISA. Assay performance was evaluated by HIV positive and negative sera and an HIV panel of BBI (PRB-601). The sensitivity and specificity of assay was determined as 96.7 [95% confidence interval: 91.3-98.9%] and 100% [95% CI: 96.1-100%], respectively. High specificity of this assay may suggest its possible use in the detection of HIV.

  4. Site-specific deletion and rearrangement of integron insert genes catalyzed by the integron DNA integrase.

    PubMed Central

    Collis, C M; Hall, R M

    1992-01-01

    Deletion of individual antibiotic resistance genes found within the variable region of integrons is demonstrated. Evidence for gene duplications and rearrangements resulting from the insertion of gene units at new locations is also presented. Deletion, duplication, and rearrangement occur only in the presence of the integron-encoded DNA integrase. These events are precise and involve loss or gain of one or more complete insert units or gene cassettes. This confirms the recent definition of gene cassettes as consisting of the gene coding sequences, all except the last 7 bases of the 59-base element found at the 3' end of the gene, and the core site located 5' to the gene (Hall et al., Mol. Microbiol. 5:1941-1959, 1991) and demonstrates that individual gene cassettes are functional units which can be independently mobilized. Both deletions and duplications can be generated by integrase-mediated cointegrate formation followed by integrase-mediated resolution involving a different pair of sites. However, deletion occurs 10 times more frequently than duplication, and we propose that the majority of deletion events are likely to involve integrase-dependent excision of the gene unit to generate a circular gene cassette. The implications of these findings in understanding the evolution of integrons and the spread of antibiotic resistance genes in bacterial populations is discussed. Images PMID:1311297

  5. A simplified mathematical model of directional DNA site-specific recombination by serine integrases

    PubMed Central

    Zhao, Jia; Stark, W. Marshall; Colloms, Sean D.; Ebenhöh, Oliver

    2017-01-01

    Serine integrases catalyse site-specific recombination to integrate and excise bacteriophage genomes into and out of their host's genome. These enzymes exhibit remarkable directionality; in the presence of the integrase alone, recombination between attP and attB DNA sites is efficient and irreversible, giving attL and attR products which do not recombine further. However, in the presence of the bacteriophage-encoded recombination directionality factor (RDF), integrase efficiently promotes recombination between attL and attR to re-form attP and attB. The DNA substrates and products of both reactions are approximately isoenergetic, and no cofactors (such as adenosine triphosphate) are required for recombination. The thermodynamic driving force for directionality of these reactions is thus enigmatic. Here, we present a minimal mathematical model which can explain the directionality and regulation of both ‘forward’ and ‘reverse’ reactions. In this model, the substrates of the ‘forbidden’ reactions (between attL and attR in the absence of RDF, attP and attB in the presence of RDF) are trapped as inactive protein–DNA complexes, ensuring that these ‘forbidden’ reactions are extremely slow. The model is in good agreement with the observed in vitro kinetics of recombination by ϕC31 integrase, and defines core features of the system necessary and sufficient for directionality. PMID:28077763

  6. Characterization of a Replication-Competent, Integrase-Defective Human Immunodeficiency Virus (HIV)/Simian Virus 40 Chimera as a Powerful Tool for the Discovery and Validation of HIV Integrase Inhibitors▿

    PubMed Central

    Daelemans, Dirk; Lu, Richard; De Clercq, Erik; Engelman, Alan

    2007-01-01

    Integrase is actively studied as an antiviral target, but many inhibitors selected from biochemical screens fail to inhibit human immunodeficiency virus (HIV) replication or primarily affect off-site targets. Here we develop and validate a replication-competent, simian virus 40-HIV integrase mutant chimera as a novel tool to classify the mechanism of action of potential integrase inhibitors. Whereas the mutant was more susceptible than the wild type to entry, reverse transcriptase, and protease inhibitors, it specifically resisted the action of integrase inhibitor L-870,810. We furthermore demonstrate inhibition of integration by GS-9137 and GS-9160 and off-site targeting by the 6-aminoquinolone antibiotic WM-5. PMID:17287285

  7. The comparative disposition and metabolism of dolutegravir, a potent HIV-1 integrase inhibitor, in mice, rats, and monkeys.

    PubMed

    Moss, Lee; Wagner, David; Kanaoka, Eri; Olson, Katie; Yueh, Yun Lan; Bowers, Gary D

    2015-01-01

    1. Plasma clearance of dolutegravir, an unboosted HIV-1 integrase inhibitor, was low in rat and monkey (0.23 and 2.12 mL/min/kg, respectively) as was the volume of distribution (0.1 and 0.28 L/kg, respectively) with terminal elimination half-life approximately 6 h. Dolutegravir was rapidly absorbed from oral solution with a high bioavailability in rat and monkey (75.6 and 87.0% respectively), but solubility or dissolution rate limited when administered as suspension. 2. Dolutegravir was highly bound (>99%) to serum proteins in rat and monkey, similar to binding to plasma and serum proteins in human. Radioactivity was associated with the plasma versus cellular components of blood across all species. 3. Following oral administration to rats, [(14)C]dolutegravir-related radioactivity was distributed to most tissues, due in part to high permeability; however, because of high plasma protein binding, tissue to blood ratios were low. In mouse, rat and monkey, the absorbed dose was extensively metabolized and secreted into bile, with the majority of the administered radioactivity eliminated in feces within 24 h. 4. The primary route of metabolism of dolutegravir was through the formation of an ether glucuronide. Additional biotransformation pathways: benzylic oxidation followed by hydrolysis to an N-dealkylated product, glucose conjugation, oxidative defluorination, and glutathione conjugation.

  8. Functional Coupling between HIV-1 Integrase and the SWI/SNF Chromatin Remodeling Complex for Efficient in vitro Integration into Stable Nucleosomes

    PubMed Central

    Lesbats, Paul; Botbol, Yair; Chevereau, Guillaume; Vaillant, Cédric; Calmels, Christina; Arneodo, Alain; Andreola, Marie-Line; Lavigne, Marc; Parissi, Vincent

    2011-01-01

    Establishment of stable HIV-1 infection requires the efficient integration of the retroviral genome into the host DNA. The molecular mechanism underlying the control of this process by the chromatin structure has not yet been elucidated. We show here that stably associated nucleosomes strongly inhibit in vitro two viral-end integration by decreasing the accessibility of DNA to integrase. Remodeling of the chromatinized template by the SWI/SNF complex, whose INI1 major component interacts with IN, restores and redirects the full-site integration into the stable nucleosome region. These effects are not observed after remodeling by other human remodeling factors such as SNF2H or BRG1 lacking the integrase binding protein INI1. This suggests that the restoration process depends on the direct interaction between IN and the whole SWI/SNF complex, supporting a functional coupling between the remodeling and integration complexes. Furthermore, in silico comparison between more than 40,000 non-redundant cellular integration sites selected from literature and nucleosome occupancy predictions also supports that HIV-1 integration is promoted in the genomic region of weaker intrinsic nucleosome density in the infected cell. Our data indicate that some chromatin structures can be refractory for integration and that coupling between nucleosome remodeling and HIV-1 integration is required to overcome this natural barrier. PMID:21347347

  9. Tracer test with As(V) under variable redox conditions controlling arsenic transport in the presence of elevated ferrous iron concentrations

    USGS Publications Warehouse

    Hohn, R.; Isenbeck-Schroter, M.; Kent, D.B.; Davis, J.A.; Jakobsen, R.; Jann, S.; Niedan, V.; Scholz, C.; Stadler, S.; Tretner, A.

    2006-01-01

    To study transport and reactions of arsenic under field conditions, a small-scale tracer test was performed in an anoxic, iron-reducing zone of a sandy aquifer at the USGS research site on Cape Cod, Massachusetts, USA. For four weeks, a stream of groundwater with added As(V) (6.7????M) and bromide (1.6??mM), was injected in order to observe the reduction of As(V) to As(III). Breakthrough of bromide (Br-), As(V), and As(III) as well as additional parameters characterizing the geochemical conditions was observed at various locations downstream of the injection well over a period of 104??days. After a short lag period, nitrate and dissolved oxygen from the injectate oxidized ferrous iron and As(V) became bound to the freshly formed hydrous iron oxides. Approximately one week after terminating the injection, anoxic conditions had been reestablished and increases in As(III) concentrations were observed within 1??m of the injection. During the observation period, As(III) and As(V) were transported to a distance of 4.5??m downgradient indicating significant retardation by sorption processes for both species. Sediment assays as well as elevated concentrations of hydrogen reflected the presence of As(V) reducing microorganisms. Thus, microbial As(V) reduction was thought to be one major process driving the release of As(III) during the tracer test in the Cape Cod aquifer. ?? 2006 Elsevier B.V. All rights reserved.

  10. Performance of Celera RUO integrase resistance assay across multiple HIV-1 subtypes.

    PubMed

    Wallis, Carole L; Viana, Raquel V; Saravanan, Shanmugam; Silva de Jesus, Carlos; Zeh, Clement; Halvas, Elias K; Mellors, John W

    2017-03-01

    HIV-1 sequence variation is a major obstacle to developing molecular based assays for multiple subtypes. This study sought to independently assess performance characteristics of the ViroSeq™ HIV-1 Integrase RUO Genotyping Kit (Celera, US) for samples of multiple different HIV-1 subtypes. 264 samples were tested in the validation, 106 from integrase inhibitor naïve patients' sent for routine HIV-1 drug resistance testing after failing a 1st- or 2nd-line regimen, and 158 samples from an external virology quality assurance program (VQA). For the latter, 53 unique VQA samples were tested in two to five different laboratories to assess assay reproducibility. For all assays, viral RNA was extracted using the ViroSeq extraction module, reverse transcribed, and amplified in a one-step reaction. Four sequencing primers were used to span codons 1-288 of integrase. The Rega subtyping tool was used for subtype assignment. Integrase polymorphisms and mutations were determined as differences from the HXB2 sequence and by the Stanford database, respectively. Sequences obtained from the different laboratories were aligned and sequence homology determined. HIV-1 RNA in the 264 samples ranged from 3.15 to 6.74logcopies/ml. Successful amplification was obtained for 97% of samples (n=256). The 8 samples that failed to amplify were subtype D (n=3), subtype C (n=1), CRF01_AE (n=1), subtype A1 (n=2), and an unassigned subtype (n=1). Of the 256 that successfully amplified samples, 203 (79%) were successfully sequenced with bidirectional coverage. Of the 53 unsuccessful samples, 13 (5%) failed sequencing and 40 (16%) did not have full bidirectional sequence, as a result of failure of sequencing primers: Primer A (n=1); Primer B (n=18); Primer C (n=1); Primer D (n=7) or short sequences (n=16). For the 135 VQA samples (30 unique samples) that were assayed by different laboratories, homology of the sequences obtained ranged from 92.1% to 100%. However, Laboratory 2 detected more mixtures

  11. Biosorption of Cr(VI) and As(V) at high concentrations by organic and inorganic wastes

    NASA Astrophysics Data System (ADS)

    María Rivas Pérez, Ivana; Paradelo Núñez, Remigio; Nóvoa Muñoz, Juan Carlos; Arias Estévez, Manuel; José Fernández Sanjurjo, María; Álvarez Rodríguez, Esperanza; Núñez Delgado, Avelino

    2016-04-01

    The potential reutilization of several wastes as biosorbents for As(V) and Cr(VI) has been assessed in batch-type experiments. The materials studied were one inorganic: mussel shell, and three organic: pine bark, oak ash and hemp waste. Batch experiments were performed in order to determine the removal capacity of the wastes under conditions of high As(V) and Cr(VI) loads. For this, 3 g of each waste material were added with 30 mL NaNO3 0.01 M dissolutions containing 0, 0.5, 1.5, 3 and 6 mmol As(V) L-1 or Cr(VI) L-1, prepared from analytical grade Na2HAsO4 or K2Cr2O7. The resulting suspensions were shaken for 24 h, centrifuged and filtered. Once each batch experiment corresponding to the sorption trials ended, each individual sample was added with 30 mL of NaNO3 0.01 M to desorb As(V) or Cr(VI), shaken for 24 h, centrifuged and filtered as in the sorption trials. Oak ash showed high sorption (>76%) and low desorption (<7%) for As(V), which was lower on mussel shell (<31%), hemp waste (<16%) and pine bark (<9.9%). In turn, pine bark showed the highest Cr(VI) sorption (>98%) with very low desorption (<0.5%), followed by oak ash (27% sorption), and hemp waste and mussel shell, that presented very low Cr(VI) sorption (<10%). Sorption data for both elements were better described by the Freundlich than by the Langmuir model. The variable results obtained for the removal of the two anionic contaminants for a given sorbent suggest that different mechanisms govern removal from the solution in each case. In summary, oak ash would be an efficient sorbent material for As(V), but not for Cr(VI), while pine bark would be the best sorbent for Cr(VI) removal.

  12. Resistance to HIV Integrase Strand Transfer Inhibitors Among Clinical Specimens in the United States, 2009–2012

    PubMed Central

    Hurt, Christopher B.; Sebastian, Joseph; Hicks, Charles B.; Eron, Joseph J.

    2014-01-01

    Background. Data on integrase inhibitor resistance come primarily from clinical trials and in vitro studies. We examined results of all clinically indicated integrase genotypic resistance tests (GRTs) performed at a US national referral lab from 2009 through 2012. Methods. Integrase sequences and demographic data were compiled with paired protease–reverse transcriptase (PR-RT) GRT results, when available. Analyses utilized the Stanford HIV Drug Resistance Database. “Major” integrase mutations included T66AIK, E92QV, F121Y, Y143CHR, S147G, Q148HKR, and N155H; multiple accessory mutations were also assessed. Results. Among 3294 sequences from 3012 patients, 471 patients had viruses with ≥1 raltegravir or elvitegravir resistance mutation (15.6%). Q148 and N155 pathways were equally represented (both n = 197); 84 had Y143 mutations. Q148 rarely occurred without accessory mutations (n = 3). Among 224 patients with serial integrase GRTs, 22 with baseline wild-type acquired a major mutation, after a median 224 days between tests (interquartile range, 148–335 days). Major mutations were observed to persist up to 462 days. Most (62%) had paired PR-RT results. Patients with integrase-resistant viruses were older and more likely to have PR-RT mutations (both P < .001). Among those with PR-RT data, 42 patients had 4-class resistance (2.3%). Sex, geographic region, and test year were not associated with integrase resistance. High-level dolutegravir resistance was predicted in 12% of patients with raltegravir- or elvitegravir-resistant viruses (2% of all patients). Conclusions. Approximately 1 in 6 US patients undergoing integrase GRT for clinical decision making harbors significant resistance, with Q148 and N155 pathways equally common. Dolutegravir is likely to have full or partial activity against most variants observed. PMID:24145878

  13. Clinical Pharmacokinetic, Pharmacodynamic and Drug-Interaction Profile of the Integrase Inhibitor Dolutegravir

    PubMed Central

    Cottrell, Mackenzie L.; Hadzic, Tanja

    2013-01-01

    Dolutegravir is a second generation integrase strand transfer inhibitor (INSTI) currently under review by the US FDA for marketing approval. Dolutegravir’s in vitro, protein adjusted 90% inhibitory concentration (IC90) for wild-type virus is 0.064 μg/ml, and it retains in vitro anti-HIV 1 activity across a broad range of viral phenotypes known to confer resistance to the currently marketed INSTIs, raltegravir and elvitegravir. Dolutegravir has a half-life (t½) of 13 to 14 hours and maintains concentrations over the in vitro, protein adjusted IC90 for more than 30 hours following a single dose. Additionally, dolutegravir has comparatively low intersubject variability compared to raltegravir and elvitegravir. A plasma exposure-response relationship has been well described, with antiviral activity strongly correlating to trough concentration (Ctrough) values. Phase III trials have assessed the antiviral activity of dolutegravir compared with efavirenz and raltegravir in antiretroviral (ARV)-naïve patients and found dolutegravir to achieve more rapid and sustained virologic suppression in both instances. Additionally, studies of dolutegravir activity in patients with known INSTI-resistant mutations have been favorable, indicating that dolutegravir retains activity in a variety of INSTI resistant phenotypes. Much like currently marketed INSTIs, dolutegravir is very well tolerated. Because dolutegravir inhibits the renal transporter, organic cation transporter (OCT) 2, reduced tubular secretion of creatinine leads to non-progressive increases in serum creatinine. These serum creatinine increases have not been associated with decreased glomerular filtration rate or progressive renal impairment. Dolutegravir’s major and minor metabolic pathways are UDP glucuronosyltransferase (UGT)1A1 and cytochrome (CYP)3A4, respectively, and it neither induces nor inhibits CYP isozymes. Thus dolutegravir has a modest drug interaction profile. However, antacids significantly

  14. Clinical pharmacokinetic, pharmacodynamic and drug-interaction profile of the integrase inhibitor dolutegravir.

    PubMed

    Cottrell, Mackenzie L; Hadzic, Tanja; Kashuba, Angela D M

    2013-11-01

    Dolutegravir is a second-generation integrase strand transfer inhibitor (INSTI) currently under review by the US Food and Drug Administration for marketing approval. The in vitro, protein-adjusted 90 % inhibitory concentration (IC90) of dolutegravir for wild-type virus is 0.064 μg/ml, and it retains in vitro anti-HIV 1 activity across a broad range of viral phenotypes that are known to confer resistance to the currently marketed INSTIs, raltegravir and elvitegravir. Dolutegravir has a terminal elimination half-life of 13-14 h and maintains concentrations over the in vitro, protein-adjusted IC90 for more than 30 h following a single dose. Additionally, dolutegravir has low inter-subject variability compared with raltegravir and elvitegravir. A plasma exposure-response relationship has been well described, with antiviral activity strongly correlating with trough concentrations. Phase III trials have assessed the antiviral activity of dolutegravir compared with efavirenz and raltegravir in antiretroviral (ARV)-naive patients and found that dolutegravir achieved more rapid and sustained virologic suppression in both instances. Additionally, studies of dolutegravir activity in patients with known INSTI-resistant mutations have been favourable, indicating that dolutegravir retains activity in a variety of INSTI-resistant phenotypes. Much like currently marketed INSTIs, dolutegravir is very well tolerated. Because dolutegravir inhibits the renal transporter organic cation transporter 2, reduced tubular secretion of creatinine leads to non-progressive increases in serum creatinine. These serum creatinine increases have not been associated with a decreased glomerular filtration rate or progressive renal impairment. Dolutegravir's major and minor metabolic pathways are uridine diphosphate glucuronosyltransferase 1A1 and cytochrome P450 (CYP)-3A4, respectively, and it neither induces nor inhibits CYP isoenzymes. Thus dolutegravir has a modest drug interaction profile

  15. Integrase residues that determine nucleotide preferences at sites of HIV-1 integration: implications for the mechanism of target DNA binding

    PubMed Central

    Serrao, Erik; Krishnan, Lavanya; Shun, Ming-Chieh; Li, Xiang; Cherepanov, Peter; Engelman, Alan; Maertens, Goedele N.

    2014-01-01

    Retroviruses favor target-DNA (tDNA) distortion and particular bases at sites of integration, but the mechanism underlying HIV-1 selectivity is unknown. Crystal structures revealed a network of prototype foamy virus (PFV) integrase residues that distort tDNA: Ala188 and Arg329 interact with tDNA bases, while Arg362 contacts the phosphodiester backbone. HIV-1 integrase residues Ser119, Arg231, and Lys258 were identified here as analogs of PFV integrase residues Ala188, Arg329 and Arg362, respectively. Thirteen integrase mutations were analyzed for effects on integrase activity in vitro and during virus infection, yielding a total of 1610 unique HIV-1 integration sites. Purine (R)/pyrimidine (Y) dinucleotide sequence analysis revealed HIV-1 prefers the tDNA signature (0)RYXRY(4), which accordingly favors overlapping flexible dinucleotides at the center of the integration site. Consistent with roles for Arg231 and Lys258 in sequence specific and non-specific binding, respectively, the R231E mutation altered integration site nucleotide preferences while K258E had no effect. S119A and S119T integrase mutations significantly altered base preferences at positions −3 and 7 from the site of viral DNA joining. The S119A preference moreover mimicked wild-type PFV selectivity at these positions. We conclude that HIV-1 IN residue Ser119 and PFV IN residue Ala188 contact analogous tDNA bases to effect virus integration. PMID:24520116

  16. Development of a G118R mutation in HIV-1 integrase following a switch to dolutegravir monotherapy leading to cross-resistance to integrase inhibitors.

    PubMed

    Brenner, Bluma G; Thomas, Réjean; Blanco, José Luis; Ibanescu, Ruxandra-Ilinca; Oliveira, Maureen; Mesplède, Thibault; Golubkov, Olga; Roger, Michel; Garcia, Federico; Martinez, Esteban; Wainberg, Mark A

    2016-07-01

    Dolutegravir shows a high barrier to resistance with no previously reported cases of acquired integrase mutations during first-line therapy. In this study, rapid development of the G118R mutation arose following a switch from first-line elvitegravir/cobicistat/tenofovir disoproxil fumarate/emtricitabine to dolutegravir monotherapy. The G118R mutation also arose in a treatment-experienced patient switched to dolutegravir monotherapy. The genetic basis for G118R selection and potential phenotypic outcome was ascertained. Genotypic analysis was performed on patients with virological failure (<1000 copies/mL) on dolutegravir-containing regimens. The Los Alamos database was queried for glycine codon 118 polymorphisms. Cell culture selections and phenotypic drug susceptibility assays assessed resistance via the G118R pathway. We report on two patients who developed viral failure while on dolutegravir monotherapy. Both patients had been on a current or previous regimen containing integrase inhibitors. Virological failure (<1000 copies/mL) emerged early within 2 months following the dolutegravir switch. The appearance of G118R in these two cases and subtype C and CRF02_AG in vitro selections were related to a rare GGA natural polymorphism at codon 118 (1.5% prevalence), facilitating a GGA to AGA transition. Cell culture selections were used to assess the in vitro progression of the G118R pathway leading to cross-resistance to all integrase inhibitors. Although resistance to dolutegravir is typically rare, genetic polymorphisms and monotherapy can facilitate the acquisition of G118R. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  17. Mutations in nonconserved domains of Ty3 integrase affect multiple stages of the Ty3 life cycle.

    PubMed

    Nymark-McMahon, M H; Sandmeyer, S B

    1999-01-01

    Ty3, a retroviruslike element of Saccharomyces cerevisiae, transposes into positions immediately upstream of RNA polymerase III-transcribed genes. The Ty3 integrase (IN) protein is required for integration of the replicated, extrachromosomal Ty3 DNA. In retroviral IN, a conserved core region is sufficient for strand transfer activity. In this study, charged-to-alanine scanning mutagenesis was used to investigate the roles of the nonconserved amino- and carboxyl-terminal regions of Ty3 IN. Each of the 20 IN mutants was defective for transposition, but no mutant was grossly defective for capsid maturation. All mutations affecting steady-state levels of mature IN protein resulted in reduced levels of replicated DNA, even when polymerase activity was not grossly defective as measured by exogenous reverse transcriptase activity assay. Thus, IN could contribute to nonpolymerase functions required for DNA production in vivo or to the stability of the DNA product. Several mutations in the carboxyl-terminal domain resulted in relatively low levels of processed 3' ends of the replicated DNA, suggesting that this domain may be important for binding of IN to the long terminal repeat. Another class of mutants produced wild-type amounts of DNA with correctly processed 3' ends. This class could include mutants affected in nuclear entry and target association. Collectively, these mutations demonstrate that in vivo, within the preintegration complex, IN performs a central role in coordinating multiple late stages of the retrotransposition life cycle.

  18. Mutations in Nonconserved Domains of Ty3 Integrase Affect Multiple Stages of the Ty3 Life Cycle

    PubMed Central

    Nymark-McMahon, M. Henrietta; Sandmeyer, Suzanne B.

    1999-01-01

    Ty3, a retroviruslike element of Saccharomyces cerevisiae, transposes into positions immediately upstream of RNA polymerase III-transcribed genes. The Ty3 integrase (IN) protein is required for integration of the replicated, extrachromosomal Ty3 DNA. In retroviral IN, a conserved core region is sufficient for strand transfer activity. In this study, charged-to-alanine scanning mutagenesis was used to investigate the roles of the nonconserved amino- and carboxyl-terminal regions of Ty3 IN. Each of the 20 IN mutants was defective for transposition, but no mutant was grossly defective for capsid maturation. All mutations affecting steady-state levels of mature IN protein resulted in reduced levels of replicated DNA, even when polymerase activity was not grossly defective as measured by exogenous reverse transcriptase activity assay. Thus, IN could contribute to nonpolymerase functions required for DNA production in vivo or to the stability of the DNA product. Several mutations in the carboxyl-terminal domain resulted in relatively low levels of processed 3′ ends of the replicated DNA, suggesting that this domain may be important for binding of IN to the long terminal repeat. Another class of mutants produced wild-type amounts of DNA with correctly processed 3′ ends. This class could include mutants affected in nuclear entry and target association. Collectively, these mutations demonstrate that in vivo, within the preintegration complex, IN performs a central role in coordinating multiple late stages of the retrotransposition life cycle. PMID:9847351

  19. Simulink-Based Simulation Architecture for Evaluating Controls for Aerospace Vehicles (SAREC-ASV)

    NASA Technical Reports Server (NTRS)

    Christhilf, David m.; Bacon, Barton J.

    2006-01-01

    The Simulation Architecture for Evaluating Controls for Aerospace Vehicles (SAREC-ASV) is a Simulink-based approach to providing an engineering quality desktop simulation capability for finding trim solutions, extracting linear models for vehicle analysis and control law development, and generating open-loop and closed-loop time history responses for control system evaluation. It represents a useful level of maturity rather than a finished product. The layout is hierarchical and supports concurrent component development and validation, with support from the Concurrent Versions System (CVS) software management tool. Real Time Workshop (RTW) is used to generate pre-compiled code for substantial component modules, and templates permit switching seamlessly between original Simulink and code compiled for various platforms. Two previous limitations are addressed. Turn around time for incorporating tabular model components was improved through auto-generation of required Simulink diagrams based on data received in XML format. The layout was modified to exploit a Simulink "compile once, evaluate multiple times" capability for zero elapsed time for use in trimming and linearizing. Trim is achieved through a Graphical User Interface (GUI) with a narrow, script definable interface to the vehicle model which facilitates incorporating new models.

  20. Sorption of As(V) on aluminosilicates treated with Fe(II) nanoparticles.

    PubMed

    Dousová, Barbora; Grygar, Tomás; Martaus, Alexandr; Fuitová, Lucie; Kolousek, David; Machovic, Vladimír

    2006-10-15

    Adsorption of arsenic on clay surfaces is important for the natural and simulated removal of arsenic species from aqueous environments. In this investigation, three samples of clay minerals (natural metakaoline, natural clinoptilolite-rich tuff, and synthetic zeolite) in both untreated and Fe-treated forms were used for the sorption of arsenate from model aqueous solution. The treatment of minerals consisted of exposing them to concentrated solution of Fe(II). Within this process the mineral surface has been laden with Fe(III) oxi(hydroxides) whose high affinity for the As(V) adsorption is well known. In all investigated systems the sorption capacity of Fe(II)-treated sorbents increased significantly in comparison to the untreated material (from about 0.5 to >20.0 mg/g, which represented more than 95% of the total As removal). The changes of Fe-bearing particles in the course of treating process and subsequent As sorption were investigated by the diffuse reflectance spectroscopy and the voltammetry of microparticles. IR spectra of treated and As(V)-saturated solids showed characteristic bands caused by Fe(III)SO(4), Fe(III)O, and AsO vibrations. In untreated As(V)-saturated solids no significant AsO vibrations were observed due to the negligible content of sorbed arsenate.

  1. Removal of As(V) and Cr(VI) from aqueous solutions using solid waste from leather industry.

    PubMed

    Oliveira, Diana Q L; Gonçalves, Maraísa; Oliveira, Luiz C A; Guilherme, Luiz R G

    2008-02-28

    This study evaluated the feasibility of using a solid waste from the leather industry as an adsorbent for removal of Cr(VI) and As(V) from aqueous media. The adsorbent material was characterized by chemical analyses, infrared spectroscopy, and scanning electronic microscopy (SEM) prior to reaction in order to assess its surface properties. Langmuir and Freundlich equations were used for analyzing the experimental data, which showed a better fit to the Langmuir model, thus suggesting a monolayer adsorption process in the surface of the adsorbent. The high amounts of Cr(VI)-133 mg g(-1) and As(V)-26 mg g(-1) adsorbed demonstrates the great potential for using this solid waste from the leather industry as a low-cost alternative to the traditionally used adsorbent materials.

  2. Catechol-substituted L-chicoric acid analogues as HIV integrase inhibitors.

    PubMed

    Lee, Jae Yeol; Yoon, Kwon Joong; Lee, Yong Sup

    2003-12-15

    HIV integrase catalyzes the integration of HIV DNA copy into the host cell DNA, which is essential for the production of progeny viruses. L-Chicoric acid and dicaffeoylquinic acids, isolated from plants, are well known potent inhibitors of HIV integrase. The common structural features of these inhibitors are caffeic acid derivatives connected to tartaric acid or quinic acid through ester bonds. In the present study, we have synthesized and tested the inhibitory activities of a new type of HIV IN inhibitors, which has catechol groups in place of caffeoyl groups in the structure of L-chicoric acid. Upon substitution of catechol groups at succinic acid, pyrrole-dicarboxylic acid, maleimide or maleic anhydride, the inhibitory activities (IC(50)=3.8-23.6 microM) were retained or remarkably increased when compared to parent compound L-chicoric acid (IC(50)=13.7 microM).

  3. Cancer-specific binary expression system activated in mice by bacteriophage HK022 Integrase

    PubMed Central

    Elias, Amer; Spector, Itay; Sogolovsky-Bard, Ilana; Gritsenko, Natalia; Rask, Lene; Mainbakh, Yuli; Zilberstein, Yael; Yagil, Ezra; Kolot, Mikhail

    2016-01-01

    Binary systems based on site-specific recombination have been used for tumor specific transcription targeting of suicide genes in animal models. In these binary systems a site specific recombinase or integrase that is expressed from a tumor specific promoter drives tumor specific expression of a cytotoxic gene. In the present study we developed a new cancer specific binary expression system activated by the Integrase (Int) of the lambdoid phage HK022. We demonstrate the validity of this system by the specific expression of a luciferase (luc) reporter in human embryonic kidney 293T (HEK293T) cells and in a lung cancer mouse model. Due to the absence viral vectors and of cytotoxicity the Int based binary system offers advantages over previously described counterparts and may therefore be developed into a safer cancer cell killing system. PMID:27117628

  4. Behaviour of silica and florisil as solid supports in the removal process of as(v) from aqueous solutions.

    PubMed

    Gabor, Andreea; Davidescu, Corneliu Mircea; Negrea, Adina; Ciopec, Mihaela; Lupa, Lavinia

    2015-01-01

    In this study two solid supports, silica and florisil, were impregnated with crown ether (dibenzo-18-crown-6) and Fe(III) ions and their efficiency was compared in the adsorption process of As(V) from aqueous solutions. The solid supports were impregnated with crown ether due to their ability to build complexes with positives ions. Fe(III) was used because of As(V) affinity for it. The impregnated solid supports were characterized by energy dispersive X-ray analysis, scanning electron microscopy, Fourier transform infrared spectroscopy, and the specific surface area. The influence of the solid : liquid ratio on the adsorption process, kinetic studies for the pseudo-first-order and pseudo-second-order, and activation energy were studied. Thermodynamic studies as well as equilibrium studies were carried out. The obtained results showed that, from the two considered materials, impregnated silica presents a higher efficiency with a good selectivity, able to remove As(V) from aqueous solutions containing trace concentrations.

  5. Preclinical profile of BI 224436, a novel HIV-1 non-catalytic-site integrase inhibitor.

    PubMed

    Fenwick, Craig; Amad, Ma'an; Bailey, Murray D; Bethell, Richard; Bös, Michael; Bonneau, Pierre; Cordingley, Michael; Coulombe, René; Duan, Jianmin; Edwards, Paul; Fader, Lee D; Faucher, Anne-Marie; Garneau, Michel; Jakalian, Araz; Kawai, Stephen; Lamorte, Louie; LaPlante, Steven; Luo, Laibin; Mason, Steve; Poupart, Marc-André; Rioux, Nathalie; Schroeder, Patricia; Simoneau, Bruno; Tremblay, Sonia; Tsantrizos, Youla; Witvrouw, Myriam; Yoakim, Christiane

    2014-06-01

    BI 224436 is an HIV-1 integrase inhibitor with effective antiviral activity that acts through a mechanism that is distinct from that of integrase strand transfer inhibitors (INSTIs). This 3-quinolineacetic acid derivative series was identified using an enzymatic integrase long terminal repeat (LTR) DNA 3'-processing assay. A combination of medicinal chemistry, parallel synthesis, and structure-guided drug design led to the identification of BI 224436 as a candidate for preclinical profiling. It has antiviral 50% effective concentrations (EC50s) of <15 nM against different HIV-1 laboratory strains and cellular cytotoxicity of >90 μM. BI 224436 also has a low, ∼2.1-fold decrease in antiviral potency in the presence of 50% human serum and, by virtue of a steep dose-response curve slope, exhibits serum-shifted EC95 values ranging between 22 and 75 nM. Passage of virus in the presence of inhibitor selected for either A128T, A128N, or L102F primary resistance substitutions, all mapping to a conserved allosteric pocket on the catalytic core of integrase. BI 224436 also retains full antiviral activity against recombinant viruses encoding INSTI resistance substitutions N155S, Q148H, and E92Q. In drug combination studies performed in cellular antiviral assays, BI 224436 displays an additive effect in combination with most approved antiretrovirals, including INSTIs. BI 224436 has drug-like in vitro absorption, distribution, metabolism, and excretion (ADME) properties, including Caco-2 cell permeability, solubility, and low cytochrome P450 inhibition. It exhibited excellent pharmacokinetic profiles in rat (clearance as a percentage of hepatic flow [CL], 0.7%; bioavailability [F], 54%), monkey (CL, 23%; F, 82%), and dog (CL, 8%; F, 81%). Based on the excellent biological and pharmacokinetic profile, BI 224436 was advanced into phase 1 clinical trials.

  6. Efficacy and Tolerability of Integrase Inhibitors in Antiretroviral-Naive Patients.

    PubMed

    D'Abbraccio, Maurizio; Busto, Annunziata; De Marco, Mario; Figoni, Mario; Maddaloni, Adelaide; Abrescia, Nicola

    2015-01-01

    Integrase strand transfer inhibitors are a new class of antiretroviral agents recently licensed for the treatment of both naive and experienced HIV-infected patients. They inhibit the catalytic activity of the HIV-encoded enzyme integrase and prevent the integration of the HIV genome into the host cell genome, so slowing the propagation of the infection. Integrase strand transfer inhibitors cause a rapid drop in viral load, exhibit very low drug interactions (except elvitegravir/cobicistat), and have low pill burden and convenient dosing frequency. Drugs in this class have been compared to others in antiretroviral-naive patients with efavirenz and with protease inhibitors. Final results of the STARTMRK trial highlighted the better virologic and immunologic performance of raltegravir over efavirenz/emtricitabine/tenofovir disoproxil co-formulation. Raltegravir was also superior to atazanavir/ritonavir and darunavir/ritonavir in the ACTG 5257 study for the combined virologic/tolerability endpoint. Elvitegravir/cobicistat/emtricitabine/tenofovir was non-inferior to efavirenz/emtricitabine/tenofovir and to atazanavir/ritonavir plus emtricitabine/tenofovir in terms of confirmed virologic response in the GS-US-236-0102 and GS-US-236-0103 studies, respectively. Finally, dolutegravir showed non-inferiority compared to raltegravir in the SPRING-2 study and was superior to efavirenz and darunavir/ritonavir in the SINGLE and FLAMINGO trials, respectively. The aim of this review is to analyze the data on efficacy and safety of integrase strand transfer inhibitors in antiretroviral-naive HIV patients and discuss the strengths and weaknesses of drugs within this class.

  7. Concise and Practical Asymmetric Synthesis of a Challenging Atropisomeric HIV Integrase Inhibitor.

    PubMed

    Fandrick, Keith R; Li, Wenjie; Zhang, Yongda; Tang, Wenjun; Gao, Joe; Rodriguez, Sonia; Patel, Nitinchandra D; Reeves, Diana C; Wu, Jiang-Ping; Sanyal, Sanjit; Gonnella, Nina; Qu, Bo; Haddad, Nizar; Lorenz, Jon C; Sidhu, Kanwar; Wang, June; Ma, Shengli; Grinberg, Nelu; Lee, Heewon; Tsantrizos, Youla; Poupart, Marc-André; Busacca, Carl A; Yee, Nathan K; Lu, Bruce Z; Senanayake, Chris H

    2015-06-08

    A practical and efficient synthesis of a complex chiral atropisomeric HIV integrase inhibitor has been accomplished. The combination of a copper-catalyzed acylation along with the implementation of the BI-DIME ligands for a ligand-controlled Suzuki cross-coupling and an unprecedented bis(trifluoromethane)sulfonamide-catalyzed tert-butylation renders the synthesis of this complex molecule robust, safe, and economical. Furthermore, the overall synthesis was conducted in an asymmetric and diastereoselective fashion with respect to the imbedded atropisomer.

  8. Intermolecular interactions in the crystal structures of potential HIV-1 integrase inhibitors.

    PubMed

    Majerz-Maniecka, Katarzyna; Musiol, Robert; Nitek, Wojciech; Oleksyn, Barbara J; Mouscadet, Jean-Francois; Le Bret, Marc; Polanski, Jaroslaw

    2006-02-15

    2-[(2,5-dichloro-4-nitro-phenylamino)-methoxy-methyl]-8-hydroxy-quinoline 1 and 2-methyl-quinoline-5,8-dione-5-oxime 2 were obtained as potential HIV-1 integrase inhibitors and analyzed by X-ray crystallography. Semiempirical theoretical calculations of energy preferred conformations were also carried out. The crystal structures of both compounds are stabilized via hydrogen bonds and pi-pi stacking interactions. The planarity of compound 1 is caused by intramolecular hydrogen bonds.

  9. Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome

    PubMed Central

    2013-01-01

    Background Phage-encoded serine integrases, such as φC31 integrase, are widely used for genome engineering. Fifteen such integrases have been described but their utility for genome engineering has not been compared in uniform assays. Results We have compared fifteen serine integrases for their utility for DNA manipulations in mammalian cells after first demonstrating that all were functional in E. coli. Chromosomal recombination reporters were used to show that seven integrases were active on chromosomally integrated DNA in human fibroblasts and mouse embryonic stem cells. Five of the remaining eight enzymes were active on extra-chromosomal substrates thereby demonstrating that the ability to mediate extra-chromosomal recombination is no guide to ability to mediate site-specific recombination on integrated DNA. All the integrases that were active on integrated DNA also promoted DNA integration reactions that were not mediated through conservative site-specific recombination or damaged the recombination sites but the extent of these aberrant reactions varied over at least an order of magnitude. Bxb1 integrase yielded approximately two-fold more recombinants and displayed about two fold less damage to the recombination sites than the next best recombinase; φC31 integrase. Conclusions We conclude that the Bxb1 and φC31 integrases are the reagents of choice for genome engineering in vertebrate cells and that DNA damage repair is a major limitation upon the utility of this class of site-specific recombinase. PMID:24139482

  10. The Promise of Dolutegravir: A Novel Second Generation Integrase Strand Transfer Inhibitor.

    PubMed

    Singh, Harmanjit; Kaur, Mandeep; Kakkar, Ashish K; Kumar, Harish

    2016-01-01

    Dolutegravir (DTG), a novel integrase strand transfer inhibitor (INSTI), is one of the newest addition to the arsenal of anti-human immunodeficiency virus (HIV) therapeutics. Dolutegravir is the first member of the class of second generation integrase strand transfer inhibitors aimed primarily to address the current unmet need for novel unboosted integrase inhibitors with convenient once-daily dosing and a superior resistance profile. During its clinical development, DTG has demonstrated noninferior or superior efficacy in both treatment-naive as well as treatmentexperienced individuals including those who have previously failed first generation INSTIs. Other potential advantages include a favorable safety profile, low propensity for drug-drug interactions, and prolonged serum half-life permitting once-daily administration in treatment-naive or treatment-experienced INSTI naive HIV patients. Twice-daily administration is recommended in individuals with established or suspected resistance to first-generation INSTIs. This review outlines the need for new HIV therapeutics and summarizes the efficacy, safety, and pharmacokinetic profile of dolutegravir.

  11. Postexposure protection of macaques from vaginal SHIV infection by topical integrase inhibitors.

    PubMed

    Dobard, Charles; Sharma, Sunita; Parikh, Urvi M; West, Rolieria; Taylor, Andrew; Martin, Amy; Pau, Chou-Pong; Hanson, Debra L; Lipscomb, Jonathan; Smith, James; Novembre, Francis; Hazuda, Daria; Garcia-Lerma, J Gerardo; Heneine, Walid

    2014-03-12

    Coitally delivered microbicide gels containing antiretroviral drugs are important for HIV prevention. However, to date, microbicides have contained entry or reverse transcriptase inhibitors that block early steps in virus infection and thus need to be given as a preexposure dose that interferes with sexual practices and may limit compliance. Integrase inhibitors block late steps after virus infection and therefore are more suitable for post-coital dosing. We first determined the kinetics of strand transfer in vitro and confirmed that integration begins about 6 hours after infection. We then used a repeat-challenge macaque model to assess efficacy of vaginal gels containing integrase strand transfer inhibitors when applied before or after simian/human immunodeficiency virus (SHIV) challenge. We showed that gel containing the strand transfer inhibitor L-870812 protected two of three macaques when applied 30 min before SHIV challenge. We next evaluated the efficacy of 1% raltegravir gel and demonstrated its ability to protect macaques when applied 3 hours after SHIV exposure (five of six protected; P < 0.05, Fisher's exact test). Breakthrough infections showed no evidence of drug resistance in plasma or vaginal secretions despite continued gel dosing after infection. We documented rapid vaginal absorption reflecting a short pharmacological lag time and noted that vaginal, but not plasma, virus load was substantially reduced in the breakthrough infection after raltegravir gel treatment. We provide a proof of concept that topically applied integrase inhibitors protect against vaginal SHIV infection when administered shortly before or 3 hours after virus exposure.

  12. Structural Properties of HIV Integrase. Lens Epithelium-derived Growth Factor Oligomers

    SciTech Connect

    Gupta, K.; Diamond, T; Hwang, Y; Bushman, F; Van Duyne, G

    2010-01-01

    Integrase (IN) is the catalytic component of the preintegration complex, a large nucleoprotein assembly critical for the integration of the retroviral genome into a host chromosome. Although partial crystal structures of human immunodeficiency virus IN alone and its complex with the integrase binding domain of the host factor PSIP1/lens epithelium-derived growth factor (LEDGF)/p75 are available, many questions remain regarding the properties and structures of LEDGF-bound IN oligomers. Using analytical ultracentrifugation, multiangle light scattering, and small angle x-ray scattering, we have established the oligomeric state, stoichiometry, and molecular shapes of IN {center_dot} LEDGF complexes in solution. Analyses of intact IN tetramers bound to two different LEDGF truncations allow for placement of the integrase binding domain by difference analysis. Modeling of the small angle x-ray scattering envelopes using existing structural data suggests domain arrangements in the IN oligomers that support and extend existing biochemical data for IN {center_dot} LEDGF complexes and lend new insights into the quaternary structure of LEDGF-bound IN tetramers. These IN oligomers may be involved in stages of the viral life cycle other than integration, including assembly, budding, and early replication.

  13. Sorption of lindane to river biofilms, suspended particles and sediments in the presence and absence of Cr(VI) and As(V)

    NASA Astrophysics Data System (ADS)

    Guo, Z.; Dong, D.; Hua, X.; Lan, X.; Liang, D.

    2013-12-01

    Few study has focused on the effects of Cr(VI) and As(V) which are often in the form of anionic groups in natural aquatic environment on the sorption of dissolved hydrophobic organic pollutants (HOPs) onto natural solid materials from water. In this study, we compared the sorption thermodynamics of lindene as a model dissolved HOP on biofilms, suspended particles and surface sediments collected from the same river in the presence and absence of Cr(VI) and As(V). Visual MINTEQ (version 3.0) was applied to calculate Cr(VI) and As(V) species in the sorption system. CrO42- (89%) was the main Cr(VI) species and HAsO42- (78%) and H2AsO4- (22%) were the As(V) species. Compared with Freundlich and the dual reactive domain model, linear model gave a better fit for the lindane sorption isotherms on the biofilms, particles and sediments (Fig.1). For the lindane sorption on the biofilms, the linear model distribution coefficient (KL) were reduced by 48.0% and 47.6% in the presence of Cr(VI) and As(V), respectively; For that on the suspended particles, KL were reduced by 6.2% and 10.8%, respectively; For the sediments, KL were reduced by 30.2% and 34.5%, respectively. In general, the presence of Cr(VI) and As(V) inhibited lindane sorption onto the three solids and inhibition level decreased in the order biofilms > sediments > particles. In contrast to the promotion of metal cations on the dissolved HOPs sorption onto natural solid materials, anionic Cr(VI) and As(V) groups suppressed the sorption. This indicated that anionic Cr(VI) and As(V) groups might occupy some sorption sites on solids and then occurred competitive sorption with lindane. Taking account of the errors in experiment, effects on the sorption between Cr(VI) and As(V) has no significant difference. This shown that the type of metal has little effect on the HOPs sorption onto natural solids. This result was consistent with that in other related studis. In addition, we inferred that the differences of component

  14. Molecular modeling study on the allosteric inhibition mechanism of HIV-1 integrase by LEDGF/p75 binding site inhibitors.

    PubMed

    Xue, Weiwei; Liu, Huanxiang; Yao, Xiaojun

    2014-01-01

    HIV-1 integrase (IN) is essential for the integration of viral DNA into the host genome and an attractive therapeutic target for developing antiretroviral inhibitors. LEDGINs are a class of allosteric inhibitors targeting LEDGF/p75 binding site of HIV-1 IN. Yet, the detailed binding mode and allosteric inhibition mechanism of LEDGINs to HIV-1 IN is only partially understood, which hinders the structure-based design of more potent anti-HIV agents. A molecular modeling study combining molecular docking, molecular dynamics simulation, and binding free energy calculation were performed to investigate the interaction details of HIV-1 IN catalytic core domain (CCD) with two recently discovered LEDGINs BI-1001 and CX14442, as well as the LEDGF/p75 protein. Simulation results demonstrated the hydrophobic domain of BI-1001 and CX14442 engages one subunit of HIV-1 IN CCD dimer through hydrophobic interactions, and the hydrophilic group forms hydrogen bonds with HIV-1 IN CCD residues from other subunit. CX14442 has a larger tert-butyl group than the methyl of BI-1001, and forms better interactions with the highly hydrophobic binding pocket of HIV-1 IN CCD dimer interface, which can explain the stronger affinity of CX14442 than BI-1001. Analysis of the binding mode of LEDGF/p75 with HIV-1 IN CCD reveals that the LEDGF/p75 integrase binding domain residues Ile365, Asp366, Phe406 and Val408 have significant contributions to the binding of the LEDGF/p75 to HIV1-IN. Remarkably, we found that binding of BI-1001 and CX14442 to HIV-1 IN CCD induced the structural rearrangements of the 140 s loop and oration displacements of the side chains of the three conserved catalytic residues Asp64, Asp116, and Glu152 located at the active site. These results we obtained will be valuable not only for understanding the allosteric inhibition mechanism of LEDGINs but also for the rational design of allosteric inhibitors of HIV-1 IN targeting LEDGF/p75 binding site.

  15. Impact of wastewater derived dissolved organic carbon on reduction, mobility, and bioavailability of As(V) and Cr(VI) in contaminated soils.

    PubMed

    Kunhikrishnan, Anitha; Choppala, Girish; Seshadri, Balaji; Wijesekara, Hasintha; Bolan, Nanthi S; Mbene, Kenneth; Kim, Won-Il

    2017-01-15

    In this work, the effects of various wastewater sources (storm water, sewage effluent, piggery effluent, and dairy effluent) on the reduction, and subsequent mobility and bioavailability of arsenate [As(V)] and chromate [Cr(VI)] were compared using both spiked and field contaminated soils. Wastewater addition to soil can increase the supply of carbon, nutrients, and stimulation of microorganisms which are considered to be important factors enhancing the reduction of metal(loid)s including As and Cr. The wastewater-induced mobility and bioavailability of As(V) and Cr(VI) were examined using leaching, earthworm, and soil microbial activity tests. The rate of reduction of As(V) was much less than that of Cr(VI) both in the presence and absence of wastewater addition. Wastewater addition increased the reduction of both As(V) and Cr(VI) compared to the control (Milli-Q water) and the effect was more pronounced in the case of Cr(VI). The leaching experiment indicated that Cr(VI) was more mobile than As(V). Wastewater addition increased the mobility and bioavailability of As(V), but had an opposite effect on Cr(VI). The difference in the mobility and bioavailability of Cr(VI) and As(V) between wastewater sources can be attributed to the difference in their dissolved organic carbon (DOC) content. The DOC provides carbon as an electron donor for the reduction of As(V) and Cr(VI) and also serves as a complexing agent thereby impacting their mobility and bioavailability. The DOC-induced reduction increased both the mobility and bioavailability of As, but it caused an opposite effect in the case of Cr.

  16. Organo-modified sericite in the remediation of an aquatic environment contaminated with As(III) or As(V).

    PubMed

    Lee, Seung Mok; Tiwari, Diwakar

    2014-01-01

    The aim of this investigation was to obtain the hybrid material precursor to the naturally and abundantly available sericite, a mica-based clay; the materials were further employed in the remediation of arsenic from aqueous solutions. The study was intended to provide a cost-effective and environmentally benign treatment technology. The hybrid organo-modified sericite was obtained using hexadecyltrimethylammonium bromide (HDTMA) and alkyldimethylbenzylammonium chloride (AMBA) organic surfactants by introducing regulated doses of HDTMA or AMBA. The materials were characterized using infrared and X-ray diffraction analytical data, whereas the surface morphology was discussed by taking its SEM images. These materials were employed to assess the pre-concentration and speciation of As(III) and As(V) from aqueous solutions. The batch reactor data showed that increasing the sorptive concentration (from 1.0 to 15.0 mg/L) and pH (i.e., pH 2.0 to 10.0) caused the percent uptake of As(III) and As(V) to decrease significantly. The kinetic data showed that a sharp initial uptake of arsenic reached its equilibrium state within about 50 min of contact time, and the sorption kinetics followed a pseudo-second-order rate law both for As(III) and As(V) sorption. A 1,000 times increase in the background electrolyte concentration, i.e., NaNO3, caused a significant decrease in As(III) removal, whereas As(V) was almost unaffected, which inferred that As(III) was adsorbed, mainly by the van der Waals or even by the electrostatic attraction, whereas As(V) was adsorbed chemically and formed "inner-sphere" complexes at the solid/solution interface. The equilibrium state modeling studies indicated that the sorption data fitted well the Freundlich and Langmuir adsorption isotherms. Henceforth, the removal capacity was calculated under these equilibrium conditions. It was noted that organo-modified sericite possessed a significantly higher removal capacity compared to its virgin sericite

  17. Advances in Shallow-Water, High-Resolution Seafloor Mapping: Integrating an Autonomous Surface Vessel (ASV) Into Nearshore Geophysical Studies

    NASA Astrophysics Data System (ADS)

    Denny, J. F.; O'Brien, T. F.; Bergeron, E.; Twichell, D.; Worley, C. R.; Danforth, W. W.; Andrews, B. A.; Irwin, B.

    2006-12-01

    The U.S. Geological Survey (USGS) has been heavily involved in geological mapping of the seafloor since the 1970s. Early mapping efforts such as GLORIA provided broad-scale imagery of deep waters (depths > 400 meters) within the Exclusive Economic Zone (EEZ). In the early 1990's, the USGS research emphasis shifted from deep- to shallow-water environments (inner continental shelf, nearshore, estuaries) to address pertinent coastal issues such as erosion, sediment availability, sediment transport, vulnerability of coastal areas to natural and anthropogenic hazards, and resource management. Geologic framework mapping in these shallow- water environments has provided valuable data used to 1) define modern sediment distribution and thickness, 2) determine underlying stratigraphic and structural controls on shoreline behavior, and 3) enable onshore-to- offshore geologic mapping within the coastal zone when coupled with subaerial techniques such as GPR and topographic LIDAR. Research in nearshore areas presents technological challenges due to the dynamics of the environment, high volume of data collected, and the geophysical limitations of operating in very shallow water. In 2004, the USGS, in collaboration with NOAA's Coastal Services Center, began a multi-year seafloor mapping effort to better define oyster habitats within Apalachicola Bay, Florida, a shallow water estuary along the northern Gulf of Mexico. The bay poses a technological challenge due to its shallow depths (< 4-m) and high turbidity that prohibits the use of bathymetric LIDAR. To address this extreme shallow water setting, the USGS incorporated an Autonomous Surface Vessel (ASV) into seafloor mapping operations, in June 2006. The ASV is configured with a chirp sub-bottom profiler (4 24 kHz), dual-frequency chirp sidescan-sonar (100/500 kHz), single-beam echosounder (235 kHz), and forward-looking digital camera, and will be used to delineate the distribution and thickness of surficial sediment, presence

  18. Synthesis, characterization and adsorptive properties of carbon with iron nanoparticles and iron carbide for the removal of As(V) from water.

    PubMed

    Gutierrez-Muñiz, O E; García-Rosales, G; Ordoñez-Regil, E; Olguin, M T; Cabral-Prieto, A

    2013-01-15

    This manuscript presents the synthesis of carbon modified with iron nanoparticles (CFe) and iron carbide (CarFe) from the pyrolyzed crown leaves of pineapple (Ananas comosus) treated with iron salts. The materials that were obtained were used for the removal of As(V) from aqueous media. The carbonaceous materials were characterized by Scanning Electron Microscopy (SEM), Transmission electron microscopy (TEM), X-ray diffraction (XRD), X-Ray Photoelectron Spectroscopy (XPS) and Mossbauer Spectroscopy. The specific area (BET), number site density and point of zero charge (pH(pzc)) were also determined. The kinetic parameters were obtained by fitting the experimental data to the pseudo-first-order and pseudo-second-order models. Different isotherm models were applied to describe the As(V) adsorption behavior. The kinetics of As(V) sorption by CFe and CarFe was well defined for the pseudo-second-order model (R(2) = 0.9994 and 0.999, respectively). The maximum As(V) uptake was 1.8 mg g(-1) for CFe and 1.4 mg g(-1) for CarFe. The results obtained indicated that both materials are equally useful for As(V) sorption. The As(V) experimental isotherm data were described by the Freundlich model for CFe and CarFe.

  19. Preconcentration determination of arsenic species by sorption of As(V) on Amberlite IRA-410 coupled with fluorescence quenching of L-cysteine capped CdS nanoparticles.

    PubMed

    Hosseini, Mohammad Saeid; Nazemi, Sahar

    2013-10-07

    A simple and accurate method for arsenic speciation analysis in natural and drinking water samples is described in which preconcentration of arsenic as As(V) was coupled with spectrofluorometric determination. The extracted As(V) species with a column containing Amberlite IRA-410 were subjected to L-cysteine capped CdS quantum dots (QDs) and the fluorescence quenching of the QDs due to reduction of As(V) by L-cysteine was considered as a signal relevant to As(V) concentration. The As(III) species were also determined after oxidation of As(III) ions to As(V) with H2O2 and measurement of the total arsenic content. In treatment with 400 mL portions of water samples containing 30 μg L(-1) As(V), the relative standard deviation was 2.8%. The detection limit of arsenic was also found to be 0.75 μg L(-1) (1 × 10(-8) M). The reliability of proposed method was confirmed using certified reference materials. The trace amounts of arsenic species were then determined in different water samples, satisfactorily.

  20. Comparison of Fe-Al-modified natural materials by an electrochemical method and chemical precipitation for the adsorption of F- and As(V).

    PubMed

    Vázquez Mejía, G; Martínez-Miranda, V; Fall, C; Linares-Hernández, I; Solache-Ríos, M

    2016-01-01

    The adsorption of fluoride and arsenic ions by modified natural materials may have an impact on the removal of F- and As(V) from waters. In this work, a zeolitic material and pozzolan (commonly known as pumicite) were modified with aluminium an iron by an electrochemical method and chemical precipitation, respectively. The adsorbents were characterized by X-ray diffraction, scanning electron microscopy with energy X-ray disperse spectroscopy analysis and the point of zero charge (pHzpc). F- and As(V) adsorption properties of both materials were investigated. Adsorption kinetic data were best fitted to pseudo-second-order model and equilibrium data to the Langmuir isotherm model. The highest F- and As(V) sorption capacities were obtained for modified zeolitic (0.866 mg/g) and pozzolan (3.35 mg/g) materials, respectively, with initial F- or As(V) concentrations of 10 mg/L. It was found that the unmodified materials did not show either adsorption of F- ions or As(V), which indicated that Al and Fe in the adsorbents are responsible for the adsorption of these ions. In general, both modified materials show similar capacities for the adsorption of F- and As(V).

  1. Integrase-independent HIV-1 infection is augmented under conditions of DNA damage and produces a viral reservoir

    SciTech Connect

    Ebina, Hirotaka Kanemura, Yuka; Suzuki, Yasutsugu; Urata, Kozue; Misawa, Naoko; Koyanagi, Yoshio

    2012-05-25

    HIV-1 possesses a viral protein, integrase (IN), which is necessary for its efficient integration in target cells. However, it has been reported that an IN-defective HIV strain is still capable of integration. Here, we assessed the ability of wild type (WT) HIV-1 to establish infection in the presence of IN inhibitors. We observed a low, yet clear infection of inhibitor-incubated cells infected with WT HIV which was identical to cells infected with IN-deficient HIV, D64A. Furthermore, the IN-independent integration could be enhanced by the pretreatment of cells with DNA-damaging agents suggesting that integration is mediated by a DNA repair system. Moreover, significantly faster viral replication kinetics with augmented viral DNA integration was observed after infection in irradiated cells treated with IN inhibitor compared to nonirradiated cells. Altogether, our results suggest that HIV DNA has integration potential in the presence of an IN inhibitor and may serve as a virus reservoir.

  2. Adeno-associated virus Rep-mediated targeting of integrase-defective retroviral vector DNA circles into human chromosome 19

    SciTech Connect

    Huang, Shuohao; Kawabe, Yoshinori; Ito, Akira; Kamihira, Masamichi

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Adeno-associated virus (AAV) is capable of targeted integration in human cells. Black-Right-Pointing-Pointer Integrase-defective retroviral vector (IDRV) enables a circular DNA delivery. Black-Right-Pointing-Pointer A targeted integration system of IDRV DNA using the AAV integration mechanism. Black-Right-Pointing-Pointer Targeted IDRV integration ameliorates the safety concerns for retroviral vectors. -- Abstract: Retroviral vectors have been employed in clinical trials for gene therapy owing to their relative large packaging capacity, alterable cell tropism, and chromosomal integration for stable transgene expression. However, uncontrollable integrations of transgenes are likely to cause safety issues, such as insertional mutagenesis. A targeted transgene integration system for retroviral vectors, therefore, is a straightforward way to address the insertional mutagenesis issue. Adeno-associated virus (AAV) is the only known virus capable of targeted integration in human cells. In the presence of AAV Rep proteins, plasmids possessing the p5 integration efficiency element (p5IEE) can be integrated into the AAV integration site (AAVS1) in the human genome. In this report, we describe a system that can target the circular DNA derived from non-integrating retroviral vectors to the AAVS1 site by utilizing the Rep/p5IEE integration mechanism. Our results showed that after G418 selection 30% of collected clones had retroviral DNA targeted at the AAVS1 site.

  3. Four-tiered pi interaction at the dimeric interface of HIV-1 integrase critical for DNA integration and viral infectivity.

    PubMed

    Al-Mawsawi, Laith Q; Hombrouck, Anneleen; Dayam, Raveendra; Debyser, Zeger; Neamati, Nouri

    2008-08-01

    HIV-1 integrase (IN) is an essential enzyme for viral infection. Here, we report an extensive pi electron orbital interaction between four amino acids, W132, M178, F181 and F185, located at the dimeric interface of IN that is critical for the strand transfer activity alone. Catalysis of nine different mutant IN proteins at these positions were evaluated. Whereas the 3'-processing activity is predominantly strong, the strand transfer activity of each enzyme was completely dependent on an intact pi electron orbital interaction at the dimeric interface. Four representative IN mutants were constructed in the context of the infectious NL4.3 HIV-1 viral clone. Whereas viruses with an intact pi electron orbital interaction at the IN dimeric interface replicated comparable to wild type, viruses containing an abolished pi interaction were non-infectious. Q-PCR analysis of viral DNA forms during viral replication revealed pleiotropic effects of most mutations. We hypothesize that the pi interaction is a critical contact point for the assembly of functional IN multimeric complexes, and that IN multimerization is required for a functional pre-integration complex. The rational design of small molecule inhibitors targeting the disruption of this pi-pi interaction should lead to powerful anti-retroviral drugs.

  4. Four-tiered {pi} interaction at the dimeric interface of HIV-1 integrase critical for DNA integration and viral infectivity

    SciTech Connect

    Al-Mawsawi, Laith Q.; Hombrouck, Anneleen; Dayam, Raveendra; Debyser, Zeger; Neamati, Nouri

    2008-08-01

    HIV-1 integrase (IN) is an essential enzyme for viral infection. Here, we report an extensive {pi} electron orbital interaction between four amino acids, W132, M178, F181 and F185, located at the dimeric interface of IN that is critical for the strand transfer activity alone. Catalysis of nine different mutant IN proteins at these positions were evaluated. Whereas the 3'-processing activity is predominantly strong, the strand transfer activity of each enzyme was completely dependent on an intact {pi} electron orbital interaction at the dimeric interface. Four representative IN mutants were constructed in the context of the infectious NL4.3 HIV-1 viral clone. Whereas viruses with an intact {pi} electron orbital interaction at the IN dimeric interface replicated comparable to wild type, viruses containing an abolished {pi} interaction were non-infectious. Q-PCR analysis of viral DNA forms during viral replication revealed pleiotropic effects of most mutations. We hypothesize that the {pi} interaction is a critical contact point for the assembly of functional IN multimeric complexes, and that IN multimerization is required for a functional pre-integration complex. The rational design of small molecule inhibitors targeting the disruption of this {pi}-{pi} interaction should lead to powerful anti-retroviral drugs.

  5. Mapping the λ Integrase bridges in the nucleoprotein Holliday junction intermediates of viral integrative and excisive recombination.

    PubMed

    Tong, Wenjun; Warren, David; Seah, Nicole E; Laxmikanthan, Gurunathan; Van Duyne, Gregory D; Landy, Arthur

    2014-08-26

    The site-specific recombinase encoded by bacteriophage λ [λ Integrase (Int)] is responsible for integrating and excising the viral chromosome into and out of the chromosome of its Escherichia coli host. In contrast to the other well-studied and highly exploited tyrosine recombinase family members, such as Cre and Flp, Int carries out a reaction that is highly directional, tightly regulated, and depends on an ensemble of accessory DNA bending proteins acting on 240 bp of DNA encoding 16 protein binding sites. This additional complexity enables two pathways, integrative and excisive recombination, whose opposite, and effectively irreversible, directions are dictated by different physiological and environmental signals. Int recombinase is a heterobivalent DNA binding protein that binds via its small amino-terminal domain to high affinity arm-type DNA sites and via its large, compound carboxyl-terminal domain to core-type DNA sites, where DNA cleavage and ligation are executed. Each of the four Int protomers, within a multiprotein 400-kDa recombinogenic complex, is thought to bind and, with the aid of DNA bending proteins, bridge one arm- and one core-type DNA site. Despite a wealth of genetic, biochemical, and functional information generated by many laboratories over the last 50 y, it has not been possible to decipher the patterns of Int bridges, an essential step in understanding the architectures responsible for regulated directionality of recombination. We used site-directed chemical cross-linking of Int in trapped Holliday junction recombination intermediates and recombination reactions with chimeric recombinases, to identify the unique and monogamous patterns of Int bridges for integrative and excisive recombination.

  6. Simian Virus 40-Based Replication of Catalytically Inactive Human Immunodeficiency Virus Type 1 Integrase Mutants in Nonpermissive T Cells and Monocyte-Derived Macrophages

    PubMed Central

    Lu, Richard; Nakajima, Noriko; Hofmann, Wolfgang; Benkirane, Monsef; Teh-Jeang, Kuan; Sodroski, Joseph; Engelman, Alan

    2004-01-01

    Integrase function is required for retroviral replication in most instances. Although certain permissive T-cell lines support human immunodeficiency virus type 1 (HIV-1) replication in the absence of functional integrase, most cell lines and primary human cells are nonpermissive for integrase mutant growth. Since unintegrated retroviral DNA is lost from cells following cell division, we investigated whether incorporating a functional origin of DNA replication into integrase mutant HIV-1 might overcome the block to efficient gene expression and replication in nonpermissive T-cell lines and primary cells. Whereas the Epstein-Barr virus (EBV) origin (oriP) did little to augment expression from an integrase mutant reporter virus in EBV nuclear antigen 1-expressing cells, simian virus 40 (SV40) oriT dramatically enhanced integrase mutant infectivity in T-antigen (Tag)-expressing cells. Incorporating oriT into the nef position of a full-length, integrase-defective virus strain yielded efficient replication in Tag-expressing nonpermissive Jurkat T cells without reversion to an integration-competent genotype. Adding Tag to integrase mutant-oriT viruses yielded 11.3-kb SV40-HIV chimeras that replicated in Jurkat cells and primary monocyte-derived macrophages. Real-time quantitative PCR analyses of Jurkat cell infections revealed that amplified copies of unintegrated DNA likely contributed to SV40-HIV integrase mutant replication. SV40-based HIV-1 integrase mutant replication in otherwise nonpermissive cells suggests alternative approaches to standard integrase-mediated retroviral gene transfer strategies. PMID:14694097

  7. Dicaffeoyltartaric acid analogues inhibit human immunodeficiency virus type 1 (HIV-1) integrase and HIV-1 replication at nontoxic concentrations.

    PubMed

    Reinke, Ryan A; King, Peter J; Victoria, Joseph G; McDougall, Brenda R; Ma, Guoxiang; Mao, Yingqun; Reinecke, Manfred G; Robinson, W Edward

    2002-08-15

    The human immunodeficiency virus type 1 (HIV-1) is a major health problem worldwide. In this study, 17 analogues of L-chicoric acid, a potent inhibitor of HIV integrase, were studied. Of these analogues, five submicromolar inhibitors of integrase were discovered and 13 compounds with activity against integrase at less than 10 microM were identified. Six demonstrated greater than 10-fold selectivity for HIV replication over cellular toxicity. Ten analogues inhibited HIV replication at nontoxic concentrations. Alteration of the linkages between the two bis-catechol rings, including the use of amides, mixed amide esters, cholate, and alkyl bridges, was explored. Amides were as active as esters but were more toxic in tissue culture. Alkyl and cholate bridges were significantly less potent against HIV-1 integrase in vitro and were inactive against HIV-1 replication. Two amino acid derivates and one digalloylderivative of L-chicoric acid (L-CA) showed improved selectivity over L-CA against integration in cell culture. These data suggest that in addition to the bis-catechols and free carboxylic acid groups reported previously, polar linkages are important constituents for optimal activity against HIV-1 integrase and that new derivatives can be developed with increased specificity for integration over HIV entry in vivo.

  8. HIV Type 1 genetic diversity and naturally occurring polymorphisms in HIV type 1 Kenyan isolates: implications for integrase inhibitors.

    PubMed

    Nyamache, Anthony Kebira; Muigai, Anne W T; Nganga, Zipporah; Khamadi, Samoel A

    2012-08-01

    Little is known about the extent and predictors of polymorphisms potentially influencing susceptibility to HIV integrase inhibitors. HIV-1 genetic diversity and drug resistance are major challenges in patient management globally. To evaluate HIV genetic diversity and drug resistance-associated mutations within a Nairobi cohort, genetic analysis of the HIV-1 pol-integrase gene regions was performed on samples collected from 42 subjects and 113 Kenyan intergrase sequences deposited in the Los Alamos HIV database. From the partial pol-integrase sequences analyzed phylogenetically, it was shown that 26 (61.9%) were subtype A1, 9 (21.4%) were subtype D, 5 (11.9%) were subtype C, 1 (2.4%) was subtype A2 and 1 (2.4%) was subtype CRF. Integrase-associated mutations were found in 12 patients, and in all 113 sequences already deposited in GenBank. One sample from this study and five from previous Kenyan integrase sequences had mutations at T97A, which is associated with reduced susceptibility to raltegravir.

  9. Site-specific recombination system based on actinophage TG1 integrase for gene integration into bacterial genomes.

    PubMed

    Hirano, Nobutaka; Muroi, Tetsurou; Kihara, Yoshihiko; Kobayashi, Ryuichi; Takahashi, Hideo; Haruki, Mitsuru

    2011-03-01

    Phage integrases are enzymes that catalyze unidirectional site-specific recombination between the attachment sites of phage and host bacteria, attP and attB, respectively. We recently developed an in vivo intra-molecular site-specific recombination system based on actinophage TG1 serine-type integrase that efficiently acts between attP and attB on a single plasmid DNA in heterologous Escherichia coli cells. Here, we developed an in vivo inter-molecular site-specific recombination system that efficiently acted between the att site on exogenous non-replicative plasmid DNA and the corresponding att site on endogenous plasmid or genomic DNA in E. coli cells, and the recombination efficiencies increased by a factor of ~10(1-3) in cells expressing TG1 integrase over those without. Moreover, integration of attB-containing incoming plasmid DNA into attP-inserted E. coli genome was more efficient than that of the reverse substrate configuration. Together with our previous result that purified TG1 integrase functions efficiently without auxiliary host factors in vitro, these in vivo results indicate that TG1 integrase may be able to introduce attB-containing circular DNAs efficiently into attP-inserted genomes of many bacterial species in a site-specific and unidirectional manner. This system thus may be beneficial to genome engineering for a wide variety of bacterial species.

  10. Peptides derived from the HIV-1 integrase promote HIV-1 infection and multi-integration of viral cDNA in LEDGF/p75-knockdown cells

    PubMed Central

    2010-01-01

    Background The presence of the cellular Lens Epithelium Derived Growth Factor p75 (LEDGF/p75) protein is essential for integration of the Human immunodeficiency virus type 1 (HIV-1) cDNA and for efficient virus production. In the absence of LEDGF/p75 very little integration and virus production can be detected, as was demonstrated using LEDGF/p75-knokdown cells. Results Here we show that the failure to infect LEDGF/p75-knockdown cells has another reason aside from the lack of LEDGF/p75. It is also due to inhibition of the viral integrase (IN) enzymatic activity by an early expressed viral Rev protein. The formation of an inhibitory Rev-IN complex in virus-infected cells can be disrupted by the addition of three IN-derived, cell-permeable peptides, designated INr (IN derived-Rev interacting peptides) and INS (IN derived-integrase stimulatory peptide). The results of the present work confirm previous results showing that HIV-1 fails to infect LEDGF/p75-knockdown cells. However, in the presence of INrs and INS peptides, relatively high levels of viral cDNA integration as well as productive virus infection were obtained following infection by a wild type (WT) HIV-1 of LEDGF/p75-knockdown cells. Conclusions It appears that the lack of integration observed in HIV-1 infected LEDGF/p75-knockdown cells is due mainly to the inhibitory effect of Rev following the formation of a Rev-IN complex. Disruption of this inhibitory complex leads to productive infection in those cells. PMID:20678206

  11. The allosteric HIV-1 integrase inhibitor BI-D affects virion maturation but does not influence packaging of a functional RNA genome.

    PubMed

    van Bel, Nikki; van der Velden, Yme; Bonnard, Damien; Le Rouzic, Erwann; Das, Atze T; Benarous, Richard; Berkhout, Ben

    2014-01-01

    The viral integrase (IN) is an essential protein for HIV-1 replication. IN inserts the viral dsDNA into the host chromosome, thereby aided by the cellular co-factor LEDGF/p75. Recently a new class of integrase inhibitors was described: allosteric IN inhibitors (ALLINIs). Although designed to interfere with the IN-LEDGF/p75 interaction to block HIV DNA integration during the early phase of HIV-1 replication, the major impact was surprisingly found on the process of virus maturation during the late phase, causing a reverse transcription defect upon infection of target cells. Virus particles produced in the presence of an ALLINI are misformed with the ribonucleoprotein located outside the virus core. Virus assembly and maturation are highly orchestrated and regulated processes in which several viral proteins and RNA molecules closely interact. It is therefore of interest to study whether ALLINIs have unpredicted pleiotropic effects on these RNA-related processes. We confirm that the ALLINI BI-D inhibits virus replication and that the produced virus is non-infectious. Furthermore, we show that the wild-type level of HIV-1 genomic RNA is packaged in virions and these genomes are in a dimeric state. The tRNAlys3 primer for reverse transcription was properly placed on this genomic RNA and could be extended ex vivo. In addition, the packaged reverse transcriptase enzyme was fully active when extracted from virions. As the RNA and enzyme components for reverse transcription are properly present in virions produced in the presence of BI-D, the inhibition of reverse transcription is likely to reflect the mislocalization of the components in the aberrant virus particle.

  12. Structure of the Brd4 ET domain bound to a C-terminal motif from γ-retroviral integrases reveals a conserved mechanism of interaction.

    PubMed

    Crowe, Brandon L; Larue, Ross C; Yuan, Chunhua; Hess, Sonja; Kvaratskhelia, Mamuka; Foster, Mark P

    2016-02-23

    The bromodomain and extraterminal domain (BET) protein family are promising therapeutic targets for a range of diseases linked to transcriptional activation, cancer, viral latency, and viral integration. Tandem bromodomains selectively tether BET proteins to chromatin by engaging cognate acetylated histone marks, and the extraterminal (ET) domain is the focal point for recruiting a range of cellular and viral proteins. BET proteins guide γ-retroviral integration to transcription start sites and enhancers through bimodal interaction with chromatin and the γ-retroviral integrase (IN). We report the NMR-derived solution structure of the Brd4 ET domain bound to a conserved peptide sequence from the C terminus of murine leukemia virus (MLV) IN. The complex reveals a protein-protein interaction governed by the binding-coupled folding of disordered regions in both interacting partners to form a well-structured intermolecular three-stranded β sheet. In addition, we show that a peptide comprising the ET binding motif (EBM) of MLV IN can disrupt the cognate interaction of Brd4 with NSD3, and that substitutions of Brd4 ET residues essential for binding MLV IN also impair interaction of Brd4 with a number of cellular partners involved in transcriptional regulation and chromatin remodeling. This suggests that γ-retroviruses have evolved the EBM to mimic a cognate interaction motif to achieve effective integration in host chromatin. Collectively, our findings identify key structural features of the ET domain of Brd4 that allow for interactions with both cellular and viral proteins.

  13. Effect of DNA modifications on DNA processing by HIV-1 integrase and inhibitor binding: role of DNA backbone flexibility and an open catalytic site.

    PubMed

    Johnson, Allison A; Sayer, Jane M; Yagi, Haruhiko; Patil, Sachindra S; Debart, Françoise; Maier, Martin A; Corey, David R; Vasseur, Jean-Jacques; Burke, Terrence R; Marquez, Victor E; Jerina, Donald M; Pommier, Yves

    2006-10-27

    Integration of the viral cDNA into host chromosomes is required for viral replication. Human immunodeficiency virus integrase catalyzes two sequential reactions, 3'-processing (3'-P) and strand transfer (ST). The first integrase inhibitors are undergoing clinical trial, but interactions of inhibitors with integrase and DNA are not well understood in the absence of a co-crystal structure. To increase our understanding of integrase interactions with DNA, we examined integrase catalysis with oligonucleotides containing DNA backbone, base, and groove modifications placed at unique positions surrounding the 3'-processing site. 3'-Processing was blocked with substrates containing constrained sugars and alpha-anomeric residues, suggesting that integrase requires flexibility of the phosphodiester backbone at the 3'-P site. Of several benzo[a]pyrene 7,8-diol 9,10-epoxide (BaP DE) adducts tested, only the adduct in the minor groove at the 3'-P site inhibited 3'-P, suggesting the importance of the minor groove contacts for 3'-P. ST occurred in the presence of bulky BaP DE DNA adducts attached to the end of the viral DNA suggesting opening of the active site for ST. Position-specific effects of these BaP DE DNA adducts were found for inhibition of integrase by diketo acids. Together, these results demonstrate the importance of DNA structure and specific contacts with the viral DNA processing site for inhibition by integrase inhibitors.

  14. Synthesis, characterization and kinetic of a surfactant-modified bentonite used to remove As(III) and As(V) from aqueous solution.

    PubMed

    Su, Jin; Huang, Huai-Guo; Jin, Xiao-Ying; Lu, Xiao-Qiao; Chen, Zu-Liang

    2011-01-15

    In this study, organobentonites were prepared by modification of bentonite with various cationic surfactants, and were used to remove As(V) and As(III) from aqueous solution. The results showed that the adsorption capacities of bentonite modified with octadecyl benzyl dimethyl ammonium (SMB3) were 0.288 mg/g for As(V) and 0.102 mg/g for As(III), which were much higher compared to 0.043 and 0.036 mg/g of un-modified bentonite (UB). The adsorption kinetics were fitted well with the pseudo-second-order model with rate constants of 46.7 × 10(-3)g/mgh for As(V) and 3.1 × 10(-3)g/mgh for As(III), respectively. The maximum adsorption capacity of As(V) derived from the Langmuir equation reached as high as 1.48 mg/g, while the maximum adsorption capacity of As(III) was 0.82 mg/g. The adsorption of As(V) and As(III) was strongly dependent on solution pH. Addition of anions did not impact on As(III) adsorption, while they clearly suppressed adsorption of As(V). In addition, this study also showed that desorbed rates were 74.61% for As(V) and 30.32% for As(III), respectively, after regeneration of SMB3 in 0.1M HCl solution. Furthermore, in order to interpret the proposed absorption mechanism, both SMB3 and UB were extensively characterized using scanning electron microscopy (SEM), X-ray diffraction (XRD) and Fourier transform infrared (FTIR) analyses.

  15. Biosorption of As(III) and As(V) from aqueous solution by macrofungus (Inonotus hispidus) biomass: equilibrium and kinetic studies.

    PubMed

    Sari, Ahmet; Tuzen, Mustafa

    2009-05-30

    The biosorption characteristics of As(III) and As(V) from aqueous solution using the macrofungus (Inonotus hispidus) biomass were investigated as a function of pH, biomass dosage, contact time, and temperature. Langmuir, Freundlich and Dubinin-Radushkevich (D-R) models were applied to describe the biosorption isotherm of the metal ions by I. hispidus biomass. Langmuir model fitted the equilibrium data better than the Freundlich isotherm. The biosorption capacity of I. hispidus for As(III) and As(V) was found to be 51.9 mg/g and 59.6 mg/g, respectively at optimum conditions of pH 6 for As(III) and pH 2 for As(V), contact time of 30 min and temperature of 20 degrees C. The metal ions were desorbed from I. hispidus using both 1M HCl and 1M HNO(3). The high stability of I. hispidus permitted 10 times of adsorption-elution process along the studies with a decrease about 11-28% in recovery of As(III) and 10-25% for As(V). The mean free energy values evaluated from the D-R model indicated that the biosorption of As(III) and As(V) onto I. hispidus biomass was taken place by chemical ion-exchange. The calculated thermodynamic parameters showed that the biosorption of As(III) and As(V) ions onto I. hispidus biomass was feasible, spontaneous and exothermic under examined conditions. Kinetic examination of the experimental data were showed that the biosorption processes of both As(III) and As(V) followed well pseudo-second-order kinetics.

  16. Notable Difference in anti-HIV Activity of Integrase Inhibitors as a Consequence of Geometric and Enantiomeric Configurations

    PubMed Central

    Okello, Maurice; Mishra, Sanjay; Nishonov, Malik; Nair, Vasu

    2013-01-01

    While some examples are known of integrase inhibitors that exhibit potent anti-HIV activity, there are very few cases reported of integrase inhibitors that show significant differences in anti-HIV activity that result from distinctions in cis-and trans-configurations as well as enantiomeric stereostructure. We describe here the design and synthesis of two enantiomeric trans-hydroxycyclopentyl carboxamides which exhibit notable difference in anti-HIV activity. This difference is explained through their binding interactions within the active site of the HIV-1 integrase intasome. The more active enantiomer 3 (EC50 25 nM) was relatively stable in human liver microsomes. Kinetic data revealed that its impact on key cytochrome P450 isozymes, as either an inhibitor or an activator, was minor, suggesting a favorable CYP profile. PMID:23746474

  17. VizieR Online Data Catalog: Photometry of stars at NAOR and ASV in 2013-2014 (Cvetkovic+, 2016)

    NASA Astrophysics Data System (ADS)

    Cvetkovic, Z.; Pavlovic, R.; Boeva, S.

    2016-10-01

    The previous seven series of CCD observations of double and multiple stars performed by the Belgrade team at the Bulgarian National Astronomical Observatory at Rozhen (NAOR) with a CCD camera attached to the 2m telescope took place in 2004, 2005, 2006, 2009, 2010, 2011, and 2012, respectively. The results have been published in Pavlovic et al. (2005SerAJ.171...49P; 2013, Cat. J/AJ/146/52) and Cvetkovic et al. (2006SerAJ.172...53C; 2007SerAJ.174...83C; 2010SerAJ.180..103C; 2011, Cat. J/AJ/142/73; 2015, Cat. J/AJ/149/150). The eighth series comprising observations of 271 double or multiple stars took place during seven nights: five nights in 2013 and two nights in 2014. The telescope is of the Ritchey-Chretien-Coude-type with a focal length of 16m as given by the manufacturer. The frames were obtained by using the CCD camera VersArray 1300B. The basic characteristics of this camera and its orientation error are given in Table1. For each double or multiple star, 10 frames were obtained: 5 in the Johnson B filter and 5 in the Johnson V filter. During the summer of 2011, the first CCD observations of double and multiple stars from the Astronomical Station on the mountain of Vidojevica (ASV) took place. The results for the previous two series have been published in Pavlovic et al. 2013 (Cat. J/AJ/146/52) and Cvetkovic et al. 2015 (Cat. J/AJ/149/150). This station belongs to the Astronomical Observatory of Belgrade. The 60cm telescope has an equatorial mounting and a Cassegrain optical system. The telescope focal length is 600cm as given by the manufacturer. We carried out CCD observations of 343 visual double or multiple stars at ASV during 21 nights: 12 nights in 2013 and 9 nights in 2014. This is the third series of observations at ASV. For these nights, we used either SBIG ST-10ME or Apogee Alta U42 CCD cameras. Its basic characteristics are summarized in Table1. For each star pair 10 frames were obtained: 5 in the Cousins/Bessel B filter and 5 in the Cousins/Bessel V

  18. Screening of potential pseudo att sites of Streptomyces phage ΦC31 integrase in the human genome.

    PubMed

    Hu, Zhi-peng; Chen, Lu-sheng; Jia, Cai-yan; Zhu, Huan-zhang; Wang, Wei; Zhong, Jiang

    2013-04-01

    ΦC31 integrase mediates site-specific recombination between two short sequences, attP and attB, in phage and bacterial genomes, which is a promising tool in gene regulation-based therapy since the zinc finger structure is probably the DNA recognizing domain that can further be engineered. The aim of this study was to screen potential pseudo att sites of ΦC31 integrase in the human genome, and evaluate the risks of its application in human gene therapy. TFBS (transcription factor binding sites) were found on the basis of reported pseudo att sites using multiple motif-finding tools, including AlignACE, BioProspector, Consensus, MEME, and Weeder. The human genome with the proposed motif was scanned to find the potential pseudo att sites of ΦC31 integrase. The possible recognition motif of ΦC31 integrase was identified, which was composed of two co-occurrence conserved elements that were reverse complement to each other flanking the core sequence TTG. In the human genome, a total of 27924 potential pseudo att sites of ΦC31 integrase were found, which were distributed in each human chromosome with high-risk specificity values in the chromosomes 16, 17, and 19. When the risks of the sites were evaluate more rigorously, 53 hits were discovered, and some of them were just the vital functional genes or regulatory regions, such as ACYP2, AKR1B1, DUSP4, etc. The results provide clues for more comprehensive evaluation of the risks of using ΦC31 integrase in human gene therapy and for drug discovery.

  19. 3D-QSAR and molecular modeling of HIV-1 integrase inhibitors

    NASA Astrophysics Data System (ADS)

    Makhija, Mahindra T.; Kulkarni, Vithal M.

    2002-03-01

    Three-dimensional quantitative structure-activity relationship (3D QSAR) methods were applied on a series of inhibitors of HIV-1 integrase with respect to their inhibition of 3'-processing and 3'-end joining steps in vitro.The training set consisted of 27 compounds belonging to the class of thiazolothiazepines. The predictive ability of each model was evaluated using test set I consisting of four thiazolothiazepines and test set II comprised of seven compounds belonging to an entirely different structural class of coumarins. Maximum Common Substructure (MCS) based method was used to align the molecules and this was compared with other known methods of alignment. Two methods of 3D QSAR: comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were analyzed in terms of their predictive abilities. CoMSIA produced significantly better results for all correlations. The results indicate a strong correlation between the inhibitory activity of these compounds and the steric and electrostatic fields around them. CoMSIA models with considerable internal as well as external predictive ability were obtained. A poor correlation obtained with hydrophobic field indicates that the binding of thiazolothiazepines to HIV-1 integrase is mainly enthalpic in nature. Further the most active compound of the series was docked into the active site using the crystal structure of integrase. The binding site was formed by the amino acid residues 64-67, 116, 148, 151-152, 155-156, and 159. The comparison of coefficient contour maps with the steric and electrostatic properties of the receptor shows high level of compatibility.

  20. Effects of Etravirine on the Pharmacokinetics of the Integrase Inhibitor S/GSK1265744

    PubMed Central

    Ford, Susan L.; Gould, Elizabeth; Chen, Shuguang; Lou, Yu; Dumont, Etienne; Spreen, William

    2013-01-01

    HIV integrase inhibitors such as raltegravir and elvitegravir halt HIV progression, but treatment-emergent resistance and cross-resistance have been observed. The nonnucleoside reverse transcriptase inhibitor etravirine (ETR) may be used in combination with integrase inhibitors in patients with drug resistance. This single-center, open-label, two-period, single-sequence crossover study evaluated the effects of ETR coadministration on the pharmacokinetic profile of S/GSK1265744, an investigational integrase inhibitor in phase 2 studies. Healthy subjects received 30 mg of S/GSK1265744 alone once daily for 10 days (period 1) and in combination with 200 mg of ETR twice daily for 14 days (period 2). Serial plasma samples for pharmacokinetic analyses were collected on day 10 during period 1 and on day 14 during period 2. All treatments were well tolerated. Etravirine had no effects on S/GSK1265744 geometric mean ratios of the area under the curve from time zero until the end of the dosing interval (1.01; 90% confidence interval [CI], 0.956 to 1.06), of the maximum observed plasma concentration (1.04; 90% CI, 0.987 to 1.09), or of the plasma concentration at the end of the dosing interval (0.999; 90% CI, 0.942 to 1.06). Etravirine pharmacokinetics (PK) parameters observed following coadministration with S/GSK1265744 were in the range of historical values reported for ETR alone in healthy subjects. These results indicate that 30 mg of S/GSK1265744 for 10 days as monotherapy followed by an additional 14 days in combination with ETR was well tolerated in healthy subjects and that no dose adjustment of S/GSK1265744 is required when it is coadministered with ETR. PMID:23114768

  1. Dolutegravir, a second-generation integrase inhibitor for the treatment of HIV-1 infection.

    PubMed

    Rathbun, R Chris; Lockhart, Staci M; Miller, Misty M; Liedtke, Michelle D

    2014-03-01

    To review the pharmacology, safety, and efficacy of dolutegravir, an integrase strand-transfer inhibitor (INSTI), and to discuss its role in the treatment of HIV-1-infected patients. PubMed articles indexed through August 2013 were identified using the search terms S/GSK1349572, dolutegravir, and integrase inhibitor. Information was also identified from the package insert, cited publication references, professional meeting abstracts, and the ClinicalTrials.gov registry. English language articleswere selected for evaluation, with preference given to safety, efficacy, and pharmacokinetic studies conducted in HIV-1-infected patients. Dolutegravir is a new INSTI approved for combination treatment in HIV-1-infected adults and adolescent children. Four phase 3 studies provide the basis for current labeling in antiretroviral-naïve and antiretroviral-experienced adults. Results from these studies demonstrate that dolutegravir is noninferior in efficacy to raltegravir in antiretroviral-naïve patients and superior in antiretroviral-experienced patients. Superiority to efavirenz and darunavir/ritonavir was also demonstrated in antiretroviral-naïve patients. Dolutegravir is well tolerated, exhibits low potential for drug-drug interactions, and has a long serum half-life, allowing it to be administered once-daily in patients without preexisting INSTI resistance. Twice-daily administration is recommended in patients with known or suspected resistance mutations to first-generation INSTIs. Mild elevations in serum creatinine occur following dolutegravir initiation from inhibition of renal organic cation transporter 2 but do not reflect changes in glomerular filtration. Dolutegravir is the first second-generation INSTI and exhibits several advantages over current integrase inhibitors and other preferred antiretrovirals. Long-term efficacy and safety are needed to define dolutegravir's role in treatment.

  2. Yeast system as a model to study Moloney murine leukemia virus integrase: expression, mutagenesis and search for eukaryotic partners.

    PubMed

    Vera, Jorge; Parissi, Vincent; García, Andrea; Zúñiga, Roberto; Andreola, Marie-Line; Caumont-Sarcos, Anne; Tarrago-Litvak, Laura; Leon, Oscar

    2005-09-01

    Moloney murine leukemia virus (M-MuLV) integrase (IN) catalyses the insertion of the viral genome into the host chromosomal DNA. The limited solubility of the recombinant protein produced in Escherichia coli led the authors to explore the use of Saccharomyces cerevisiae for expression of M-MuLV IN. IN was expressed in yeast and purified by chromatography on nickel-NTA agarose. IN migrated as a single band in SDS-PAGE and did not contain IN degradation products. The enzyme was about twofold more active than the enzyme purified from E. coli and was free of nucleases. Using the yeast system, the substitution of the putative catalytic amino acid Asp184 by alanine was also analysed. The mutated enzyme was inactive in the in vitro assays. This is the first direct demonstration that mutation of Asp184 inactivates M-MuLV IN. Finally, S. cerevisiae was used as a model to assess the ability of M-MuLV IN to interact with eukaryotic protein partners. The expression of an active M-MuLV IN in yeast strains deficient in RAD52 induced a lethal effect. This phenotype could be attributed to cellular damage, as suggested by the viability of cells expressing inactive D184A IN. Furthermore, when active IN was expressed in a yeast strain lacking the ySNF5 transcription factor, the lethal effect was abolished, suggesting the involvement of ySNF5 in the cellular damage induced by IN. These results indicate that S. cerevisiae could be a useful model to study the interaction of IN with cellular components in order to identify potential counterparts of the natural host.

  3. Isolation and Molecular Characterization of As(V) Respiration / As Resistance Bacteria From Arsenic-Contaminated Groundwater in Blackfoot Disease Region in Taiwan

    NASA Astrophysics Data System (ADS)

    Wei, C.; Hsiao, S.; Liu, C.; Liao, C.; Chang, F.; Liao, V. H.

    2006-12-01

    Arsenic concerns range from local to international. The contamination of groundwater with arsenic is a major concern to public health in many countries. Arsenic problems in groundwater in Taiwan were first recognized during the 1960s and then Taiwan becomes the classic area for the study of Blackfoot disease and a number of other typical health problems including cancers. However, both the biogeochemistry of the groundwater and the mineral sources of arsenic in Taiwan are poorly defined at present. Increasing evidence suggest that the biogeochemical cycle of arsenic is significant dependent on microbial transformations which affect the distribution and the mobility of arsenic species in the environment. Known arsenic transforming bacteria possess diverse mechanisms for either oxidizing As(III) or reducing As(V), including energy generation and detoxification. To date, although a number of phylogenetically diverse As(V)-reducing bacteria have been isolated, studies on molecular basis of As(V) respiration are limited. In this study, the isolation and molecular characterization of relationship between As(V) respiration and As resistance were investigated. Forty-nine strains were isolated in the presence of 10 mM As(V) under anaerobic condition from arsenic-contaminated groundwater in Blackfoot disease region in Taiwan. ArrA and ArsR were used as marker genes for As(V) respiration and As resistance, respectively, by means of PCR. Of these isolated strains, one designed as L6510 was selected for further investigation because it appears to contain both ArrA and ArsR genes. Moreover, L6510 was able to grow under aerobic and anaerobic conditions. Resistance tests showed that L6510 was able to resist the high concentrations of As(V) and As(III) when grown in LB medium. Together, L6510 might possess both As(V) respiration and As resistance pathways. Further investigations including As(V) respiration, phylogenetic analysis, growth characteristics, and transposon mutagenesis

  4. 3-Hydroxypyrimidine-2,4-diones as an Inhibitor Scaffold of HIV Integrase

    PubMed Central

    Tang, Jing; Maddali, Kasthuraiah; Sham, Yuk Y.; Vince, Robert; Pommier, Yves; Wang, Zhengqiang

    2011-01-01

    Integrase (IN) represents a clinically validated target for the development of antivirals against human immunodeficiency virus (HIV). Inhibitors with a novel structure core are essential for combating resistance associated with known IN inhibitors (INIs). We have previously disclosed a novel dual inhibitor scaffold of HIV IN and reverse transcriptase (RT). Here we report the complete structure-activity relationship (SAR), molecular modeling and resistance profile of this inhibitor type on IN inhibition. These studies support an antiviral mechanism of dual inhibition against both IN and RT and validate 3-hydroxypyrimidine-2,4-diones as an IN inhibitor scaffold. PMID:21381765

  5. Substrate mimicry—overcoming HIV-1 integrase resistance mutations | Center for Cancer Research

    Cancer.gov

    HIV integrase (IN) strand transfer inhibitors (INSTIs) are among the newest anti-AIDS drugs; however, mutant forms of IN can confer resistance. We developed noncytotoxic naphthyridine-containing INSTIs that retain low nanomolar IC50 values against HIV-1 variants harboring all of the major INSTI-resistant mutations. We found by analyzing crystal structures of inhibitors bound to the IN from the prototype foamy virus (PFV) that the most successful inhibitors show striking mimicry of the bound viral DNA prior to 3'-processing and the bound host DNA prior to strand transfer.

  6. Transmitted resistance to HIV integrase strand-transfer inhibitors: right on schedule.

    PubMed

    Hurt, Christopher B

    2011-01-01

    Transmitted drug resistance (TDR), the primary acquisition of an HIV variant already resistant to antiretrovirals, affects approximately 15% of all new infections in the United States. Historically, from the time initial agents in the reverse transcriptase, protease and entry inhibitor classes were introduced, it took 3-5 years before the first case reports of TDR appeared. With the description of the first two cases of transmitted integrase stand-transfer inhibitor resistance, it is only a matter of time before the prevalence of TDR affecting this newest antiretroviral class reaches a level warranting baseline resistance testing for all patients entering care.

  7. Quantifying the effect of nanoparticles on As(V) ecotoxicity exemplified by nano-Fe2 O3 (magnetic) and nano-Al2 O3.

    PubMed

    Hu, Ji; Wang, Demin; Forthaus, Brett E; Wang, Jianmin

    2012-12-01

    Nano-Fe(2) O(3) (magnetic) and nano-Al(2) O(3) are increasingly used in biomedical fields for cellular labeling and drug delivery, and in the industry as catalysts. However, they may also serve as carriers of toxic contaminants and negatively impact the environment and human health. In the present study, Ceriodaphnia dubia was used to assess the toxicity of these nanoparticles (NPs) with and without the presence of background arsenic(V) (As[V]). The results showed that although these NPs alone did not exhibit significant toxicity in the experimental concentration range, they could significantly enhance the toxicity of As(V). The accumulation of As(V) on nano-Fe(2) O(3) (m) and nano-Al(2) O(3) , as well as the uptake of these NPs in C. dubia, were verified. An inverse relationship between the median lethal concentration (LC50) of the soluble As(V) and the accumulation of NPs in C. dubia was developed. Therefore, the uptake of As(V)-loaded NPs in C. dubia played a key role in enhancing As(V) toxicity. Copyright © 2012 SETAC.

  8. Competitive adsorption of As(III), As(V), Sb(III) and Sb(V) onto ferrihydrite in multi-component systems: Implications for mobility and distribution.

    PubMed

    Qi, Pengfei; Pichler, Thomas

    2017-05-15

    The simultaneous adsorption behavior and competitive interactions between As(III), As(V), Sb(III) and Sb(V) by ferrihydrite were evaluated in multi-component (binary, ternary, quaternary) systems. In binary systems, Sb(III) had a stronger inhibitory influence on As(III) adsorption than Sb(V) did, and As(V) had a stronger inhibitory effect on Sb(V) adsorption than As(III) did. In ternary systems, NO3(-), PO4(3-) and SO4(2-) did not compete with the adsorption of As(III) and Sb(III). NO3(-) and SO4(2-) also had no distinct effect on the adsorption of As(V) and Sb(V), while PO4(3-) competed with As(V) and Sb(V) for surface sites. In quaternary systems, the simultaneous adsorption behavior of the four redox species was pH dependent. Sb(III) always showed the strongest adsorption affinity regardless of pH. At pH 3.5 As(III) showed the lowest affinity could be due to the presence and negative effect of Sb(III) and As(V). The Freundlich model provided a good fit for the simultaneous adsorption data under quaternary conditions. The study of competitive/simultaneous adsorption of the four possible redox species onto ferrihydrite contributed to a better understanding of their distribution, mobility and fate in the environment.

  9. Biosorption of As(III) and As(V) on the surface of TW/MnFe2O4 composite from wastewater: kinetics, mechanistic and thermodynamics

    NASA Astrophysics Data System (ADS)

    Podder, M. S.; Majumder, C. B.

    2016-10-01

    In the present study, TW/MnFe2O4 composite (MTW) was synthesized and estimated as an effective biosorbent for removing As (III) and As(V) from wastewater. Physicochemical analysis of composite was performed through SEM-EDX. 86.615 and 83.478% removal efficiency were obtained by composite dosage of 2 g/L at contact time 120 min at temperature 30 °C and pH 7.0 and 4.0 for As(III) and As(V), respectively. Kinetic results study showed that Brouers-Weron-Sotolongo and Ritchie second-order for As(III) and Brouers-Weron-Sotolongo model for As(V) were capable to describe an accurate explanation of adsorption kinetic. Applicability of mechanistic models in the current study exposed that the rate-controlling step in the biosorption of both As(III) and As(V) on the surface of composite was film diffusion rather than intraparticle diffusion. The estimated thermodynamic parameters ΔG 0, ΔH 0 and ΔS 0 revealed that the biosorption of both As(III) and As(V) on the composite was feasible, spontaneous and exothermic.

  10. Caffeoylglycolic and caffeoylamino acid derivatives, halfmers of L-chicoric acid, as new HIV-1 integrase inhibitors.

    PubMed

    Lee, Seung Uk; Shin, Cha-Gyun; Lee, Chong-Kyo; Lee, Yong Sup

    2007-10-01

    Human immunodeficiency virus (HIV) integrase (IN) catalyzes the integration of HIV DNA copy into the host cell DNA. L-Chicoric acid (1) has been found to be one of the most potent HIV-1 integrase inhibitor. Caffeoylglycolic and caffeoylamino acid derivatives' halfmeric structures of L-chicoric acid 2 were synthesized for the purpose of simplifying the structure of L-chicoric acid. Among synthesized, compounds 2c and 3f showed HIV-1 IN inhibitory activities with IC(50) values of 10.5 and 12.0 microM, respectively, comparable to that of parent compound L-chicoric acid (IC(50)=15.7 microM).

  11. Discovery of 2-Pyridinone Aminals: A Prodrug Strategy to Advance a Second Generation of HIV-1 Integrase Strand Transfer Inhibitors.

    PubMed

    Raheem, Izzat T; Walji, Abbas M; Klein, Daniel; Sanders, John M; Powell, David A; Abeywickrema, Pravien; Barbe, Guillaume; Bennet, Amrith; Childers, Karla; Christensen, Melodie; Clas, Sophie Dorothee; Dubost, David; Embrey, Mark; Grobler, Jay; Hafey, Michael J; Hartingh, Timothy J; Hazuda, Daria J; Kuethe, Jeffrey T; McCabe Dunn, Jamie; Miller, Michael D; Moore, Keith P; Nolting, Andrew; Pajkovic, Natasa; Patel, Sangita; Peng, Zuihui; Rada, Vanessa; Rearden, Paul; Schreier, John D; Sisko, John; Steele, Thomas G; Truchon, Jean-François; Wai, John; Xu, Min; Coleman, Paul J

    2015-10-22

    The search for new molecular constructs that resemble the critical two-metal binding pharmacophore required for HIV integrase strand transfer inhibition represents a vibrant area of research within drug discovery. Here we present the discovery of a new class of HIV integrase strand transfer inhibitors based on the 2-pyridinone core of MK-0536. These efforts led to the identification of two lead compounds with excellent antiviral activity and preclinical pharmacokinetic profiles to support a once-daily human dose prediction. Dose escalating PK studies in dog revealed significant issues with limited oral absorption and required an innovative prodrug strategy to enhance the high-dose plasma exposures of the parent molecules.

  12. The optimization of As(V) removal over mesoporous alumina by using response surface methodology and adsorption mechanism.

    PubMed

    Han, Caiyun; Pu, Hongping; Li, Hongying; Deng, Lian; Huang, Si; He, Sufang; Luo, Yongming

    2013-06-15

    The Box-Behnken Design of the response surface methodology was employed to optimize four most important adsorption parameters (initial arsenic concentration, pH, adsorption temperature and time) and to investigate the interactive effects of these variables on arsenic(V) adsorption capacity of mesoporous alumina (MA). According to analysis of variance (ANOVA) and response surface analyses, the experiment data were excellent fitted to the quadratic model, and the interactive influence of initial concentration and pH on As(V) adsorption capacity was highly significant. The predicted maximum adsorption capacity was about 39.06 mg/g, and the corresponding optimal parameters of adsorption process were listed as below: time 720 min, temperature 52.8 °C, initial pH 3.9 and initial concentration 130 mg/L. Based on the results of arsenate species definition, FT-IR and pH change, As(V) adsorption mechanisms were proposed as follows: (1) at pH 2.0, H₃AsO₄ and H₂AsO₄(-) were adsorbed via hydrogen bond and electrostatic interaction, respectively; (2) at pH 6.6, arsenic species (H₂AsO₄(-) and HAsO₄(2-)) were removed via adsorption and ion exchange, (3) at pH 10.0, HAsO₄(2-) was adsorbed by MA via ion exchange together with adsorption, while AsO₄(3-) was removed by ion exchange. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Tetravalent manganese feroxyhyte: a novel nanoadsorbent equally selective for As(III) and As(V) removal from drinking water.

    PubMed

    Tresintsi, Sofia; Simeonidis, Konstantinos; Estradé, Sonia; Martinez-Boubeta, Carlos; Vourlias, George; Pinakidou, Fani; Katsikini, Maria; Paloura, Eleni C; Stavropoulos, George; Mitrakas, Manassis

    2013-09-03

    The development of a single-phase Fe/Mn oxy-hydroxide (δ-Fe0.76Mn0.24OOH), highly efficient at adsorbing both As(III) and As(V), is reported. Its synthesis involves the coprecipitation of FeSO4 and KMnO4 in a kilogram-scale continuous process, in acidic and strongly oxidizing environments. The produced material was identified as a manganese feroxyhyte in which tetravalent manganese is homogeneously distributed into the crystal unit, whereas a second-order hollow spherical morphology is favored. According to this structuration, the oxy-hydroxide maintains the high adsorption capacity for As(V) of a single Fe oxy-hydroxide combined with enhanced As(III) removal based on the oxidizing mediation of Mn(IV). Ion-exchange between arsenic species and sulfates as well as the strongly positive surface charge further facilitate arsenic adsorption. Batch adsorption tests performed in natural-like water indicate that Mn(IV)-feroxyhyte can remove 11.7 μg As(V)/mg and 6.7 μg As(III)/mg at equilibrium pH 7, before residual concentration overcomes the regulation limit of 10 μg As/L for drinking water. The improved efficiency of this material, its low cost, and the possibility for scaling-up its production to industry indicate the high practical impact and environmental importance of this novel adsorbent.

  14. Pyrroloaryls and pyrroloheteroaryls: Inhibitors of the HIV fusion/attachment, reverse transcriptase and integrase.

    PubMed

    Patel, Rahul V; Park, Se Won

    2015-09-01

    Heterocyclic compounds execute a very important role in drug design and discovery. This article provides the basic milestones of the research for pyrroloaryl and pyrroloheteroaryl based components targeting HIV viral replication cycle. Anti-HIV activity is elaborated for several classes of pyrrolo-compounds as pyrrolopyridines, pyrrolopyrimidines, pyrrolopyridazines, pyrrolobenzodiazepinones, pyrrolobenzothiazepines, pyrrolobenzoxazepinones, pyrrolophenanthridines, pyrroloquinoxalines, pyrrolotriazines, pyrroloquinolines, pyrrolopyrazinones, pyrrolothiatriazines, arylthiopyrroles and pyrrolopyrazolones targeting two essential HIV enzymes, reverse transcriptase and integrase as well as attachment/fusion of HIV virons to the host CD-4 cell. Such attempts were resulted in a discovery of highly potent anti-HIV agents suitable for clinical trials, for example, BMS-378806, BMS-585248, BMS-626529, BMS-663068, BMS-488043 and BMS-663749, etc. as anti-HIV attachment agents, triciribine, QX432, BI-1 and BI-2 as HIV RT inhibitors which are in preclinical or clinical development. Mechanism of action of compounds presented in this article towards the suppression of HIV attachment/fusion as well as against the activities of HIV enzymes reverse transcriptase and integrase has been discussed. Relationships of new compounds' molecular framework and HIV viral target has been overviewed in order to facilitate further construction of promising anti-HIV agents in future drug discovery process.

  15. Identification of novel HIV-1 integrase inhibitors using shape-based screening, QSAR, and docking approach.

    PubMed

    Gupta, Pawan; Garg, Prabha; Roy, Nilanjan

    2012-05-01

    The objective of this study is to identify novel HIV-1 integrase (IN) inhibitors. Here, shape-based screening and QSAR have been successfully implemented to identify the novel inhibitors for HIV-1 IN, and in silico validation is performed by docking studies. The 2D QSAR model of benzodithiazine derivatives was built using genetic function approximation (GFA) method with good internal (cross-validated r(2)  = 0.852) and external prediction (). Best docking pose of highly active molecule of the benzodithiazine derivatives was used as a template for shape-based screening of ZINC database. Toxicity prediction was also performed using Deductive Estimation of Risk from Existing Knowledge (DEREK) program to filter non-toxic molecules. Inhibitory activities of screened non-toxic molecules were predicted using derived QSAR models. Active, non-toxic screened molecules were also docked into the active site of HIV-1 IN using AutoDock and dock program. Some molecules docked similarly as highly active molecule of the benzodithiazine derivatives. These molecules also followed the same docking interactions in both the programs. Finally, four benzodithiazine derivatives were identified as novel HIV-1 integrase inhibitors based on QSAR predictions and docking interactions. ADME properties of these molecules were also computed using Discovery Studio. © 2012 John Wiley & Sons A/S.

  16. Expanding the diversity of oenococcal bacteriophages: insights into a novel group based on the integrase sequence.

    PubMed

    Jaomanjaka, Fety; Ballestra, Patricia; Dols-lafargue, Marguerite; Le Marrec, Claire

    2013-09-02

    Temperate bacteriophages are a contributor of the genetic diversity in the lactic acid bacterium Oenococcus oeni. We used a classification scheme for oenococcal prophages based on integrase gene polymorphism, to analyze a collection of Oenococcus strains mostly isolated in the area of Bordeaux, which represented the major lineages identified through MLST schemes in the species. Genome sequences of oenococcal prophages were clustered into four integrase groups (A to D) which were related to the chromosomal integration site. The prevalence of each group was determined and we could show that members of the intB- and intC-prophage groups were rare in our panel of strains. Our study focused on the so far uncharacterized members of the intD-group. Various intD viruses could be easily isolated from wine samples, while intD lysogens could be induced to produce phages active against two permissive O. oeni isolates. These data support the role of this prophage group in the biology of O. oeni. Global alignment of three relevant intD-prophages revealed significant conservation and highlighted a number of unique ORFs that may contribute to phage and lysogen fitness. © 2013 Elsevier B.V. All rights reserved.

  17. Virtual screening of the SAMPL4 blinded HIV integrase inhibitors dataset

    NASA Astrophysics Data System (ADS)

    Colas, Claire; Iorga, Bogdan I.

    2014-04-01

    Several combinations of docking software and scoring functions were evaluated for their ability to predict the binding of a dataset of potential HIV integrase inhibitors. We found that different docking software were appropriate for each one of the three binding sites considered (LEDGF, Y3 and fragment sites), and the most suitable two docking protocols, involving Glide SP and Gold ChemScore, were selected using a training set of compounds identified from the structural data available. These protocols could successfully predict respectively 20.0 and 23.6 % of the HIV integrase binders, all of them being present in the LEDGF site. When a different analysis of the results was carried out by removing all alternate isomers of binders from the set, our predictions were dramatically improved, with an overall ROC AUC of 0.73 and enrichment factor at 10 % of 2.89 for the prediction obtained using Gold ChemScore. This study highlighted the ability of the selected docking protocols to correctly position in most cases the ortho-alkoxy-carboxylate core functional group of the ligands in the corresponding binding site, but also their difficulties to correctly rank the docking poses.

  18. Latest advances in the efficacy, tolerability, and monotherapy of integrase inhibitors.

    PubMed

    Tang, Qi; Lu, Hongzhou

    2017-01-01

    More than 30 drugs for antiretroviral therapy (ART), including integrase inhibitors (INIs), have been approved by the U.S. Food and Drug Administration (FDA) as of 2017. Integrase is the third essential enzyme in the cycle of human immunodeficiency virus (HIV) replication. INIs can effectively inhibit the replication of HIV and HIV is less prone to develop resistance to INIs clinically. Previous studies based on 7 phase III clinic trials indicate that INIs have satisfactory efficacy and tolerability in patients infected with HIV. The latest advances in INIs indicate that: i) dolutegravir (DTG)-based regimens are more efficacious, tolerable, and safer forms of first-, second-, and third-Line ART; ii) current studies have indicated that DTG monotherapy fails both virologically and clinically; and iii) whether the most cost-effective treatment for DTG is to replace efavirenz (EFV) as a first-line ART, to replace protease inhibitors (PIs) in second-line ART, or to replace both as a monotherapy is unclear. Given these circumstances, further study of INIs in terms of drug interactions, dose reduction, drug convenience, and drug costs is warranted.

  19. Computational and synthetic approaches for developing Lavendustin B derivatives as allosteric inhibitors of HIV-1 integrase

    PubMed Central

    Agharbaoui, Fatima E.; Hoyte, Ashley C.; Ferro, Stefania; Gitto, Rosaria; Buemi, Maria Rosa; Fuchs, James R.; Kvaratskhelia, Mamuka; De Luca, Laura

    2017-01-01

    Through structure-based virtual screening and subsequent activity assays of selected natural products, Lavendustin B was previously identified as an inhibitor of HIV-1 integrase (IN) interaction with its cognate cellular cofactor, lens epithelium-derived growth factor (LEDGF/p75). In order to improve the inhibitory potency we have employed in silico-based approaches. Particularly, a series of new analogues was designed and docked into the LEDGF/p75 binding pocket of HIV-1 IN. To identify promising leads we used the Molecular Mechanics energies combined with the Generalized Born and Surface Area continuum solvation (MM-GBSA) method, molecular dynamics simulations and analysis of hydrogen bond occupancies. On the basis of these studies, six analogues of Lavendustine B, containing the benzylamino-hydroxybenzoic scaffold, were selected for synthesis and structure activity-relationship (SAR) studies. Our results demonstrated a good correlation between computational and experimental data, and all six analogues displayed an improved potency for inhibiting IN binding to LEDGF/p75 in vitro to respect to the parent compound Lavendustin B. Additionally, these analogs show to inhibit weakly LEDGF/p75-independent IN catalytic activity suggesting a multimodal allosteric mechanism of action. Nevertheless, for the synthesized compounds similar profiles for HIV-1 inhibition and cytoxicity were highlighted. Taken together, our studies elucidated the mode of action of Lavendustin B analogs and provided a path for their further development as a new promising class of HIV-1 integrase inhibitors. PMID:27517812

  20. [Research progress of dual inhibitors targeting HIV-1 reverse transcriptase and integrase].

    PubMed

    Liu, Hong; Zhan, Peng; Liu, Xin-Yong

    2013-04-01

    Both reverse transcriptase (RT) and integrase (IN) play crucial roles in the life cycle of HIV-1, which are also key targets in the area of anti-HIV drug research. Reverse transcriptase inhibitors are involved in the most employed drugs used to treat AIDS patients and HIV-infected people, while one of the integrase inhibitors has already been approved by US FDA to appear on the market. Great achievement has been made in the research on both, separately. Recently, much more attention of medicinal chemistry researchers has been attracted to the strategies of multi-target drugs. Compounds with excellent potency against both HIV RT and IN, evidently defined as dual inhibitors targeting both enzymes, have been obtained through considerable significant exploration, which can be classified into two categories according to different strategies. Combinatorial chemistry approach together with high throughput screening methods and multi-target-based virtual screening strategy have been useful tools for identifying selective anti-HIV compounds for long times; Rational drug design based on pharmacophore combination has also led to remarkable results. In this paper, latest progress of both categories in the discovery and structural modification will be covered, with a view to contribute to the career of anti-HIV research.

  1. HIV-1 protease and HIV-1 integrase inhibitory substances from Eclipta prostrata.

    PubMed

    Tewtrakul, Supinya; Subhadhirasakul, Sanan; Cheenpracha, Sarot; Karalai, Chatchanok

    2007-11-01

    The bioassay-guided fractionation for anti-HIV-1 integrase activity led to the isolation of six compounds from the whole plant extract of Eclipta prostrata extract. They were identified as 5-hydroxymethyl-(2,2':5',2'')-terthienyl tiglate (1), 5-hydroxymethyl-(2,2':5',2'')-terthienyl agelate (2), 5-hydroxymethyl-(2,2':5',2'')-terthienyl acetate (3), ecliptal (4), orobol (5) and wedelolactone (6). Of these, compound 6 showed the highest activity against HIV-1 integrase (IN) with an IC50 value of 4.0+/-0.2 microm, followed by compound 5 (IC50=8.1+/-0.5 microm), whereas the four terthiophene compounds (1-4) were inactive (IC50>100 microm). Regarding HIV-1 protease (PR) inhibitory activity, compound 1 exhibited appreciable activity against HIV-1 PR with an IC50 of 58.3+/-0.8 microm, followed by compound 4 (IC50=83.3+/-1.6 microm) and compound 3 (IC50=93.7+/-0.8 microm), while compounds 2, 5 and 6 were inactive against HIV-1 PR (IC50>100 microm). This is the first report of anti-HIV-1 IN activities for wedelolactone (6), a coumarin derivative, and orobol (5), an isoflavone derivative. This study supports the use of E. prostrata in AIDS patients, which is in accord with its traditional use by Thai traditional doctors for curing blood related diseases. Copyright (c) 2007 John Wiley & Sons, Ltd.

  2. Computational and synthetic approaches for developing Lavendustin B derivatives as allosteric inhibitors of HIV-1 integrase.

    PubMed

    Agharbaoui, Fatima E; Hoyte, Ashley C; Ferro, Stefania; Gitto, Rosaria; Buemi, Maria Rosa; Fuchs, James R; Kvaratskhelia, Mamuka; De Luca, Laura

    2016-11-10

    Through structure-based virtual screening and subsequent activity assays of selected natural products, Lavendustin B was previously identified as an inhibitor of HIV-1 integrase (IN) interaction with its cognate cellular cofactor, lens epithelium-derived growth factor (LEDGF/p75). In order to improve the inhibitory potency we have employed in silico-based approaches. Particularly, a series of new analogues was designed and docked into the LEDGF/p75 binding pocket of HIV-1 IN. To identify promising leads we used the Molecular Mechanics energies combined with the Generalized Born and Surface Area continuum solvation (MM-GBSA) method, molecular dynamics simulations and analysis of hydrogen bond occupancies. On the basis of these studies, six analogues of Lavendustine B, containing the benzylamino-hydroxybenzoic scaffold, were selected for synthesis and structure activity-relationship (SAR) studies. Our results demonstrated a good correlation between computational and experimental data, and all six analogues displayed an improved potency for inhibiting IN binding to LEDGF/p75 in vitro to respect to the parent compound Lavendustin B. Additionally, these analogs show to inhibit weakly LEDGF/p75-independent IN catalytic activity suggesting a multimodal allosteric mechanism of action. Nevertheless, for the synthesized compounds similar profiles for HIV-1 inhibition and cytoxicity were highlighted. Taken together, our studies elucidated the mode of action of Lavendustin B analogs and provided a path for their further development as a new promising class of HIV-1 integrase inhibitors.

  3. A phage integrase directs efficient site-specific integration in human cells

    PubMed Central

    Groth, Amy C.; Olivares, Eric C.; Thyagarajan, Bhaskar; Calos, Michele P.

    2000-01-01

    The integrase from the Streptomyces phage φC31 carries out efficient recombination between the attP site in the phage genome and the attB site in the host bacterial chromosome. In this paper, we show that the enzyme also functions in human cells. A plasmid assay system was constructed that measured intramolecular integration of attP into attB. This assay was used to demonstrate that in the presence of the φC31 integrase, precise unidirectional integration occurs with an efficiency of 100% in Escherichia coli and >50% in human cells. This assay system was also used to define the minimal sizes of attB and attP at 34 bp and 39 bp, respectively. Furthermore, precise and efficient intermolecular integration of an incoming plasmid bearing attP into an established Epstein–Barr virus plasmid bearing attB was documented in human cells. This work is a demonstration of efficient, site-specific, unidirectional integration in mammalian cells. These observations form the basis for site-specific integration strategies potentially useful in a broad range of genetic engineering applications. PMID:10801973

  4. Rapid metabolic pathway assembly and modification using serine integrase site-specific recombination.

    PubMed

    Colloms, Sean D; Merrick, Christine A; Olorunniji, Femi J; Stark, W Marshall; Smith, Margaret C M; Osbourn, Anne; Keasling, Jay D; Rosser, Susan J

    2014-02-01

    Synthetic biology requires effective methods to assemble DNA parts into devices and to modify these devices once made. Here we demonstrate a convenient rapid procedure for DNA fragment assembly using site-specific recombination by C31 integrase. Using six orthogonal attP/attB recombination site pairs with different overlap sequences, we can assemble up to five DNA fragments in a defined order and insert them into a plasmid vector in a single recombination reaction. C31 integrase-mediated assembly is highly efficient, allowing production of large libraries suitable for combinatorial gene assembly strategies. The resultant assemblies contain arrays of DNA cassettes separated by recombination sites, which can be used to manipulate the assembly by further recombination. We illustrate the utility of these procedures to (i) assemble functional metabolic pathways containing three, four or five genes; (ii) optimize productivity of two model metabolic pathways by combinatorial assembly with randomization of gene order or ribosome binding site strength; and (iii) modify an assembled metabolic pathway by gene replacement or addition.

  5. Discovery and optimization of 2-pyridinone aminal integrase strand transfer inhibitors for the treatment of HIV.

    PubMed

    Schreier, John D; Embrey, Mark W; Raheem, Izzat T; Barbe, Guillaume; Campeau, Louis-Charles; Dubost, David; McCabe Dunn, Jamie; Grobler, Jay; Hartingh, Timothy J; Hazuda, Daria J; Klein, Daniel; Miller, Michael D; Moore, Keith P; Nguyen, Natalie; Pajkovic, Natasa; Powell, David A; Rada, Vanessa; Sanders, John M; Sisko, John; Steele, Thomas G; Wai, John; Walji, Abbas; Xu, Min; Coleman, Paul J

    2017-02-20

    HIV integrase strand transfer inhibitors (InSTIs) represent an important class of antiviral therapeutics with proven efficacy and excellent tolerability for the treatment of HIV infections. In 2007, Raltegravir became the first marketed strand transfer inhibitor pioneering the way to a first-line therapy for treatment-naïve patients. Challenges with this class of therapeutics remain, including frequency of the dosing regimen and the genetic barrier to resistance. To address these issues, research towards next-generation integrase inhibitors has focused on imparting potency against RAL-resistent mutants and improving pharmacokinetic profiles. Herein, we detail medicinal chemistry efforts on a novel class of 2-pyridinone aminal InSTIs, inpsired by MK-0536, which led to the discovery of important lead molecules for our program. Systematic optimization carried out at the amide and aminal positions on the periphery of the core provided the necessary balance of antiviral activity and physiochemical properties. These efforts led to a novel aminal lead compound with the desired virological profile and preclinical pharmacokinetic profile to support a once-daily human dose prediction.

  6. The use of artificial neural network for modelling of phycoremediation of toxic elements As(III) and As(V) from wastewater using Botryococcus braunii

    NASA Astrophysics Data System (ADS)

    Podder, M. S.; Majumder, C. B.

    2016-02-01

    In the present study, a thorough investigation has been done on the removal efficiency of both As(III) and As (V) from synthetic wastewater by phycoremediation of Botryococcus braunii algal biomass. Artificial neural networks (ANNs) are practised for predicting % phycoremediation efficiency of both As(III) and As(V) ions. The influence of several parameters for example initial pH, inoculum size, contact time and initial arsenic concentration (either As(III) or As(V)) was examined systematically. The maximum phycoremediation of As(III) and As(V) was found to be 85.22% and 88.15% at pH 9.0, equilibrium time of 144 h by using algal inoculum size of 10% (v/v) and initial arsenic concentration of 50 mg/L. The data acquired from laboratory scale experimental set up was utilized for training a three-layer feed-forward back propagation (BP) with Levenberg-Marquardt (LM) training algorithm having 4:5:1 architecture. A comparison between the experimental data and model outputs provided a high correlation coefficient (R2all_ANN equal to 0.9998) and exhibited that the model was capable for predicting the phycoremediation of both As(III) and As(V) from wastewater. The network topology was optimized by changing number of neurons in hidden layers. ANNs are efficient to model and simulate highly non-liner multivariable relationships. Absolute error and Standard deviation (SD) with respect to experimental output were calculated for ANN model outputs. The comparison of phycoremediation efficiencies of both As(III) and As(V) between experimental results and ANN model outputs exhibited that ANN model can determine the behaviour of As(III) and As(V) elimination process under various circumstances.

  7. Bacterial ability in AsIII oxidation and AsV reduction: Relation to arsenic tolerance, P uptake, and siderophore production.

    PubMed

    Ghosh, Piyasa; Rathinasabapathi, Bala; Teplitski, Max; Ma, Lena Q

    2015-11-01

    The relationship between bacterial ability in arsenic transformation, siderophore production, and P uptake was investigated using six arsenic-resistant bacteria isolated from the rhizosphere of arsenic-hyperaccumulator Pteris vittata. Bacterial strains of PG5 and 12 were better arsenite (AsIII) oxidizers (31-46 vs. 6.2-21% of 1 mM AsIII) whereas PG 6, 9, 10 and 16 were better arsenate (AsV) reducers (58-95 vs. 7.5-46% of 1 mM AsV). Increase in AsV concentration from 0 to 1 mM induced 3.0-8.4 times more P uptake by bacteria but increase in P concentration from 0.1 to 1 mM reduced AsV uptake by 17-71%, indicating that P and AsV were taken up by P transporters. Bacteria producing more siderophores (PG5 and 12; >73 μM equiv) showed greater AsIII oxidation and AsIII resistance than those producing less siderophore (PG 6, 9, 10 and 16; <23 μM equiv). This observation was further supported by results obtained from mutants of Pseudomonas fluorescens impaired in siderophore production, as they were 23-25% less tolerant to AsIII than the wild-type. Arsenic-resistant bacteria increased their arsenic tolerance by retaining less arsenic in cells via efficient AsIII oxidation and AsV reduction, which were impacted by P uptake and siderophore production.

  8. The use of artificial neural network for modelling of phycoremediation of toxic elements As(III) and As(V) from wastewater using Botryococcus braunii.

    PubMed

    Podder, M S; Majumder, C B

    2016-02-15

    In the present study, a thorough investigation has been done on the removal efficiency of both As(III) and As (V) from synthetic wastewater by phycoremediation of Botryococcus braunii algal biomass. Artificial neural networks (ANNs) are practised for predicting % phycoremediation efficiency of both As(III) and As(V) ions. The influence of several parameters for example initial pH, inoculum size, contact time and initial arsenic concentration (either As(III) or As(V)) was examined systematically. The maximum phycoremediation of As(III) and As(V) was found to be 85.22% and 88.15% at pH9.0, equilibrium time of 144h by using algal inoculum size of 10% (v/v) and initial arsenic concentration of 50mg/L. The data acquired from laboratory scale experimental set up was utilized for training a three-layer feed-forward back propagation (BP) with Levenberg-Marquardt (LM) training algorithm having 4:5:1 architecture. A comparison between the experimental data and model outputs provided a high correlation coefficient (R(2)all_ANN equal to 0.9998) and exhibited that the model was capable for predicting the phycoremediation of both As(III) and As(V) from wastewater. The network topology was optimized by changing number of neurons in hidden layers. ANNs are efficient to model and simulate highly non-liner multivariable relationships. Absolute error and Standard deviation (SD) with respect to experimental output were calculated for ANN model outputs. The comparison of phycoremediation efficiencies of both As(III) and As(V) between experimental results and ANN model outputs exhibited that ANN model can determine the behaviour of As(III) and As(V) elimination process under various circumstances. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. DNA binding and synapsis by the large C-terminal domain of ϕC31 integrase

    PubMed Central

    McEwan, Andrew R.; Rowley, Paul A.; Smith, Margaret C. M.

    2009-01-01

    The integrase (Int) from phage ϕC31 acts on the phage and host-attachment sites, attP and attB, to form an integrated prophage flanked by attL and attR. Excision (attL × attR recombination) is prevented, in the absence of accessory factors, by a putative coiled-coil motif in the C-terminal domain (CTD). Int has a serine recombinase N-terminal domain, required for synapsis of recombination substrates and catalysis. We show here that the coiled-coil motif mediates protein–protein interactions between CTDs, but only when bound to DNA. Although the histidine-tagged CTD (hCTD) was monomeric in solution, hCTD bound cooperatively to three of the recombination substrates (attB, attL and attR). Furthermore, when provided with attP and attB, hCTD brought these substrates together in a synaptic complex. Substitutions in the coiled-coil motif that greatly reduce Int integration activity, L460P and Y475H, prevented CTD–CTD interactions and led to defective DNA binding and no detectable DNA synapsis. A substitution, E449K, in full length Int confers the ability to perform excision in addition to integration as it has gained the ability to synapse attL × attR. hCTDE449K was similar to hCTD in DNA binding but unable to form the CTD synapse suggesting that the CTD synapse is not essential but could be part of the mechanism that controls directionality. PMID:19515935

  10. Cell-permeable stapled peptides based on HIV-1 integrase inhibitors derived from HIV-1 gene products.

    PubMed

    Nomura, Wataru; Aikawa, Haruo; Ohashi, Nami; Urano, Emiko; Métifiot, Mathieu; Fujino, Masayuki; Maddali, Kasthuraiah; Ozaki, Taro; Nozue, Ami; Narumi, Tetsuo; Hashimoto, Chie; Tanaka, Tomohiro; Pommier, Yves; Yamamoto, Naoki; Komano, Jun A; Murakami, Tsutomu; Tamamura, Hirokazu

    2013-10-18

    HIV-1 integrase (IN) is an enzyme which is indispensable for the stable infection of host cells because it catalyzes the insertion of viral DNA into the genome and thus is an attractive target for the development of anti-HIV agents. Earlier, we found Vpr-derived peptides with inhibitory activity against HIV-1 IN. These Vpr-derived peptides are originally located in an α-helical region of the parent Vpr protein. Addition of an octa-arginyl group to the inhibitory peptides caused significant inhibition against HIV replication associated with an increase in cell permeability but also relatively high cytotoxicity. In the current study, stapled peptides, a new class of stabilized α-helical peptidomimetics were adopted to enhance the cell permeability of the above lead peptides. A series of stapled peptides, which have a hydrocarbon link formed by a ruthenium-catalyzed ring-closing metathesis reaction between successive turns of α-helix, were designed, synthesized, and evaluated for biological activity. In cell-based assays some of the stapled peptides showed potent anti-HIV activity comparable with that of the original octa-arginine-containing peptide (2) but with lower cytotoxicity. Fluorescent imaging experiments revealed that these stapled peptides are significantly cell permeable, and CD analysis showed they form α-helical structures, whereas the unstapled congeners form β-sheet structures. The application of this stapling strategy to Vpr-derived IN inhibitory peptides led to a remarkable increase in their potency in cells and a significant reduction of their cytotoxicity.

  11. Feline leukemia virus integrase and capsid packaging functions do not change the insertion profile of standard Moloney retroviral vectors.

    PubMed

    Métais, J-Y; Topp, S; Doty, R T; Borate, B; Nguyen, A-D; Wolfsberg, T G; Abkowitz, J L; Dunbar, C E

    2010-06-01

    Adverse events linked to perturbations of cellular genes by vector insertion reported in gene therapy trials and animal models have prompted attempts to better understand the mechanisms directing viral vector integration. The integration profiles of vectors based on MLV, ASLV, SIV and HIV have all been shown to be non-random, and novel vectors with a safer integration pattern have been sought. Recently, we developed a producer cell line called CatPac that packages standard MoMLV vectors with feline leukemia virus (FeLV) gag, pol and env gene products. We now report the integration profile of this vector, asking if the FeLV integrase and capsid proteins could modify the MoMLV integration profile, potentially resulting in a less genotoxic pattern. We transduced rhesus macaque CD34+ hematopoietic progenitor cells with CatPac or standard MoMLV vectors, and determined their integration profile by LAM-PCR. We obtained 184 and 175 unique integration sites (ISs) respectively for CatPac and standard MoMLV vectors, and these were compared with 10 000 in silico-generated random IS. The integration profile for CatPac vector was similar to MoMLV and equally non-random, with a propensity for integration near transcription start sites and in highly dense gene regions. We found an IS for CatPac vector localized 715 nucleotides upstream of LMO-2, the gene involved in the acute lymphoblastic leukemia developed by X-SCID patients treated by gene therapy using MoMLV vectors. In conclusion, we found that replacement of MoMLV env, gag and pol gene products with FeLV did not alter the basic integration profile. Thus, there appears to be no safety advantage for this packaging system. However, considering the stability and efficacy of CatPac vectors, further development is warranted, using potentially safer vector backbones, for instance those with a SIN configuration.

  12. Development of magnetic graphene oxide adsorbent for the removal and preconcentration of As(III) and As(V) species from environmental water samples.

    PubMed

    Rashidi Nodeh, Hamid; Wan Ibrahim, Wan Aini; Ali, Imran; Sanagi, Mohd Marsin

    2016-05-01

    New-generation adsorbent, Fe3O4@SiO2/GO, was developed by modification of graphene oxide (GO) with silica-coated (SiO2) magnetic nanoparticles (Fe3O4). The synthesized adsorbent was characterized using Fourier transform infrared spectroscopy, X-ray diffractometry, energy-dispersive X-ray spectroscopy, and field emission scanning electron microscopy. The developed adsorbent was used for the removal and simultaneous preconcentration of As(III) and As(V) from environmental waters prior to ICP-MS analysis. Fe3O4@SiO2/GO provided high adsorption capacities, i.e., 7.51 and 11.46 mg g(-1) for As(III) and As(V), respectively, at pH 4.0. Adsorption isotherm, kinetic, and thermodynamic were investigated for As(III) and As(V) adsorption. Preconcentration of As(III) and As(V) were studied using magnetic solid-phase extraction (MSPE) method at pH 9.0 as the adsorbent showed selective adsorption for As(III) only in pH range 7-10. MSPE using Fe3O4@SiO2/GO was developed with good linearities (0.05-2.0 ng mL(-1)) and high coefficient of determination (R (2) = 0.9992 and 0.9985) for As(III) and As(V), respectively. The limits of detection (LODs) (3× SD/m, n = 3) obtained were 7.9 pg mL(-1) for As(III) and 28.0 pg mL(-1) for As(V). The LOD obtained is 357-1265× lower than the WHO maximum permissible limit of 10.0 ng mL(-1). The developed MSPE method showed good relative recoveries (72.55-109.71 %) and good RSDs (0.1-4.3 %, n = 3) for spring water, lake, river, and tap water samples. The new-generation adsorbent can be used for the removal and simultaneous preconcentration of As(III) and As(V) from water samples successfully. The adsorbent removal for As(III) is better than As(V).

  13. Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31.

    PubMed Central

    Groth, Amy C; Fish, Matthew; Nusse, Roel; Calos, Michele P

    2004-01-01

    The phiC31 integrase functions efficiently in vitro and in Escherichia coli, yeast, and mammalian cells, mediating unidirectional site-specific recombination between its attB and attP recognition sites. Here we show that this site-specific integration system also functions efficiently in Drosophila melanogaster in cultured cells and in embryos. Intramolecular recombination in S2 cells on transfected plasmid DNA carrying the attB and attP recognition sites occurred at a frequency of 47%. In addition, several endogenous pseudo attP sites were identified in the fly genome that were recognized by the integrase and used as substrates for integration in S2 cells. Two lines of Drosophila were created by integrating an attP site into the genome with a P element. phiC31 integrase injected into embryos as mRNA functioned to promote integration of an attB-containing plasmid into the attP site, resulting in up to 55% of fertile adults producing transgenic offspring. A total of 100% of these progeny carried a precise integration event at the genomic attP site. These experiments demonstrate the potential for precise genetic engineering of the Drosophila genome with the phiC31 integrase system and will likely benefit research in Drosophila and other insects. PMID:15126397

  14. Nanobody-based chimeric receptor gene integration in Jurkat cells mediated by PhiC31 integrase

    SciTech Connect

    Iri-Sofla, Farnoush Jafari; Rahbarizadeh, Fatemeh; Ahmadvand, Davoud; Rasaee, Mohammad J.

    2011-11-01

    The crucial role of T lymphocytes in anti-tumor immunity has led to the development of novel strategies that can target and activate T cells against tumor cells. Recombinant DNA technology has been used to generate non-MHC-restricted chimeric antigen receptors (CARs). Here, we constructed a panel of recombinant CAR that harbors the anti-MUC1 nanobody and the signaling and co-signaling moieties (CD3{zeta}/CD28) with different spacer regions derived from human IgG3 with one or two repeats of the hinge sequence or the hinge region of Fc{gamma}RII. The PhiC31 integrase system was employed to investigate if the recombination efficiency could be recruited for high and stable expression of T cell chimeric receptor genes. The effect of nuclear localization signal (NLS) and two different promoters (CMV and CAG) on efficacy of PhiC31 integrase in human T cell lines was evaluated. The presence of integrase in combination with NLS, mediated up to 7.6 and 8.5 fold increases in CAR expression in ZCHN-attB and ZCHHN-attB cassette integrated T cells, respectively. Our results showed that highly efficient and stable transduction of the Jurkat cell line by PhiC31 integrase is a feasible modality for generating anti-cancer chimeric T cells for use in cancer immunotherapy.

  15. Impacts of Shewanella putrefaciens strain CN-32 cells and extracellular polymeric substances on the sorption of As(V) and As(III) on Fe(III)-(hydr)oxides.

    PubMed

    Huang, Jen-How; Elzinga, Evert J; Brechbuehl, Yves; Voegelin, Andreas; Kretzschmar, Ruben

    2011-04-01

    We investigated the effects of Shewanella putrefaciens cells and extracellular polymeric substances on the sorption of As(III) and As(V) to goethite, ferrihydrite, and hematite at pH 7.0. Adsorption of As(III) and As(V) at solution concentrations between 0.001 and 20 μM decreased by 10 to 45% in the presence of 0.3 g L(-1) EPS, with As(III) being affected more strongly than As(V). Also, inactivated Shewanella cells induced desorption of As(V) from the Fe(III)-(hydr)oxide mineral surfaces. ATR-FTIR studies of ternary As(V)-Shewanella-hematite systems indicated As(V) desorption concurrent with attachment of bacterial cells at the hematite surface, and showed evidence of inner-sphere coordination of bacterial phosphate and carboxylate groups at hematite surface sites. Competition between As(V) and bacterial phosphate and carboxylate groups for Fe(III)-(oxyhydr)oxide surface sites is proposed as an important factor leading to increased solubility of As(V). The results from this study have implications for the solubility of As(V) in the soil rhizosphere and in geochemical systems undergoing microbially mediated reduction and indicate that the presence of sorbed oxyanions may affect Fe-reduction and biofilm development at mineral surfaces.

  16. In vitro activity of dolutegravir against wild-type and integrase inhibitor-resistant HIV-2.

    PubMed

    Smith, Robert A; Raugi, Dana N; Pan, Charlotte; Sow, Papa Salif; Seydi, Moussa; Mullins, James I; Gottlieb, Geoffrey S

    2015-02-05

    Dolutegravir recently became the third integrase strand transfer inhibitor (INSTI) approved for use in HIV-1-infected individuals. In contrast to the extensive dataset for HIV-1, in vitro studies and clinical reports of dolutegravir for HIV-2 are limited. To evaluate the potential role of dolutegravir in HIV-2 treatment, we compared the susceptibilities of wild-type and INSTI-resistant HIV-1 and HIV-2 strains to the drug using single-cycle assays, spreading infections of immortalized T cells, and site-directed mutagenesis. HIV-2 group A, HIV-2 group B, and HIV-1 isolates from INSTI-naïve individuals were comparably sensitive to dolutegravir in the single-cycle assay (mean EC50 values = 1.9, 2.6, and 1.3 nM, respectively). Integrase substitutions E92Q, Y143C, E92Q + Y143C, and Q148R conferred relatively low levels of resistance to dolutegravir in HIV-2ROD9 (2- to 6-fold), but Q148K, E92Q + N155H, T97A + N155H and G140S + Q148R resulted in moderate resistance (10- to 46-fold), and the combination of T97A + Y143C in HIV-2ROD9 conferred high-level resistance (>5000-fold). In contrast, HIV-1NL4-3 mutants E92Q + N155H, G140S + Q148R, and T97A + Y143C showed 2-fold, 4-fold, and no increase in EC50, respectively, relative to the parental strain. The resistance phenotypes for E92Q + N155H, and G140S + Q148R HIV-2ROD9 were also confirmed in spreading infections of CEM-ss cells. Our data support the use of dolutegravir in INSTI-naïve HIV-2 patients but suggest that, relative to HIV-1, a broader array of replacements in HIV-2 integrase may enable cross-resistance between dolutegravir and other INSTI. Clinical studies are needed to evaluate the efficacy of dolutegravir in HIV-2-infected individuals, including patients previously treated with raltegravir or elvitegravir.

  17. HIV Type 1 Integrase Natural Polymorphisms in Viral Variants Circulating in FSU Countries.

    PubMed

    Lapovok, Ilya; Laga, Vita; Kazennova, Elena; Bobkova, Marina

    2017-08-15

    Natural variability of integrase (IN) across HIV-1 variants may influence the emergence of resistant viruses. The most apparent explanation of these fact is the IN polymorphism and the associated differences in codon usage, which, in turn, influence the probability and the terms of DRMs acquisition. Possible mechanisms by which polymorphisms affect DRMs emergence remain disputed and should still be clarified because these substitutions may be associated with a reduced activity of some INSTIs and may impact on ART regimen choice depending of HIV-1 subtype. The aim of this work was to assess the prevalence of naturally occurring polymorphisms within the HIV-1 integrase gene, which might influence the susceptibility to INSTIs, among the patients from Russia and former USSR countries, according to HIV-1 subtypes. A study involved 506 HIV-1 IN sequences of INSTI-naive patients from Russia, Ukraine, Armenia, Kyrgyzstan, Kazakhstan, Uzbekistan, Belarus, and Georgia. Among them, 194 sequences were newly obtained in this study and 312 were downloaded from Los-Alamos database. The proviral DNA was sequenced using an in-house PCR protocol designed on the basis of a well-conserved integrase region in order to detect all HIV-1 variants. The phylogenetic analyses based on IN population sequencing found subtype A6 being the most prevalent (259) (51.2%) in the collection studied, followed by subtype G (36) (7.1%), AG-recombinants (148) (29.3%), subtype B (50) (9.9%), and CRF03_AB (5) (1,0%). The major INSTI resistance-associated mutations (DRMs) were found only in two A6 samples. The prevalence of minor/accessory substitutions depended on HIV-1 variants, while the most notable findings were L74I in subtype A6 (93.1%) and E157Q in subtype B (44.0%). Most of minor DRMs and polymorphic substitutions were concentrated in the central catalytic domain of the IN molecule. Both the DDE triad and HHCC zinc binding motifs were fully conserved. The results of the study suggest a very low

  18. Chemical reactive features of novel amino acids intercalated layered double hydroxides in As(III) and As(V) adsorption.

    PubMed

    Shen, Liang; Jiang, Xiuli; Chen, Zheng; Fu, Dun; Li, Qingbiao; Ouyang, Tong; Wang, Yuanpeng

    2017-06-01

    Layered double hydroxides (LDHs) intercalated with amino acids such as methionine (Met) were synthesized as new adsorbents to remediate arsenic-polluted water. This Zn2Al-Met-LDHs, identified with the formula of Zn0.7Al0.3(OH)2(Met)0.3·0.32H2O, has good thermal stability. Adsorption experiments with Zn2Al-Met-LDHs showed that the residual arsenic in solution could be reduced below the regulation limit, and this adsorption process fitted Langmuir isotherm and the pseudo-second-order kinetics well. A remarkably high removal efficiency and the maximum adsorption capacity for As(III) were achieved, 96.7% and 94.1 mg/g, respectively, at 298 K. The desorption efficiency of As(III) from the arsenic-saturated Zn2Al-Met-LDHs (<8.7%), far less than that of As(V), promises a specific and reliable uptake of As(III) in sorts of solutions. More importantly, a complete and in-depth spectra analysis through FTIR, XPS and NMR was conducted to explain the excellent performance of Zn2Al-Met-LDHs in arsenic removal. Herein, two special chemical reactions were proposed as the dominant mechanisms, i.e., hydrogen bonding between the carboxyl group of the host Met and the hydroxyl group of As(III) or As(V), and the formation of a chelate ring between the guest As(III) and the S, N bidentate ligands of the intercalated Met in the LDHs.

  19. Nature and reactivity of layered double hydroxides formed by coprecipitating Mg, Al and As(V): Effect of arsenic concentration, pH, and aging.

    PubMed

    Sommella, Alessia; Caporale, Antonio G; Denecke, Melissa A; Mangold, Stefan; Pigna, Massimo; Santoro, Anna; Terzano, Roberto; Violante, Antonio

    2015-12-30

    Arsenic (As) co-precipitation is one of the major processes controlling As solubility in soils and waters. When As is co-precipitated with Al and Mg, the possible formation of layered double hydroxides (LDHs) and other nanocomposites can stabilize As in their structures thus making this toxic element less available. We investigated the nature and reactivity of Mg-Al-arsenate [As(V)] co-precipitated LDHs formed in solution affected by As concentration, pH, and aging. At the beginning of the co-precipitation process, poorly crystalline LDH and non-crystalline Al(Mg)-oxides form. Prolonged aging of the samples promotes crystallization of LDHs, evidenced by an increase in As K XANES intensities and XRD peak intensities. During aging Al- and/or Mg-oxides are likely transformed by dissolution/re-precipitation processes into more crystalline but still defective LDHs. Surface area, chemical composition, reactivity of the precipitates, and anion exchange properties of As(V) in the co-precipitates are influenced by pH, aging, and As concentration. This study demonstrates that (i) As(V) retards or inhibits the formation and transformation of LDHs and (ii) more As(V) is removed from solution if co-precipitated with Mg and Al than by sorption onto well crystallized LDHs. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Successive extraction of As(V), Cu(II) and P(V) ions from water using spent coffee powder as renewable bioadsorbents

    PubMed Central

    Hao, Linlin; Wang, Peng; Valiyaveettil, Suresh

    2017-01-01

    For the first time, renewable and easy accessible pre-bleached spent coffee powder coated with polyethylenimine (PEI) and ferric ions (Coffee-PEI-Fe) was used for the successive adsorption of As(V), Cu(II) and P(V) ions from spiked water samples. Fully characterized coffee-PEI-Fe was employed for batch mode experiments. Kinetic regression analysis showed that the adsorption processes of As(V) and P(V) anions follows a pseudo-second-order model, while the adsorption of Cu(II) ions fit with a pseudo-first-order model. The maximum adsorption capacities estimated by Langmuir model for As(V), Cu(II) and P(V) ions were 83.3, 200.1, and 50.2 mg/g, respectively. The simulated results revealed that the internal diffusion is the rate-determining step for the adsorptions of As(V) and Cu(II) ions, while film diffusion is the mass transfer resistance for the adsorption of P(V) ions on the surface of coffee-PEI-Fe. The successive adsorptions of adsorbates were achieved through electrostatic attraction between adsorbent surface and adsorbates. The dynamic column adsorption behavior of the adsorbent was described by Thomas model, which showed a good agreement with the experimental values (qexp). The results presented in this paper could be used for developing efficient adsorbent from renewable materials for water purification. PMID:28220853

  1. Equilibrium and kinetics studies on As(V) and Sb(V) removal by Fe2+ -doped Mg-Al layered double hydroxides.

    PubMed

    Kameda, Tomohito; Kondo, Eisuke; Yoshioka, Toshiaki

    2015-03-15

    Mg-Al layered double hydroxides (Mg-Al LDHs) doped with Fe(2+) adsorbed As(V) [Formula: see text] and Sb(V) [Formula: see text] from an aqueous solution through anion exchange with Cl(-) intercalated in the LDH interlayer. Fe(2+)-doped Mg-Al LDH exhibited superior As(V) removal compared with Mg-Al LDH. The oxidation of Fe(2+) doped in the Mg-Al LDH host layer to Fe(3+) increased the positive layer charge of the LDH, thus increasing the anion-uptake capacity owing to stronger electrostatic attractive force between the positively charged layer and the anion. However, Fe(2+)-doped Mg-Al LDH was not superior to Mg-Al LDH in terms of Sb(V) removal. This was attributed to the preferential intercalation of OH(-) over [Formula: see text] . The As(V) and Sb(V) removal by LDH followed Langmuir-type adsorption, which proceeded via a pseudo-first-order reaction. The equilibrium and kinetics studies confirm that the adsorption of As(V) and Sb(V) by Fe(2+)-doped Mg-Al LDH was the result of chemical adsorption, involving the anion exchange of [Formula: see text] and [Formula: see text] with the intercalated Cl(-).

  2. Removal of As(V), Cr(III) and Cr(VI) from aqueous environments by poly(acrylonitril-co-acrylamidopropyl-trimethyl ammonium chloride)-based hydrogels.

    PubMed

    Dudu, Tuba Ersen; Sahiner, Mehtap; Alpaslan, Duygu; Demirci, Sahin; Aktas, Nahit

    2015-09-15

    Cationic poly(Acrylonitril-co-Acrylamidopropyl-trimethyl Ammonium Chloride) (p(AN-co-APTMACl)) hydrogels in bulk were synthesized by using acrylonitrile (AN) and 3-acrylamidopropyl-trimethyl ammonium chloride (APTMACl) as monomers. The prepared hydrogels were exposed to amidoximation reaction to replace hydrophobic nitrile groups with hydrophilic amidoxime groups that have metal ion binding ability. Those replacements were increased the hydrogels absorption capacity for As(V) and Cr(VI). Langmuir and Freundlich isotherms equations were utilized to obtain the best-fitted isotherm model for the absorption of the ions at different metal ion concentrations. The absorption data of As(V) ion were fitted well to Freundlich isotherm while those of Cr(VI) and Cr(III) ions were fitted well to Langmuir isotherm. The maximum absorption of poly(3-acrylamidopropyl-trimethyl ammonium chloride (p(APTMACl)) and amid-p(AN-co-APTMACl) macro gels were 22.39 mg and 21.83 mg for As(V), and 30.65 mg and 18.16 mg for Cr(VI) ion per unit gram dried gel, respectively. Kinetically, the absorption behaviors of Cr(III) and Cr(VI) ions were fitted well to a pseudo 2nd-order kinetic model and those of As(V) ions were fitted well to a pseudo 1st order kinetic model.

  3. Speciation of AsIII and AsV in fruit juices by dispersive liquid–liquid microextraction and hydride generation-atomic fluorescence spectrometry

    USDA-ARS?s Scientific Manuscript database

    A new procedure was developed to speciate and quantify As(III) and As(V) in fruit juices. At pH 3.0, As(III) and ammonium pyrrolidine dithiocarbamate (APDC) formed a complex, which was extracted into carbon tetrachloride by dispersive liquid–liquid microextraction (DLLME) and subsequently quantified...

  4. Impact of carbon nanotubes on the toxicity of inorganic arsenic [AS(III) and AS(V)] to Daphnia magna: The role of certain arsenic species.

    PubMed

    Wang, Xinghao; Qu, Ruijuan; Allam, Ahmed A; Ajarem, Jamaan; Wei, Zhongbo; Wang, Zuoyao

    2016-07-01

    As a type of emerging nanomaterial, hydroxylated multiwalled carbon nanotubes (OH-MWCNTs) may interact with other pollutants in the aquatic environments and further influence their toxicity, transport, and fate. Thus, evaluation of toxicity to arsenic in the presence of CNTs needs to receive much more attention. The present study was conducted to explore the underlying mechanisms of OH-MWCNT-induced arsenic (As[III] and As[V]) toxicity changes in the aquatic organism Daphnia magna at different pH levels. The most toxic species for As(III) and As(V) to D. magna were found to be H2 AsO3 (-) and H2 AsO4 (-) . It appeared that the pH values were of greatest importance when the biological toxicity of As(III) and As(V) was compared. Furthermore, the effects of OH-MWCNTs on arsenic toxicity to D. magna indicated that the presence of OH-MWCNTs could enhance the toxicity of arsenic. The interactions of arsenic with OH-MWCNTs were further investigated by conducting adsorption experiments. The adsorption capacity of As(V) by OH-MWCNTs was found to be higher than that of As(III). To conclude, adsorption of certain arsenic species onto OH-MWCNTs is crucial for a reliable interpretation of enhanced toxicity. Environ Toxicol Chem 2016;35:1852-1859. © 2016 SETAC.

  5. Spectroscopic characterization and solubility investigation on the effects of As(V) on mineral structure tooeleite (Fe₆(AsO₃)₂SO₄(OH)₂·H₂O).

    PubMed

    Liu, Jing; Deng, Shiming; Zhao, Fenghua; Cheng, Hongfei; Frost, Ray L

    2015-01-05

    Tooeleite is an unique ferric arsenite sulfate mineral, which has the potential significance of directly fixing As(III) as mineral trap. The tooeleite and various precipitates were hydrothermally synthesized under the different of initial As(III)/As(V) molar ratios and characterized by XRD, FTIR, XPS and SEM. The crystallinity of tooeleite decreases with the amount of As(V). The precipitate is free of any crystalline tooeleite at the level of that XRD could detect when the ratio of As(III)/As(V) of 7:3 and more. The characteristic bands of tooeleite are observed in 772, 340, 696 and 304 cm(-1), which are assigned to the ν₁, ν₂, ν₃ and ν₄ vibrations of AsO₃(3-). These intensities of bands gradually decreases with the presence of As(V) and its increasing. An obviously wide band is observed in 830 cm(-1), which is the ν₁ vibration of AsO₄. The result of XPS reveals that the binding energies of As₃d increase from 44.0 eV to 45.5 eV, which indicates that the amount of As(V) in the precipitates increases. The concentrations of arsenic released of these precipitates are 350-650 mg/L. The stability of tooeleite decreases by comparison when the presence of coexisting As(V) ions. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. TISSUE DISTRIBUTION AND URINARY EXCRETION OF INORGANIC ARSENIC AND ITS METHYLATED METABOLITES IN C57BL/6 MICE FOLLOWING SUBCHRONIC EXPOSURE TO ARSENATE (ASV) IN DRINKING WATER

    EPA Science Inventory

    The relationship of exposure and tissue concentration of parent chemical and metabolites over prolonged exposure is a critical issue for chronic toxicities mediated by metabolite(s) rather than parent chemical alone. This is an issue for AsV because its trivalent metabolites hav...

  7. TISSUE DISTRIBUTION AND URINARY EXCRETION OF INORGANIC ARSENIC AND ITS METHYLATED METABOLITES IN C57BL/6 MICE FOLLOWING SUBCHRONIC EXPOSURE TO ARSENATE (ASV) IN DRINKING WATER

    EPA Science Inventory

    The relationship of exposure and tissue concentration of parent chemical and metabolites over prolonged exposure is a critical issue for chronic toxicities mediated by metabolite(s) rather than parent chemical alone. This is an issue for AsV because its trivalent metabolites hav...

  8. Successive extraction of As(V), Cu(II) and P(V) ions from water using spent coffee powder as renewable bioadsorbents

    NASA Astrophysics Data System (ADS)

    Hao, Linlin; Wang, Peng; Valiyaveettil, Suresh

    2017-02-01

    For the first time, renewable and easy accessible pre-bleached spent coffee powder coated with polyethylenimine (PEI) and ferric ions (Coffee-PEI-Fe) was used for the successive adsorption of As(V), Cu(II) and P(V) ions from spiked water samples. Fully characterized coffee-PEI-Fe was employed for batch mode experiments. Kinetic regression analysis showed that the adsorption processes of As(V) and P(V) anions follows a pseudo-second-order model, while the adsorption of Cu(II) ions fit with a pseudo-first-order model. The maximum adsorption capacities estimated by Langmuir model for As(V), Cu(II) and P(V) ions were 83.3, 200.1, and 50.2 mg/g, respectively. The simulated results revealed that the internal diffusion is the rate-determining step for the adsorptions of As(V) and Cu(II) ions, while film diffusion is the mass transfer resistance for the adsorption of P(V) ions on the surface of coffee-PEI-Fe. The successive adsorptions of adsorbates were achieved through electrostatic attraction between adsorbent surface and adsorbates. The dynamic column adsorption behavior of the adsorbent was described by Thomas model, which showed a good agreement with the experimental values (qexp). The results presented in this paper could be used for developing efficient adsorbent from renewable materials for water purification.

  9. Successive extraction of As(V), Cu(II) and P(V) ions from water using spent coffee powder as renewable bioadsorbents.

    PubMed

    Hao, Linlin; Wang, Peng; Valiyaveettil, Suresh

    2017-02-21

    For the first time, renewable and easy accessible pre-bleached spent coffee powder coated with polyethylenimine (PEI) and ferric ions (Coffee-PEI-Fe) was used for the successive adsorption of As(V), Cu(II) and P(V) ions from spiked water samples. Fully characterized coffee-PEI-Fe was employed for batch mode experiments. Kinetic regression analysis showed that the adsorption processes of As(V) and P(V) anions follows a pseudo-second-order model, while the adsorption of Cu(II) ions fit with a pseudo-first-order model. The maximum adsorption capacities estimated by Langmuir model for As(V), Cu(II) and P(V) ions were 83.3, 200.1, and 50.2 mg/g, respectively. The simulated results revealed that the internal diffusion is the rate-determining step for the adsorptions of As(V) and Cu(II) ions, while film diffusion is the mass transfer resistance for the adsorption of P(V) ions on the surface of coffee-PEI-Fe. The successive adsorptions of adsorbates were achieved through electrostatic attraction between adsorbent surface and adsorbates. The dynamic column adsorption behavior of the adsorbent was described by Thomas model, which showed a good agreement with the experimental values (qexp). The results presented in this paper could be used for developing efficient adsorbent from renewable materials for water purification.

  10. The ars Detoxification System Is Advantageous but Not Required for As(V) Respiration by the Genetically Tractable Shewanella Species Strain ANA-3

    PubMed Central

    Saltikov, Chad W.; Cifuentes, Ana; Venkateswaran, Kasthuri; Newman, Dianne K.

    2003-01-01

    Arsenate [As(V); HAsO42−] respiration by bacteria is poorly understood at the molecular level largely due to a paucity of genetically tractable organisms with this metabolic capability. We report here the isolation of a new As(V)-respiring strain (ANA-3) that is phylogenetically related to members of the genus Shewanella and that also provides a useful model system with which to explore the molecular basis of As(V) respiration. This gram-negative strain stoichiometrically couples the oxidation of lactate to acetate with the reduction of As(V) to arsenite [As(III); HAsO2]. The generation time and lactate molar growth yield (Ylactate) are 2.8 h and 10.0 g of cells mol of lactate−1, respectively, when it is grown anaerobically on lactate and As(V). ANA-3 uses a wide variety of terminal electron acceptors, including oxygen, soluble ferric iron, oxides of iron and manganese, nitrate, fumarate, the humic acid functional analog 2,6-anthraquinone disulfonate, and thiosulfate. ANA-3 also reduces As(V) to As(III) in the presence of oxygen and resists high concentrations of As(III) (up to 10 mM) when grown under either aerobic or anaerobic conditions. ANA-3 possesses an ars operon (arsDABC) that allows it to resist high levels of As(III); this operon also confers resistance to the As-sensitive strains Shewanella oneidensis MR-1 and Escherichia coli AW3110. When the gene encoding the As(III) efflux pump, arsB, is inactivated in ANA-3 by a polar mutation that also eliminates the expression of arsC, which encodes an As(V) reductase, the resulting As(III)-sensitive strain still respires As(V); however, the generation time and the Ylactate value are two- and threefold lower, respectively, than those of the wild type. These results suggest that ArsB and ArsC may be useful for As(V)-respiring bacteria in environments where As concentrations are high, but that neither is required for respiration. PMID:12732551

  11. The ars detoxification system is advantageous but not required for As(V) respiration by the genetically tractable Shewanella species strain ANA-3.

    PubMed

    Saltikov, Chad W; Cifuentes, Ana; Venkateswaran, Kasthuri; Newman, Dianne K

    2003-05-01

    Arsenate [As(V); HAsO(4)(2-)] respiration by bacteria is poorly understood at the molecular level largely due to a paucity of genetically tractable organisms with this metabolic capability. We report here the isolation of a new As(V)-respiring strain (ANA-3) that is phylogenetically related to members of the genus Shewanella and that also provides a useful model system with which to explore the molecular basis of As(V) respiration. This gram-negative strain stoichiometrically couples the oxidation of lactate to acetate with the reduction of As(V) to arsenite [As(III); HAsO(2)]. The generation time and lactate molar growth yield (Y(lactate)) are 2.8 h and 10.0 g of cells mol of lactate(-1), respectively, when it is grown anaerobically on lactate and As(V). ANA-3 uses a wide variety of terminal electron acceptors, including oxygen, soluble ferric iron, oxides of iron and manganese, nitrate, fumarate, the humic acid functional analog 2,6-anthraquinone disulfonate, and thiosulfate. ANA-3 also reduces As(V) to As(III) in the presence of oxygen and resists high concentrations of As(III) (up to 10 mM) when grown under either aerobic or anaerobic conditions. ANA-3 possesses an ars operon (arsDABC) that allows it to resist high levels of As(III); this operon also confers resistance to the As-sensitive strains Shewanella oneidensis MR-1 and Escherichia coli AW3110. When the gene encoding the As(III) efflux pump, arsB, is inactivated in ANA-3 by a polar mutation that also eliminates the expression of arsC, which encodes an As(V) reductase, the resulting As(III)-sensitive strain still respires As(V); however, the generation time and the Y(lactate) value are two- and threefold lower, respectively, than those of the wild type. These results suggest that ArsB and ArsC may be useful for As(V)-respiring bacteria in environments where As concentrations are high, but that neither is required for respiration.

  12. Addition of E138K to R263K in HIV integrase increases resistance to dolutegravir, but fails to restore activity of the HIV integrase enzyme and viral replication capacity

    PubMed Central

    Mesplède, Thibault; Osman, Nathan; Wares, Melissa; Quashie, Peter K.; Hassounah, Said; Anstett, Kaitlin; Han, Yingshan; Singhroy, Diane N.; Wainberg, Mark A.

    2014-01-01

    Background The results of several clinical trials suggest that the integrase inhibitor dolutegravir may be less prone than other drugs to the emergence of HIV drug resistance mutations in treatment-naive patients. We have shown that the R263K mutation commonly emerged during tissue culture selection studies with dolutegravir and conferred low levels of resistance to this drug while simultaneously diminishing both HIV replication capacity and integrase enzymatic activity. E138K has been identified as a secondary mutation for dolutegravir in selection studies and has also been observed as a secondary mutation in the clinic for the integrase inhibitors raltegravir and elvitegravir. Methods We used biochemical cell-free strand-transfer assays and tissue culture assays to characterize the effects of the E138K/R263K combination of mutations on resistance to dolutegravir, integrase enzyme activity and HIV-1 replication capacity. Results We show here that the addition of the E138K substitution to R263K increased the resistance of HIV-1 to dolutegravir but failed to restore viral replication capacity, integrase strand-transfer activity and integration within cellular DNA. We also show that the addition of E138K to R263K did not increase the resistance to raltegravir or elvitegravir. The addition of the E138K substitution to R263K was also less detrimental to integrase strand-transfer activity and integration than a different secondary mutation at position H51Y that had also been selected in culture. Conclusions The E138K substitution failed to restore the defect in viral replication capacity that is associated with R263K, confirming previous selection studies that failed to identify compensatory mutation(s) for the latter primary mutation. This study suggests that the R263K resistance pathway may represent an evolutionary dead end for HIV in treatment-naive individuals who are treated with dolutegravir and will need to be confirmed by the long-term use of dolutegravir in

  13. Addition of E138K to R263K in HIV integrase increases resistance to dolutegravir, but fails to restore activity of the HIV integrase enzyme and viral replication capacity.

    PubMed

    Mesplède, Thibault; Osman, Nathan; Wares, Melissa; Quashie, Peter K; Hassounah, Said; Anstett, Kaitlin; Han, Yingshan; Singhroy, Diane N; Wainberg, Mark A

    2014-10-01

    The results of several clinical trials suggest that the integrase inhibitor dolutegravir may be less prone than other drugs to the emergence of HIV drug resistance mutations in treatment-naive patients. We have shown that the R263K mutation commonly emerged during tissue culture selection studies with dolutegravir and conferred low levels of resistance to this drug while simultaneously diminishing both HIV replication capacity and integrase enzymatic activity. E138K has been identified as a secondary mutation for dolutegravir in selection studies and has also been observed as a secondary mutation in the clinic for the integrase inhibitors raltegravir and elvitegravir. We used biochemical cell-free strand-transfer assays and tissue culture assays to characterize the effects of the E138K/R263K combination of mutations on resistance to dolutegravir, integrase enzyme activity and HIV-1 replication capacity. We show here that the addition of the E138K substitution to R263K increased the resistance of HIV-1 to dolutegravir but failed to restore viral replication capacity, integrase strand-transfer activity and integration within cellular DNA. We also show that the addition of E138K to R263K did not increase the resistance to raltegravir or elvitegravir. The addition of the E138K substitution to R263K was also less detrimental to integrase strand-transfer activity and integration than a different secondary mutation at position H51Y that had also been selected in culture. The E138K substitution failed to restore the defect in viral replication capacity that is associated with R263K, confirming previous selection studies that failed to identify compensatory mutation(s) for the latter primary mutation. This study suggests that the R263K resistance pathway may represent an evolutionary dead end for HIV in treatment-naive individuals who are treated with dolutegravir and will need to be confirmed by the long-term use of dolutegravir in the clinic. © The Author 2014. Published

  14. Newer tetracycline derivatives: synthesis, anti-HIV, antimycobacterial activities and inhibition of HIV-1 integrase.

    PubMed

    Sriram, Dharmarajan; Yogeeswari, Perumal; Senchani, Geetha; Banerjee, Debjani

    2007-04-15

    A series of new tetracycline derivatives has been synthesized by reacting appropriate tetracyclines, formaldehyde and secondary amino (piperazino) function of fluoroquinolones using microwave irradiation with the yield ranging from 41 evaluated for its anti-HIV, antimycobacterial activities and HIV-1 integrase (IN) enzyme inhibition studies. Among the synthesized compounds, compound 10 was found to be the most promising compound active against HIV-1 replication with EC(50) of 5.2 microM and was nontoxic to the CEM cells until 200 microM, and MIC of 0.2 microg/mL against Mycobacterium tuberculosis, with moderate inhibition of both 3'-processing and strand transfer steps of HIV-1 IN.

  15. Progress in HIV-1 Integrase Inhibitors: A Review of their Chemical Structure Diversity

    PubMed Central

    Hajimahdi, Zahra; Zarghi, Afshin

    2016-01-01

    HIV-1 integrase (IN) enzyme, one of the three main enzymes of HIV-1, catalyzed the insertion of the viral DNA into the genome of host cells. Because of the lack of its homologue in human cells and its essential role in HIV-1 replication, IN inhibition represents an attractive therapeutic target for HIV-1 treatment. Since identification of IN as a promising therapeutic target, a major progress has been made, which has facilitated and led to the approval of three drugs. This review focused on the structural features of the most important IN inhibitors and categorized them structurally in 10 scaffolds. We also briefly discussed the structural and functional properties of HIV-1 IN and binding modes of IN inhibitors. The SAR analysis of the known IN inhibitors provides some useful clues to the possible future discovery of novel IN inhibitors. PMID:28243261

  16. 6,7-Dihydroxy-1-oxoisoindoline-4-sulfonamide-containing HIV-1 Integrase Inhibitors

    PubMed Central

    Zhao, Xue Zhi; Maddali, Kasthuraiah; Smith, Steven J.; Métifiot, Mathieu; Johnson, Barry C.; Marchand, Christophe; Hughes, Stephen H.; Pommier, Yves; Burke, Terrence R.

    2012-01-01

    Although an extensive body of scientific and patent literature exists describing the development of HIV-1 integrase (IN) inhibitors, Merck’s raltegravir and Gilead’s elvitegravir remain the only IN inhibitors FDA-approved for the treatment of AIDS. The emergence of raltegravir-resistant strains of HIV-1 containing mutated forms of IN underlies the need for continued efforts to enhance the efficacy of IN inhibitors against resistant mutants. We have previously described bicyclic 6,7-dihydroxyoxoisoindolin-1-ones that show good IN inhibitory potency. This report describes the effects of introducing substituents into the 4- and 5- positions of the parent 6,7-dihydroxyoxoisoindolin-1-one platform. We have developed several sulfonamide-containing analogs that enhance potency in cell-based HIV assays by more than two orders-of-magnitude and we describe several compounds that are more potent than raltegravir against the clinically relevant Y143R IN mutant. PMID:23149229

  17. Successful Prevention of Transmission of Integrase Resistance in the Swiss HIV Cohort Study.

    PubMed

    Scherrer, Alexandra U; Yang, Wan-Lin; Kouyos, Roger D; Böni, Jürg; Yerly, Sabine; Klimkait, Thomas; Aubert, Vincent; Cavassini, Matthias; Battegay, Manuel; Hauser, Christoph; Calmy, Alexandra; Schmid, Patrick; Bernasconi, Enos; Günthard, Huldrych F

    2016-08-01

    The prevalence of integrase strand transfer inhibitor (INSTI)-transmitted drug resistance (TDR) may increase with the increasing use of INSTIs. We analyzed the prevalence of INSTI TDR in the Swiss HIV Cohort Study (2008-2014). In 1 of 1316 drug-naive samples (0.1%), a major INSTI TDR mutation was detected. Prevalence was stable, although INSTIs were increasingly used. We showed that this is in contrast to the introduction of previous drug classes, in which more treatment failures with resistant strains occurred and TDR was observed more rapidly. We demonstrated on a population-level that it is possible to avoid TDR to a new drug class for years. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  18. A Method for Rapid Genetic Integration into Plasmodium falciparum Utilizing Mycobacteriophage Bxb1 Integrase

    PubMed Central

    Adjalley, Sophie H.; Lee, Marcus C.S.; Fidock, David A.

    2011-01-01

    Genetic manipulation of the human malaria parasite Plasmodium falciparum has presented substantial challenges for research efforts aimed at better understanding the complex biology of this highly virulent organism. The development of methods to perform gene disruption, allelic replacement or transgene expression has provided important insights into the function of parasite genes. However, genomic integration studies have been hindered by low transfection and recombination efficiencies, and are complicated by the propensity of this parasite to maintain episomal replicating plasmids. We have developed a fast and efficient site-specific system of integrative recombination into the P. falciparum genome, which is catalyzed by the mycobacteriophage Bxb1 serine integrase. This system has the advantage of providing greater genetic and phenotypic homogeneity within transgenic lines as compared to earlier methods based on episomal replication of plasmids. Herein, we present this methodology. PMID:20676977

  19. Inhibition of Feline leukemia virus replication by the integrase inhibitor Raltegravir.

    PubMed

    Cattori, Valentino; Weibel, Beatrice; Lutz, Hans

    2011-08-26

    The oncogenic gammaretrovirus Feline leukemia virus (FeLV) has been the leading cause of death among domestic cats until the introduction of efficient diagnostics and vaccines in the late 1980s. So far, no efficient treatment for viremic animals is available. Hence, use of the FeLV model to evaluate antiretroviral therapies applied to HIV is a timely task. The efficacy of the integrase inhibitor Raltegravir, which is widely used for the treatment of HIV in humans, has been assessed in vitro for the FeLV-A/Glasgow-1 strain. EC(50) values for FeLV-A inhibition in feline cell lines are in the range of that observed for HIV and xenotropic murine leukemia virus-related gammaretrovirus. Therefore, Raltegravir may be a potential therapeutical agent for felids with progressive FeLV infection.

  20. HIV/HCV-coinfection: which role can new antiretrovirals such as integrase inhibitors play?

    PubMed Central

    2009-01-01

    End-stage liver disease has become one of the most frequent causes of death in HIV/HCV-coinfected patients. The role of new antiretrovirals in the progression of liver fibrosis has yet to be defined. However with significant toxicities and drug-to-drug interactions of nucleoside reverse transcriptase inhibitors in combination with ribavirin, with drug to drug interaction of HIV protease inhibitors with HCV protease inhibitors and calcineurin-inhibitors, new antiretrovirals lacking these interactions represent attractive alternatives in the setting of anti-HCV therapy or post liver transplantation. In the following review we want to focus on the new class of HIV integrase inhibitors and discuss present data with regard to special issues of HIV and HCV co-infection. PMID:19959415

  1. Efficient ΦC31 integrase-mediated site-specific germline transformation of Anopheles gambiae.

    PubMed

    Pondeville, Emilie; Puchot, Nicolas; Meredith, Janet M; Lynd, Amy; Vernick, Kenneth D; Lycett, Gareth J; Eggleston, Paul; Bourgouin, Catherine

    2014-07-01

    Current transgenic methodology developed for mosquitoes has not been applied widely to the major malaria vector Anopheles gambiae, which has proved more difficult to genetically manipulate than other mosquito species and dipteran insects. In this protocol, we describe ΦC31-mediated site-specific integration of transgenes into the genome of A. gambiae. The ΦC31 system has many advantages over 'classical' transposon-mediated germline transformation systems, because it allows integration of large transgenes at specific, characterized genomic locations. Starting from a general protocol, we have optimized steps from embryo collection to co-injection of transgene-containing plasmid and in vitro-produced ΦC31 integrase mRNA. We also provide tips for screening transgenic larvae. The outlined procedure provides robust transformation in A. gambiae, resulting in homozygous transgenic lines in ∼2-3 months.

  2. HIV-1 Integrase Binds the Viral RNA Genome and Is Essential during Virion Morphogenesis.

    PubMed

    Kessl, Jacques J; Kutluay, Sebla B; Townsend, Dana; Rebensburg, Stephanie; Slaughter, Alison; Larue, Ross C; Shkriabai, Nikoloz; Bakouche, Nordine; Fuchs, James R; Bieniasz, Paul D; Kvaratskhelia, Mamuka

    2016-08-25

    While an essential role of HIV-1 integrase (IN) for integration of viral cDNA into human chromosome is established, studies with IN mutants and allosteric IN inhibitors (ALLINIs) have suggested that IN can also influence viral particle maturation. However, it has remained enigmatic as to how IN contributes to virion morphogenesis. Here, we demonstrate that IN directly binds the viral RNA genome in virions. These interactions have specificity, as IN exhibits distinct preference for select viral RNA structural elements. We show that IN substitutions that selectively impair its binding to viral RNA result in eccentric, non-infectious virions without affecting nucleocapsid-RNA interactions. Likewise, ALLINIs impair IN binding to viral RNA in virions of wild-type, but not escape mutant, virus. These results reveal an unexpected biological role of IN binding to the viral RNA genome during virion morphogenesis and elucidate the mode of action of ALLINIs.

  3. Efficiency of Recombination Reactions Catalyzed by Class 1 Integron Integrase IntI1

    PubMed Central

    Collis, Christina M.; Recchia, Gavin D.; Kim, Mi-Jurng; Stokes, H. W.; Hall, Ruth M.

    2001-01-01

    The class 1 integron integrase, IntI1, recognizes two distinct types of recombination sites, attI sites, found in integrons, and members of the 59-be family, found in gene cassettes. The efficiencies of the integrative version of the three possible reactions, i.e., between two 59-be, between attI1 and a 59-be, or between two attI1 sites, were compared. Recombination events involving two attI1 sites were significantly less efficient than the reactions in which a 59-be participated, and the attI1 × 59-be reaction was generally preferred over the 59-be × 59-be reaction. Recombination of attI1 with secondary sites was less efficient than the 59-be × secondary site reaction. PMID:11274113

  4. Integrase-mediated spacer acquisition during CRISPR-Cas adaptive immunity.

    PubMed

    Nuñez, James K; Lee, Amy S Y; Engelman, Alan; Doudna, Jennifer A

    2015-03-12

    Bacteria and archaea insert spacer sequences acquired from foreign DNAs into CRISPR loci to generate immunological memory. The Escherichia coli Cas1-Cas2 complex mediates spacer acquisition in vivo, but the molecular mechanism of this process is unknown. Here we show that the purified Cas1-Cas2 complex integrates oligonucleotide DNA substrates into acceptor DNA to yield products similar to those generated by retroviral integrases and transposases. Cas1 is the catalytic subunit and Cas2 substantially increases integration activity. Protospacer DNA with free 3'-OH ends and supercoiled target DNA are required, and integration occurs preferentially at the ends of CRISPR repeats and at sequences adjacent to cruciform structures abutting AT-rich regions, similar to the CRISPR leader sequence. Our results demonstrate the Cas1-Cas2 complex to be the minimal machinery that catalyses spacer DNA acquisition and explain the significance of CRISPR repeats in providing sequence and structural specificity for Cas1-Cas2-mediated adaptive immunity.

  5. Resistance to the anti-human immunodeficiency virus type 1 compound L-chicoric acid results from a single mutation at amino acid 140 of integrase.

    PubMed

    King, P J; Robinson, W E

    1998-10-01

    L-Chicoric acid is an inhibitor of human immunodeficiency virus type 1 (HIV-1) integrase in vitro and of HIV-1 replication in tissue culture. Following 3 months of selection in the presence of increasing concentrations of L-chicoric acid, HIV-1 was completely resistant to the compound. Introduction of the mutant integrase containing a single glycine-to-serine amino acid change at position 140 into the native, L-chicoric acid-sensitive virus demonstrated that this change was sufficient to confer resistance to L-chicoric acid. These results confirm through natural selection previous biochemical studies showing that L-chicoric acid inhibits integrase and that the drug is likely to interact at residues near the catalytic triad in the integrase active site.

  6. Regulation, Integrase-Dependent Excision, and Horizontal Transfer of Genomic Islands in Legionella pneumophila

    PubMed Central

    Lautner, Monika; Schunder, Eva; Herrmann, Vroni

    2013-01-01

    Legionella pneumophila is a Gram-negative freshwater agent which multiplies in specialized nutrient-rich vacuoles of amoebae. When replicating in human alveolar macrophages, Legionella can cause Legionnaires' disease. Recently, we identified a new type of conjugation/type IVA secretion system (T4ASS) in L. pneumophila Corby (named trb-tra). Analogous versions of trb-tra are localized on the genomic islands Trb-1 and Trb-2. Both can exist as an episomal circular form, and Trb-1 can be transferred horizontally to other Legionella strains by conjugation. In our current work, we discovered the importance of a site-specific integrase (Int-1, lpc2818) for the excision and conjugation process of Trb-1. Furthermore, we identified the genes lvrRABC (lpc2813 to lpc2816) to be involved in the regulation of Trb-1 excision. In addition, we demonstrated for the first time that a Legionella genomic island (LGI) of L. pneumophila Corby (LpcGI-2) encodes a functional type IV secretion system. The island can be transferred horizontally by conjugation and is integrated site specifically into the genome of the transconjugants. LpcGI-2 generates three different episomal forms. The predominant episomal form, form A, is generated integrase dependently (Lpc1833) and transferred by conjugation in a pilT-dependent manner. Therefore, the genomic islands Trb-1 and LpcGI-2 should be classified as integrative and conjugative elements (ICEs). Coculture studies of L. pneumophila wild-type and mutant strains revealed that the int-1 and lvrRABC genes (located on Trb-1) as well as lpc1833 and pilT (located on LpcGI-2) do not influence the in vivo fitness of L. pneumophila in Acanthamoeba castellanii. PMID:23354744

  7. Usefulness of Integrase resistance testing in proviral HIV-1 DNA in patients with Raltegravir prior failure.

    PubMed

    Fernández-Caballero, Jose Ángel; Chueca, Natalia; Álvarez, Marta; Mérida, María Dolores; López, Josefa; Sánchez, José Antonio; Vinuesa, David; Martínez, María Ángeles; Hernández, José; García, Federico

    2016-05-13

    In our study, we have hypothesized that proviral DNA may show the history of mutations that emerged at previous failures to a Raltegravir containing regimen, in patients who are currently undetectable and candidates to simplification to a Dolutegravir containing regimen, in order to decide on once a day or twice a day dosing. We have performed a pilot, observational, retrospective, non interventional study, including 7 patients infected by HIV-1, all with a history of previous failure to a RAL containing regimen, that were successfully salvaged and had reached viral suppression. A genotypic viral Integrase region study was available for each patient at the moment of RAL failure. After an average (IQR) time of 48 months (29-53) Integrase resistance mutations in proviral DNA were studied. All the patients were infected by HIV-1 B subtypes, with a mean age of 55 (range 43 to 56), originating from Spain, and 4 were women. Median viral load (log) and CD4 count at the moment of the study on proviral DNA was of 1.3 log cp/ml (range 0-1.47) and 765.5 cells/μL (range; 436.75-1023.75). The median time (IQR) between previous failure to RAL and the study on proviral DNA was 48 (29-53) months. At Raltegravir failure, N155H was detected in four patients, and other secondary mutations were detected in five patients (71.4 %). In proviral DNA, N155H was detected by population sequencing in three patients (42.8 %), and UDS demonstrated a 9.77 % relative abundance of N155H in the remaining patient. Sanger sequencing correctly identified all the secondary mutations. This is a pilot study that demonstrates the possibility of properly identifying N155H and some secondary mutations 29-53 months after failure.

  8. Resistance mutations against dolutegravir in HIV integrase impair the emergence of resistance against reverse transcriptase inhibitors.

    PubMed

    Oliveira, Maureen; Mesplède, Thibault; Quashie, Peter K; Moïsi, Daniela; Wainberg, Mark A

    2014-03-27

    Among 1222 antiretroviral-naive patients who received dolutegravir (DTG) as part of first-line therapy, none has developed resistance against this compound after 48-96 weeks of follow-up. Moreover, only four occurrences of virological failure with resistance mutations have been documented in previously drug-experienced patients who received DTG as a first time integrase inhibitor as a component of a second-line regimen. The R263K integrase resistance mutation was observed in two of these individuals who received suboptimal background regimens. We have previously selected mutations at position R263K, G118R, H51Y, and E138K as being associated with low-level resistance to DTG. Now, we sought to investigate the facility with which resistance on the part of R263K-containing viruses might develop. We tested the ability of DTG-resistant viruses containing either the R263K or G118R and/or H51Y mutations to develop further resistance against several reverse transcriptase inhibitors during in-vitro selection experiments. Our results show that DTG-resistant viruses are impaired in their ability to acquire further resistance to each of nevirapine and lamivudine as a consequence of their relative inability to develop resistance mutations associated with these two compounds. Our findings provide an explanation for the fact that no individual has yet progressed to virological failure with resistance mutations associated with dolutegravir in clinical trials in which patients received dolutegravir together with an optimized background regimen.

  9. Marine integrons containing novel integrase genes, attachment sites, attI, and associated gene cassettes in polluted sediments from Suez and Tokyo Bays.

    PubMed

    Elsaied, Hosam; Stokes, Hatch W; Kitamura, Keiko; Kurusu, Yasurou; Kamagata, Yoichi; Maruyama, Akihiko

    2011-07-01

    In order to understand the structure and biological significance of integrons and associated gene cassettes in marine polluted sediments, metagenomic DNAs were extracted from sites at Suez and Tokyo Bays. PCR amplicons containing new integrase genes, intI, linked with novel gene cassettes, were recovered and had sizes from 1.8 to 2.5 kb. This approach uncovered, for the first time, the structure and diversity of both marine integron attachment site, attI, and the first gene cassette, the most efficiently expressed integron-associated gene cassette. The recovered 13 and 20 intI phylotypes, from Suez and Tokyo Bay samples, respectively, showed a highly divergence, suggesting a difference in integron composition between the sampling sites. Some intI phylotypes showed similarity with that from Geobacter metallireducens, belonging to Deltaproteobacteria, the dominant class in both sampling sites, as determined by 16S rRNA gene analysis. Thirty distinct families of putative attI site, as determined by the presence of an attI-like simple site, were recovered. A total of 146 and 68 gene cassettes represented Suez and Tokyo Bay unsaturated cassette pools, respectively. Gene cassettes, including a first cassette, from both sampling sites encoded two novel families of glyoxalase/bleomycin antibiotic-resistance protein. Gene cassettes from Suez Bay encoded proteins similar to haloacid dehalogenases, protein disulfide isomerases and death-on-curing and plasmid maintenance system killer proteins. First gene cassettes from Tokyo Bay encoded a xenobiotic-degrading protein, cardiolipin synthetase, esterase and WD40-like β propeller protein. Many of the first gene cassettes encoded proteins with no ascribable function but some of them were duplicated and possessed signal functional sites, suggesting efficient adaptive functions to their bacterial sources. Thus, each sampling site had a specific profile of integrons and cassette types consistent with the hypothesis that the

  10. Structure-activity relationships: analogues of the dicaffeoylquinic and dicaffeoyltartaric acids as potent inhibitors of human immunodeficiency virus type 1 integrase and replication.

    PubMed

    King, P J; Ma, G; Miao, W; Jia, Q; McDougall, B R; Reinecke, M G; Cornell, C; Kuan, J; Kim, T R; Robinson, W E

    1999-02-11

    The dicaffeoylquinic acids (DCQAs) and dicaffeoyltartaric acids (DCTAs) are potent and selective inhibitors of human immunodeficiency virus type 1 (HIV-1) integrase. They also inhibit HIV-1 replication at nontoxic concentrations. Since integrase is an excellent target for anti-HIV therapy, structure-activity relationships were employed to synthesize compounds with: (1) improved potency against HIV-1 integrase, (2) improved anti-HIV effect in tissue culture, and (3) increased selectivity as indicated by low cellular toxicity. Thirty-four analogues of the DCTAs and DCQAs were synthesized and tested for cell toxicity, anti-HIV activity, and inhibition of HIV-1 integrase. Seventeen of the 34 analogues had potent activity against HIV-1 integrase ranging from 0. 07 to >10 microM. Seventeen analogues that were synthesized or purchased had no inhibitory activity against integrase at concentrations of 25 microM. Of the biologically active analogues, 7 of the 17 inhibited HIV replication at nontoxic concentrations. The most potent compounds were D-chicoric acid, meso-chicoric acid, bis(3,4-dihydroxydihydrocinnamoyl)-L-tartaric acid, digalloyl-L-tartaric acid, bis(3,4-dihydroxybenzoyl)-L-tartaric acid, dicaffeoylglyceric acid, and bis(3, 4-dihydroxyphenylacetyl)-L-tartaric acid. Anti-HIV activity of the active compounds in tissue culture ranged from 35 to 0.66 microM. Structure-activity relationships demonstrated that biscatechol moieties were absolutely required for inhibition of integrase, while at least one free carboxyl group was required for anti-HIV activity. These data demonstrate that analogues of the DCTAs and the DCQAs can be synthesized which have improved activity against HIV integrase.

  11. In vivo and in vitro characterization of site-specific recombination of a novel serine integrase from the temperate phage EFC-1

    SciTech Connect

    Yoon, Bohyun; Kim, Inki; Nam, Ja-Ae; Chang, Hyo-Ihl; Ha, Chang Hoon

    2016-04-22

    EFC-1 integrase is a site-specific recombinase that belongs to the large family of serine recombinase. In previously study, we isolated the temperate phage EFC-1, and characterized its genomic sequence. Within its genome, Orf28 was predicted encode a 464 amino acid of a putative integrase gene. In this study, EFC-1 integrase was characterized in vitro and in vivo. In vitro assay was performed using purified His-tag fusion integrase. Also, to identify which serine is involved in the catalytic domain, we used site-directed mutagenesis and analyzed by a recombination assay in vitro. In vivo assay involved PCR and confocal microscopy in HEK293 cells, and determined the minimal lengths of attP and attB sites. According to our results, the EFC-1 integrase-mediated recombination was site-specific and unidirectional system in vitro and in vivo. Serine 21 of EFC-1 integrase plays a major role in the catalytic domain, and minimal sizes of attB and attP was defined 48 and 54 bp. Our finding may help develop a useful tool for gene therapy and gene delivery system. - Highlights: • EFC-1 integrase-mediated recombination was site-specific and unidirectional system. • Serine 21 of EFC-1 integrase plays a major role in the catalytic domain. • The functional minimal sizes of attB and attP was defined 48 and 54 bp.

  12. Detection of drug resistance-associated mutations in human immunodeficiency virus type 1 integrase derived from drug-naive individuals in Surabaya, Indonesia.

    PubMed

    Kotaki, Tomohiro; Khairunisa, Siti Qamariyah; Sukartiningrum, Septhia Dwi; Witaningrum, Adiana Mutamsari; Rusli, Musofa; Diansyah, M Noor; Arfijanto, M Vitanata; Rahayu, Retno Pudji; Nasronudin; Kameoka, Masanori

    2014-05-01

    Although human immunodeficiency virus type 1 (HIV-1) infection causes serious health problems in Indonesia, information in regard to drug resistance is limited. We performed a genotypic study on HIV-1 integrase derived from drug-naive individuals in Surabaya, Indonesia. Sequencing analysis revealed that no primary mutations associated with drug resistance to integrase inhibitors were detected; however, secondary mutations, V72I, L74I/M, V165I, V201I, I203M, and S230N, were detected in more than 5% of samples. In addition, V201I was conserved among all samples. Most integrase genes were classified into CRF01_AE genes. Interestingly, 40% of the CRF01_AE genes had an unusual insertion in the C-terminus of integrase. These mutations and insertions were considered natural polymorphisms since these mutations coincided with previous reports, and integrase inhibitors have not been used in Indonesia. Our results indicated that further studies may be required to assess the impact of these mutations on integrase inhibitors prior to their introduction into Indonesia.

  13. A Novel Assay for Screening Inhibitors Targeting HIV Integrase LEDGF/p75 Interaction Based on Ni2+ Coated Magnetic Agarose Beads

    PubMed Central

    Dawei, Zhang; Hongqiu, He; Mengmeng, Liu; Zhixia, Meng; Shunxing, Guo

    2016-01-01

    HIV-1 integrase (IN) plays an essential role in viral replication and thus serves as an important target for chemotherapeutic intervention against HIV-1 infection. However, the current three clinical IN inhibitors, raltegravir, elvitegravir and dolutegravir share the same inhibitory mechanism, resulting in a common clinical resistance profile which have emerged in infected patients receiving treatment. Therefore, it is important to develop small molecule inhibitors that impair IN function with distinct mechanisms of action. In this work, a magnetic-beads based biochemical assay targeting the protein-protein interaction (PPI) between HIV IN and the cellular cofactor LEDGF/p75 was developed for identification of HIV-1 IN inhibitors. Furthermore, a library containing 1000 US. Food and Drug Administration (FDA)-approved drugs currently used for human medication was screened to identify inhibitors targeting the PPI. The assay was proved to be quite robust and with the novel assay we successfully identified dexlansoprazole (IC50 of 4.8 μM), a FDA-approved proton pump inhibitor, as a potential inhibitor for the PPI between IN and LEDGF/p75, which bound to the LEDGF/p75 partner with a kinetic dissociation (Kd) constant of 330 nM ± 2.6 nM. PMID:27633629

  14. Development and Identification of a Novel Anti-HIV-1 Peptide Derived by Modification of the N-Terminal Domain of HIV-1 Integrase

    PubMed Central

    Sala, Marina; Spensiero, Antonia; Esposito, Francesca; Scala, Maria C.; Vernieri, Ermelinda; Bertamino, Alessia; Manfra, Michele; Carotenuto, Alfonso; Grieco, Paolo; Novellino, Ettore; Cadeddu, Marta; Tramontano, Enzo; Schols, Dominique; Campiglia, Pietro; Gomez-Monterrey, Isabel M.

    2016-01-01

    The viral enzyme integrase (IN) is essential for the replication of human immunodeficiency virus type 1 (HIV-1) and represents an important target for the development of new antiretroviral drugs. In this study, we focused on the N-terminal domain (NTD), which is mainly involved into protein oligomerization process, for the development and synthesis of a library of overlapping peptide sequences, with specific length and specific offset covering the entire native protein sequence NTD IN 1–50. The most potent fragment, VVAKEIVAH (peptide 18), which includes a His residue instead of the natural Ser at position 39, inhibits the HIV-1 IN activity with an IC50 value of 4.5 μM. Amino acid substitution analysis on this peptide revealed essential residues for activity and allowed us to identify two nonapeptides (peptides 24 and 25), that show a potency of inhibition similar to the one of peptide 18. Interestingly, peptide 18 does not interfere with the dynamic interplay between IN subunits, while peptides 24 and 25 modulated these interactions in different manners. In fact, peptide 24 inhibited the IN-IN dimerization, while peptide 25 promoted IN multimerization, with IC50 values of 32 and 4.8 μM, respectively. In addition, peptide 25 has shown to have selective anti-infective cell activity for HIV-1. These results confirmed peptide 25 as a hit for further development of new chemotherapeutic agents against HIV-1. PMID:27375570

  15. A novel co-crystal structure affords the design of gain-of-function lentiviral integrase mutants in the presence of modified PSIP1/LEDGF/p75.

    PubMed

    Hare, Stephen; Shun, Ming-Chieh; Gupta, Saumya Shree; Valkov, Eugene; Engelman, Alan; Cherepanov, Peter

    2009-01-01

    Lens epithelium derived growth factor (LEDGF), also known as PC4 and SFRS1 interacting protein 1 (PSIP1) and transcriptional co-activator p75, is the cellular binding partner of lentiviral integrase (IN) proteins. LEDGF accounts for the characteristic propensity of Lentivirus to integrate within active transcription units and is required for efficient viral replication. We now present a crystal structure containing the N-terminal and catalytic core domains (NTD and CCD) of HIV-2 IN in complex with the IN binding domain (IBD) of LEDGF. The structure extends the known IN-LEDGF interface, elucidating primarily charge-charge interactions between the NTD of IN and the IBD. A constellation of acidic residues on the NTD is characteristic of lentiviral INs, and mutations of the positively charged residues on the IBD severely affect interaction with all lentiviral INs tested. We show that the novel NTD-IBD contacts are critical for stimulation of concerted lentiviral DNA integration by LEDGF in vitro and for its function during the early steps of HIV-1 replication. Furthermore, the new structural details enabled us to engineer a mutant of HIV-1 IN that primarily functions only when presented with a complementary LEDGF mutant. These findings provide structural basis for the high affinity lentiviral IN-LEDGF interaction and pave the way for development of LEDGF-based targeting technologies for gene therapy.

  16. A C-terminal "Tail" Region in the Rous Sarcoma Virus Integrase Provides High Plasticity of Functional Integrase Oligomerization during Intasome Assembly.

    PubMed

    Pandey, Krishan K; Bera, Sibes; Shi, Ke; Aihara, Hideki; Grandgenett, Duane P

    2017-03-24

    The retrovirus integrase (IN) inserts the viral cDNA into the host DNA genome. Atomic structures of five different retrovirus INs complexed with their respective viral DNA or branched viral/target DNA substrates have indicated these intasomes are composed of IN subunits ranging from tetramers, to octamers, or to hexadecamers. IN precursors are monomers, dimers, or tetramers in solution. But how intasome assembly is controlled remains unclear. Therefore, we sought to unravel the functional mechanisms in different intasomes. We produced kinetically stabilized Rous sarcoma virus (RSV) intasomes with human immunodeficiency virus type 1 strand transfer inhibitors that interact simultaneously with IN and viral DNA within intasomes. We examined the ability of RSV IN dimers to assemble two viral DNA molecules into intasomes containing IN tetramers in contrast to one possessing IN octamers. We observed that the last 18 residues of the C terminus ("tail" region) of IN (residues 1-286) determined whether an IN tetramer or octamer assembled with viral DNA. A series of truncations of the tail region indicated that these 18 residues are critical for the assembly of an intasome containing IN octamers but not for an intasome containing IN tetramers. The C-terminally truncated IN (residues 1-269) produced an intasome that contained tetramers but failed to produce an intasome with octamers. Both intasomes have similar catalytic activities. The results suggest a high degree of plasticity for functional multimerization and reveal a critical role of the C-terminal tail region of IN in higher order oligomerization of intasomes, potentially informing future strategies to prevent retroviral integration.

  17. The inhibition process of HIV-1 integrase by diketoacids molecules: Understanding the factors governing the better efficiency of dolutegravir.

    PubMed

    El Khoury, Léa; Piquemal, Jean-Philip; Fermandjian, Serge; Maroun, Richard G; Gresh, Nohad; Hobaika, Zeina

    2017-07-01

    The Human Immunodeficiency Virus-1 integrase is responsible for the covalent insertion of a newly synthesized double-stranded viral DNA into the host cells, and is an emerging target for antivirus drug design. Raltegravir (RAL) and elvitegravir (EVG) are the first two integrase strand transfer inhibitors used in therapy. However, treated patients eventually develop detrimental resistance mutations. By contrast, a recently approved drug, dolutegravir (DTG), presents a high barrier to resistance. This study aims to understand the increased efficiency of DTG upon focusing on its interaction properties with viral DNA. The results showed DTG to be involved in more extended interactions with viral DNA than EVG. Such interactions involve the halobenzene and scaffold of DTG and EVG and bases 5'G(-4)3', 3'A(3)5'and 3'C(4)5'. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Real-time monitoring of disintegration activity of catalytic core domain of HIV-1 integrase using molecular beacon.

    PubMed

    Zhang, Da-wei; Zhao, Ming-ming; He, Hong-qiu; Guo, Shun-xing

    2013-09-15

    HIV-1 integrase, an essential enzyme for retroviral replication, is a validated target for anti-HIV therapy development. The catalytic core domain of integrase (IN-CCD) is capable of catalyzing disintegration reaction. In this work, a hairpin-shaped disintegration substrate was designed and validated by enzyme-linked immunosorbent assay; a molecular beacon-based assay was developed for disintegration reaction of IN-CCD. Results showed that the disintegration substrate could be recognized and catalyzed by IN-CCD, and the disintegration reaction can be monitored according to the increase of fluorescent signal. The assay can be applied to real-time detection of disintegration with advantages of simplicity, high sensitivity, and excellent specificity. Copyright © 2013 Elsevier Inc. All rights reserved.

  19. Analysis of HIV Integrase Resistance in Black Men Who Have Sex with Men in the United States.

    PubMed

    Chen, Iris; Zhang, Yinfeng; Cummings, Vanessa; Cloherty, Gavin A; Connor, Matthew; Beauchamp, Geetha; Griffith, Sam; Rose, Scott; Gallant, Joel; Scott, Hyman M; Shoptaw, Steven; Del Rio, Carlos; Kuo, Irene; Mannheimer, Sharon; Tieu, Hong-Van; Hurt, Christopher B; Fields, Sheldon D; Wheeler, Darrell P; Mayer, Kenneth H; Koblin, Beryl A; Eshleman, Susan H

    2017-04-06

    Resistance to reverse transcriptase and protease inhibitors was frequently detected in HIV from black men who have sex with men (MSM) enrolled in the HIV prevention trials network (HPTN) 061 study. In this study, integrase strand transfer inhibitor (INSTI) resistance was analyzed in black MSM enrolled in HPTN 061 (134 infected at enrollment and 23 seroconverters) and a follow-up study, HPTN 073 (eight seroconverters). The ViroSeq HIV-1 Integrase Genotyping Kit (Abbott Molecular) was used for analysis. Major INSTI resistance mutations were not detected in any of the samples. HIV from 14 (8.4%) of the 165 men, including 4 (12.9%) of 31 seroconverters, had accessory or polymorphic INSTI-associated mutations. The most frequently detected mutation was E157Q. These findings are promising because INSTI-based regimens are now recommended for first-line antiretroviral treatment and because long-acting cabotegravir is being evaluated for pre-exposure prophylaxis.

  20. Optimization of rhodanine scaffold for the development of protein-protein interaction inhibitors.

    PubMed

    Ferro, Stefania; De Luca, Laura; Agharbaoui, Fatima Ezzahra; Christ, Frauke; Debyser, Zeger; Gitto, Rosaria

    2015-07-01

    Searching for novel protein-protein interactions inhibitors (PPIs) herein we describe the identification of a new series of rhodanine derivatives. The selection was performed by means virtual-screening, docking studies, Molecular Dynamic (MD) simulations and synthetic approaches. All the new obtained compounds were tested in order to evaluate their ability to inhibit the interaction between the HIV-1 integrase (IN) enzyme and the nuclear protein lens epithelium growth factor LEDGF/p75.

  1. Three-dimensional hierarchical flower-like Mg-Al-layered double hydroxides: highly efficient adsorbents for As(v) and Cr(vi) removal

    NASA Astrophysics Data System (ADS)

    Yu, Xin-Yao; Luo, Tao; Jia, Yong; Xu, Ren-Xia; Gao, Chao; Zhang, Yong-Xing; Liu, Jin-Huai; Huang, Xing-Jiu

    2012-05-01

    3D hierarchical flower-like Mg-Al-layered double hydroxides (Mg-Al-LDHs) were synthesized by a simple solvothermal method in a mixed solution of ethylene glycol (EG) and water. The formation mechanism of the flower-like Mg-Al-LDHs was proposed. After calcination, the flower-like morphology could be completely preserved. With relatively high specific surface areas, Mg-Al-LDHs and calcined Mg-Al-LDHs with 3D hierarchical nanostructures were tested for their application in water purification. When tested as adsorbents in As(v) and Cr(vi) removal, the as-prepared calcined Mg-Al-LDHs showed excellent performance, and the adsorption capacities of calcined Mg-Al-LDHs for As(v) and Cr(vi) were better than those of Mg-Al-LDHs. The adsorption isotherms, kinetics and mechanisms for As(v) and Cr(vi) onto calcined Mg-Al-LDHs were also investigated. The high uptake capability of the as-prepared novel 3D hierarchical calcined Mg-Al-LDHs make it a potentially attractive adsorbent in water purification. Also, this facile strategy may be extended to synthesize other LDHs with 3D hierarchical nanostructures, which may find many other applications due to their novel structural features.3D hierarchical flower-like Mg-Al-layered double hydroxides (Mg-Al-LDHs) were synthesized by a simple solvothermal method in a mixed solution of ethylene glycol (EG) and water. The formation mechanism of the flower-like Mg-Al-LDHs was proposed. After calcination, the flower-like morphology could be completely preserved. With relatively high specific surface areas, Mg-Al-LDHs and calcined Mg-Al-LDHs with 3D hierarchical nanostructures were tested for their application in water purification. When tested as adsorbents in As(v) and Cr(vi) removal, the as-prepared calcined Mg-Al-LDHs showed excellent performance, and the adsorption capacities of calcined Mg-Al-LDHs for As(v) and Cr(vi) were better than those of Mg-Al-LDHs. The adsorption isotherms, kinetics and mechanisms for As(v) and Cr(vi) onto calcined

  2. Synthesis, anti-HIV activity, integrase enzyme inhibition and molecular modeling of catechol, hydroquinone and quinol labdane analogs.

    PubMed

    Pawar, Rohan; Das, Tiyasa; Mishra, Sanjay; Nutan; Pancholi, Boskey; Gupta, Satish K; Bhat, Sujata V

    2014-01-01

    Labdane analogs with o-quinol, catechol and hydroquinone moiety have been synthesized using Diels-Alder reaction of methyl 3,4-dioxocyclohexa-1,5-diene-carboxylate, 3,4-dioxocyclohexa-1,5-diene-carboxylic acid and 3,6-dioxocyclohexa-1,4-dienecarboxylic acid with mono terpene 1,3-dienes, namely ocimene and myrcene. The resulting molecules and their derivatives were evaluated for their anti-HIV-1 activity using TZM-bl cell based virus infectivity assay. Two molecules 13 and 18 showed anti-HIV activity with IC50 values 5.0 (TI=11) and 4.6 (TI=46)μM, respectively. The compounds 17, 18 and 20 showed efficacy against HIV-1 integrase activity and showed inhibition with IC50 13.4, 11.1 and 11.5μM, respectively. The HIV-1 integrase inhibition activity of these synthetic molecules was comparable with integric acid, the natural fungal metabolite. Molecular modeling studies for the HIV-1 integrase inhibition of these active synthetic molecules indicated the binding to the active site residues of the enzyme.

  3. HIV-2 Integrase Polymorphisms and Longitudinal Genotypic Analysis of HIV-2 Infected Patients Failing a Raltegravir-Containing Regimen

    PubMed Central

    Cavaco-Silva, Joana; Abecasis, Ana; Miranda, Ana Cláudia; Poças, José; Narciso, Jorge; Águas, Maria João; Maltez, Fernando; Almeida, Isabel; Germano, Isabel; Diniz, António; Gonçalves, Maria de Fátima; Gomes, Perpétua; Cunha, Celso; Camacho, Ricardo Jorge

    2014-01-01

    To characterize the HIV-2 integrase gene polymorphisms and the pathways to resistance of HIV-2 patients failing a raltegravir-containing regimen, we studied 63 integrase strand transfer inhibitors (INSTI)-naïve patients, and 10 heavily pretreated patients exhibiting virological failure while receiving a salvage raltegravir-containing regimen. All patients were infected by HIV-2 group A. 61.4% of the integrase residues were conserved, including the catalytic motif residues. No INSTI-major resistance mutations were detected in the virus population from naïve patients, but two amino acids that are secondary resistance mutations to INSTIs in HIV-1 were observed. The 10 raltegravir-experienced patients exhibited resistance mutations via three main genetic pathways: N155H, Q148R, and eventually E92Q - T97A. The 155 pathway was preferentially used (7/10 patients). Other mutations associated to raltegravir resistance in HIV-1 were also observed in our HIV-2 population (V151I and D232N), along with several novel mutations previously unreported. Data retrieved from this study should help build a more robust HIV-2-specific algorithm for the genotypic interpretation of raltegravir resistance, and contribute to improve the clinical monitoring of HIV-2-infected patients. PMID:24681625

  4. HIV-2 integrase polymorphisms and longitudinal genotypic analysis of HIV-2 infected patients failing a raltegravir-containing regimen.

    PubMed

    Cavaco-Silva, Joana; Abecasis, Ana; Miranda, Ana Cláudia; Poças, José; Narciso, Jorge; Águas, Maria João; Maltez, Fernando; Almeida, Isabel; Germano, Isabel; Diniz, António; Gonçalves, Maria de Fátima; Gomes, Perpétua; Cunha, Celso; Camacho, Ricardo Jorge

    2014-01-01

    To characterize the HIV-2 integrase gene polymorphisms and the pathways to resistance of HIV-2 patients failing a raltegravir-containing regimen, we studied 63 integrase strand transfer inhibitors (INSTI)-naïve patients, and 10 heavily pretreated patients exhibiting virological failure while receiving a salvage raltegravir-containing regimen. All patients were infected by HIV-2 group A. 61.4% of the integrase residues were conserved, including the catalytic motif residues. No INSTI-major resistance mutations were detected in the virus population from naïve patients, but two amino acids that are secondary resistance mutations to INSTIs in HIV-1 were observed. The 10 raltegravir-experienced patients exhibited resistance mutations via three main genetic pathways: N155H, Q148R, and eventually E92Q - T97A. The 155 pathway was preferentially used (7/10 patients). Other mutations associated to raltegravir resistance in HIV-1 were also observed in our HIV-2 population (V151I and D232N), along with several novel mutations previously unreported. Data retrieved from this study should help build a more robust HIV-2-specific algorithm for the genotypic interpretation of raltegravir resistance, and contribute to improve the clinical monitoring of HIV-2-infected patients.

  5. A highly efficient site-specific integration strategy using combination of homologous recombination and the ΦC31 integrase.

    PubMed

    Ou, Hailong; Huang, Ying; Ma, Qingwen; Ren, Zhaorui; Huang, Shuzhen; Zeng, Fanyi; Zeng, Yitao

    2013-09-20

    The introduction of double-strand breaks (DSBs) at target sites could greatly enhance homologous recombination, and engineered nucleases, such as zinc finger and transcription activator-like effector nucleases, have been successfully developed for making such breaks. In this study, we present a highly efficient site-specific integration strategy based on homologous recombination and ΦC31 integrase. An attB sequence was introduced at the homologous arm of an insertion targeting vector. DSBs at the target locus and donor were then simultaneously generated by the ΦC31 integrase when co-transfected with the donor vector, consequently stimulating homologous recombination. The results demonstrated that our strategy is feasible and the efficiency at the BF4 target site, which we previously identified in the bovine genome, was as high as 93%. The frequency at another site (BF10) was almost two-fold greater in comparison to the vector without homologous arms. This technology requires no sophisticated nuclease design efforts, and the off-target effect is reduced by ΦC31 integrase compared to the use of engineered nucleases, thereby offering a simple and safe way to effectively express a donor gene at a desired locus. This development has great potential value, especially in transgenesis or gene therapy applications. Copyright © 2013 Elsevier B.V. All rights reserved.

  6. Incorporation of SemiSpan SuperSonic Transport (S4T) Aeroservoelastic Models into SAREC-ASV Simulation

    NASA Technical Reports Server (NTRS)

    Christhilf, David M.; Pototzky, Anthony S.; Stevens, William L.

    2010-01-01

    The Simulink-based Simulation Architecture for Evaluating Controls for Aerospace Vehicles (SAREC-ASV) was modified to incorporate linear models representing aeroservoelastic characteristics of the SemiSpan SuperSonic Transport (S4T) wind-tunnel model. The S4T planform is for a Technology Concept Aircraft (TCA) design from the 1990s. The model has three control surfaces and is instrumented with accelerometers and strain gauges. Control laws developed for wind-tunnel testing for Ride Quality Enhancement, Gust Load Alleviation, and Flutter Suppression System functions were implemented in the simulation. The simulation models open- and closed-loop response to turbulence and to control excitation. It provides time histories for closed-loop stable conditions above the open-loop flutter boundary. The simulation is useful for assessing the potential impact of closed-loop control rate and position saturation. It also provides a means to assess fidelity of system identification procedures by providing time histories for a known plant model, with and without unmeasured turbulence as a disturbance. Sets of linear models representing different Mach number and dynamic pressure conditions were implemented as MATLAB Linear Time Invariant (LTI) objects. Configuration changes were implemented by selecting which LTI object to use in a Simulink template block. A limited comparison of simulation versus wind-tunnel results is shown.

  7. Comparison of a plant based natural surfactant with SDS for washing of As(V) from Fe rich soil.

    PubMed

    Mukhopadhyay, Soumyadeep; Mohd, Ali Hashim; Sahu, Jaya Narayan; Yusoff, Ismail; Sen, Gupta Bhaskar

    2013-11-01

    This study explores the possible application of a biodegradable plant based surfactant, obtained from Sapindus mukorossi, for washing low levels of arsenic (As) from an iron (Fe) rich soil. Natural association of As(V) with Fe(III) makes the process difficult. Soapnut solution was compared to anionic surfactant sodium dodecyl sulfate (SDS) in down-flow and a newly introduced suction mode for soil column washing. It was observed that soapnut attained up to 86% efficiency with respect to SDS in removing As. Full factorial design of experiment revealed a very good fit of data. The suction mode generated up to 83 kPa pressure inside column whilst down-flow mode generated a much higher pressure of 214 kPa, thus making the suction mode more efficient. Micellar solubilisation was found to be responsible for As desorption from the soil and it followed 1st order kinetics. Desorption rate coefficient of suction mode was found to be in the range of 0.005 to 0.01, much higher than down-flow mode values. Analysis of the FT-IR data suggested that the soapnut solution did not interact chemically with As, offering an option for reusing the surfactant. Soapnut can be considered as a soil washing agent for removing As even from soil with high Fe content.

  8. Chelation Motifs Affecting Metal-dependent Viral Enzymes: N′-acylhydrazone Ligands as Dual Target Inhibitors of HIV-1 Integrase and Reverse Transcriptase Ribonuclease H Domain

    PubMed Central

    Carcelli, Mauro; Rogolino, Dominga; Gatti, Anna; Pala, Nicolino; Corona, Angela; Caredda, Alessia; Tramontano, Enzo; Pannecouque, Christophe; Naesens, Lieve; Esposito, Francesca

    2017-01-01

    Human immunodeficiency virus type 1 (HIV-1) infection, still represent a serious global health emergency. The chronic toxicity derived from the current anti-retroviral therapy limits the prolonged use of several antiretroviral agents, continuously requiring the discovery of new antiviral agents with innovative strategies of action. In particular, the development of single molecules targeting two proteins (dual inhibitors) is one of the current main goals in drug discovery. In this contest, metal-chelating molecules have been extensively explored as potential inhibitors of viral metal-dependent enzymes, resulting in some important classes of antiviral agents. Inhibition of HIV Integrase (IN) is, in this sense, paradigmatic. HIV-1 IN and Reverse Transcriptase-associated Ribonuclease H (RNase H) active sites show structural homologies, with the presence of two Mg(II) cofactors, hence it seems possible to inhibit both enzymes by means of chelating ligands with analogous structural features. Here we present a series of N′-acylhydrazone ligands with groups able to chelate the Mg(II) hard Lewis acid ions in the active sites of both the enzymes, resulting in dual inhibitors with micromolar and even nanomolar activities. The most interesting identified N′-acylhydrazone analog, compound 18, shows dual RNase H-IN inhibition and it is also able to inhibit viral replication in cell-based antiviral assays in the low micromolar range. Computational modeling studies were also conducted to explore the binding attitudes of some model ligands within the active site of both the enzymes. PMID:28373864

  9. L-chicoric acid inhibits human immunodeficiency virus type 1 integration in vivo and is a noncompetitive but reversible inhibitor of HIV-1 integrase in vitro.

    PubMed

    Reinke, Ryan A; Lee, Deborah J; McDougall, Brenda R; King, Peter J; Victoria, Joseph; Mao, Yingqun; Lei, Xiangyang; Reinecke, Manfred G; Robinson, W Edward

    2004-09-01

    The human immunodeficiency virus (HIV) integrase (IN) must covalently join the viral cDNA into a host chromosome for productive HIV infection. l-Chicoric acid (l-CA) enters cells poorly but is a potent inhibitor of IN in vitro. Using quantitative real-time polymerase chain reaction (PCR), l-CA inhibits integration at concentrations from 500 nM to 10 microM but also inhibits entry at concentrations above 1 microM. Using recombinant HIV IN, steady-state kinetic analyses with l-CA were consistent with a noncompetitive or irreversible mechanism of inhibition. IN, in the presence or absence of l-CA, was successively washed. Inhibition of IN diminished, demonstrating that l-CA was reversibly bound to the protein. These data demonstrate that l-CA is a noncompetitive but reversible inhibitor of IN in vitro and of HIV integration in vivo. Thus, l-CA likely interacts with amino acids other than those which bind substrate.

  10. The integrase cofactor LEDGF/p75 associates with Iws1 and Spt6 for postintegration silencing of HIV-1 gene expression in latently infected cells.

    PubMed

    Gérard, Annabelle; Ségéral, Emmanuel; Naughtin, Monica; Abdouni, Ahmed; Charmeteau, Bénédicte; Cheynier, Rémi; Rain, Jean-Christophe; Emiliani, Stéphane

    2015-01-14

    The persistence of a latent reservoir containing transcriptionally silent, but replication-competent, integrated provirus is a serious challenge to HIV eradication. HIV integration is under the control of LEDGF/p75, the cellular cofactor of viral integrase. Investigating possible postintegration roles for LEDGF/p75, we find that LEDGF/p75 represses HIV expression in latently infected cells. LEDGF/p75 associated with two proteins involved in the control of gene expression and chromatin structure, Spt6 and Iws1, to form a stable complex. Iws1 plays a role in the establishment of latent infection, whereas Spt6 functions to recruit Iws1 and LEDGF/p75 to the silenced provirus and maintains histone occupancy at the HIV promoter. In latently infected cells, depletion of the complex results in reactivation of HIV expression Altogether, our results indicate that a complex containing LEDGF/p75, Iws1, and Spt6 participates in regulating postintegration steps of HIV latency. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Packageable antiviral therapeutics against human immunodeficiency virus type 1: virion-targeted virus inactivation by incorporation of a single-chain antibody against viral integrase into progeny virions.

    PubMed

    Okui, N; Sakuma, R; Kobayashi, N; Yoshikura, H; Kitamura, T; Chiba, J; Kitamura, Y

    2000-03-01

    To determine their activities as an antiviral agent packageable within virions and suitable for continued expression in cells, we tested a single-chain antibody (scAb) against human immunodeficiency virus type 1 (HIV-1) integrase and its three fusion proteins: fused to viral protein R (scab-Vpr), a double-cassette of the WXXF motif binding to Vpr (scAb-WXXF), and viral major capsid protein (scAb-CA), respectively. Cotransfection of human 293T cells with expression plasmid for scAb-Vpr or -WXXF along with HIV-1 clone pLAI resulted in the production of a normal amount of progeny virions with infectivity decreased by more than 10(3)-fold. Immunoblot analyses showed that scAb-Vpr or -WXXF was associated with virions, whereas scAb or scAb-CA was not, suggesting that scAb-Vpr or -WXXF was incorporated into virions. The incorporation of scAb-WXXF appeared to be Vpr dependent, because the fusion protein was associated with the wild-type but not with Vpr-truncated HIV-1 virions. Since G418-selected HeLa clones carrying expression plasmid for scAb-WXXF were obtained much more frequently than those for scAb-Vpr, scAb-WXXF was inferred to be less toxic to cells than scAb-Vpr. These results suggest that scAb-WXXF may serve as a novel class of antiviral therapeutic that inactivates progeny HIV virions from within.

  12. Adsorption of As(III), As(V) and Cu(II) on zirconium oxide immobilized alginate beads in aqueous phase.

    PubMed

    Kwon, Oh-Hun; Kim, Jong-Oh; Cho, Dong-Wan; Kumar, Rahul; Baek, Seung Han; Kurade, Mayur B; Jeon, Byong-Hun

    2016-10-01

    A composite adsorbent to remove arsenite [As(III)], arsenate [As(V)], and copper [Cu(II)] from aqueous phase was synthesized by immobilizing zirconium oxide on alginate beads (ZOAB). The composition (wt%) of ZOAB (Zr-34.0; O-32.7; C-21.3; Ca-1.0) was confirmed by energy dispersive X-ray (EDX) analysis. Sorption studies were conducted on single and binary sorbate systems, and the effects of contact time, initial adsorbate concentration, and pH on the adsorption performance of ZOAB (pHPZC = 4.3) were monitored. The sorption process for As(III)/As(V) and Cu(II) reached an equilibrium state within 240 h and 24 h, respectively, with maximum sorption capacities of 32.3, 28.5, and 69.9 mg g(-1), respectively. The addition of Cu(II) was favorable for As(V) sorption in contrast to As(III). In the presence of 48.6 mg L(-1) Cu(II), the sorption capacity of As(V) increased from 1.5 to 3.8 mg g(-1) after 240 h. The sorption data for As(III)/As(V) and Cu(II) conformed the Freundlich and Langmuir isotherm models, respectively. The adsorption of As(III), As(V), and Cu(II) followed pseudo second order kinetics. The effect of arsenic species on Cu(II) sorption was insignificant. The results of present study demonstrated that the synthesized sorbent could be useful for the simultaneous removal of both anionic and cationic contaminants from wastewaters.

  13. Simultaneous removal of As(V) and Cr(VI) from water by macroporous anion exchanger supported nanoscale hydrous ferric oxide composite.

    PubMed

    Hua, Ming; Yang, Bowen; Shan, Chao; Zhang, Weiming; He, Shiya; Lv, Lu; Pan, Bingcai

    2017-03-01

    As(V) and Cr(VI) are both highly toxic anionic pollutants and commonly co-exist in some industrial effluents and contaminated waters. In this study, simultaneous removal of them was efficiently achieved by employing a composite adsorbent (HFO-201) fabricated by immobilizing nanoscale hydrous ferric oxide (HFO) within a macroporous anion exchanger D201. The HFO-201 composite possesses two types of adsorption sites, i.e. the quaternary ammonium groups fixed on the D201 matrix and the embedded HFO nanoparticles. In the binary solution, the adsorption kinetic processes of both As(V) and Cr(VI) by HFO-201 were well fitted with the pseudo-first order kinetic model. Furthermore, HFO-201 exhibited a significantly higher adsorption capacity toward As(V) than D201 and an identical adsorption capacity toward Cr(VI) to D201. During the removal process, As(V) was captured by both the electrostatic attraction from the fixed quaternary ammonium groups and the formation of inner-sphere complex with the embedded HFO nanoparticles. Whereas, Cr(VI) was primarily adsorbed by the fixed ammonium groups. Fixed-bed treatment of As(V)/Cr(VI) binary synthetic water by HFO-201 resulted in elimination of As (from 1.0 to below 0.01 mg/L) and Cr (from 5.0 to below 0.05 mg/L), with the treatment capacity of 1700 bed volume (BV). Moreover, the exhausted HFO-201 was amenable to efficient in situ regeneration with a binary NaOH-NaCl solution for repeated use without any significant capacity loss.

  14. Insights into the subsurface transport of As(V) and Se(VI) in produced water from hydraulic fracturing using soil samples from Qingshankou Formation, Songliao Basin, China.

    PubMed

    Chen, Season S; Sun, Yuqing; Tsang, Daniel C W; Graham, Nigel J D; Ok, Yong Sik; Feng, Yujie; Li, Xiang-Dong

    2017-04-01

    Produced water is a type of wastewater generated from hydraulic fracturing, which may pose a risk to the environment and humans due to its high ionic strength and the presence of elevated concentrations of metals/metalloids that exceed maximum contamination levels. The mobilization of As(V) and Se(VI) in produced water and selected soils from Qingshankou Formation in the Songliao Basin in China were investigated using column experiments and synthetic produced water whose quality was representative of waters arising at different times after well creation. Temporal effects of produced water on metal/metalloid transport and sorption/desorption were investigated by using HYDRUS-1D transport modelling. Rapid breakthrough and long tailings of As(V) and Se(VI) transport were observed in Day 1 and Day 14 solutions, but were reduced in Day 90 solution probably due to the elevated ionic strength. The influence of produced water on the hydrogeological conditions (i.e., change between equilibrium and non-equilibrium transport) was evidenced by the change of tracer breakthrough curves before and after the leaching of produced water. This possibly resulted from the sorption of polyacrylamide (PAM (-CH2CHCONH2-)n) onto soil surfaces, through its use as a friction reducer in fracturing solutions. The sorption was found to be reversible in this study. Minimal amounts of sorbed As(V) were desorbed whereas the majority of sorbed Se(VI) was readily leached out, to an extent which varied with the composition of the produced water. These results showed that the mobilization of As(V) and Se(VI) in soil largely depended on the solution pH and ionic strength. Understanding the differences in metal/metalloid transport in produced water is important for proper risk management. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Removal of As(III) and As(V) from aqueous solutions using nanoscale zero valent iron-reduced graphite oxide modified composites.

    PubMed

    Wang, Can; Luo, Hanjin; Zhang, Zilong; Wu, Yan; Zhang, Jian; Chen, Shaowei

    2014-03-15

    Nanoscale zero valent iron (NZVI) has high adsorption capacity of As(III) and As(V), but it is limited in practical use due to its small particle size and aggregation effect. Reduce graphite oxide (RGO) has been used as a support because of its high surface area. In order to utilize the advantage of NZVI and RGO as well as to avoid the disadvantage of NZVI, we loaded NZVI onto RGO via chemical reactions in this study. The adsorption capacity of As(III) and As(V), as determined from the Langmuir adsorption isotherms in batch experiments, was 35.83mgg(-1) and 29.04mgg(-1), respectively. And the adsorption kinetics fitted well with pseudo-second-order model. The residual concentration was found to meet the standard of WHO after the samples were treated with 0.4gL(-1) NZVI-RGO when the initial concentration of As(III) and As(V) were below 8ppm and 3ppm. Especially, when the initial concentration of As(III) was below 3ppm, the residual concentration was within 1ppb; whereas, the residual concentration was undetected when the initial concentration of As(III) was 1ppm. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Fabrication of magnetic biochar as a treatment medium for As(V) via pyrolysis of FeCl3-pretreated spent coffee ground.

    PubMed

    Cho, Dong-Wan; Yoon, Kwangsuk; Kwon, Eilhann E; Biswas, Jayanta Kumar; Song, Hocheol

    2017-10-01

    This study investigated the preparation of magnetic biochar from N2- and CO2-assisted pyrolysis of spent coffee ground (SCG) for use as an adsorption medium for As(V), and the effects of FeCl3 pretreatment of SCG on the material properties and adsorption capability of the produced biochar. Pyrolysis of FeCl3-pretreated SCG in CO2 atmosphere produced highly porous biochar with its surface area ∼70 times greater than that produced in N2 condition. However, despite the small surface area, biochar produced in N2 showed greater As(V) adsorption capability. X-ray diffraction and X-ray photoelectron spectrometer analyses identified Fe3C and Fe3O4 as dominant mineral phases in N2 and CO2 conditions, with the former being much more adsorptive toward As(V). The overall results suggest functional biochar can be facilely fabricated by necessary pretreatment to expand the applicability of biochar for specific purposes. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Rapid on-site separation of As(III) and As(V) in waters using a disposable thiol-modified sand cartridge.

    PubMed

    Du, Jingjing; Che, Dongsheng; Zhang, Jianfeng; Jing, Chuanyong

    2014-08-01

    The rapid redox transformation of arsenic (As) species in waters presents a great environmental challenge in the accurate determination of its concentration and toxicity. The motivation of the present study was therefore to develop a method for rapid on-site separation of As(V) and As(III) in various aqueous matrices. The authors synthesized a thiol-modified sand (T-sand) that selectively removed As(III) but did not adsorb As(V). The novel application of this T-sand in a disposable cartridge was able to successfully separate As(V) (37-970 µg L(-1) ) and As(III) (not detected to 488 µg L(-1) ) in 23 groundwater samples collected in areas with naturally occurring As. The As speciation results determined with T-sand separation in the field were consistent with those obtained using high-performance liquid chromatography-atomic fluorescence spectrometry. Furthermore, the T-sand cartridge was applicable in a wide variety of matrices, including groundwater, leachants of the toxicity-characteristic leaching procedure, and extracts from the California waste extraction test; sequential extraction test; and in vitro gastrointestinal extraction. This easy-to-use separation method is especially suitable for routine field monitoring of As speciation.

  18. As(III) and As(V) sorption on iron-modified non-pyrolyzed and pyrolyzed biomass from Petroselinum crispum (parsley).

    PubMed

    Jiménez-Cedillo, M J; Olguín, M T; Fall, C; Colin-Cruz, A

    2013-03-15

    The sorption of As(III) and As(V) from aqueous solutions onto iron-modified Petroselinum crispum (PCFe) and iron-modified carbonaceous material from the pyrolysis of P. crispum (PCTTFe) was investigated. The modified sorbents were characterized with scanning electron microscopy. The sorbent elemental composition was determined with energy-dispersive X-ray spectroscopy (EDS). The principal functional groups from the sorbents were determined with FT-IR. The specific surfaces and points of zero charge (pzc) of the materials were also determined. As(III) and As(V) sorption onto the modified sorbents were performed in a batch system. After the sorption process, the As content in the liquid and solid phases was determined with atomic absorption and neutron activation analyses, respectively. After the arsenic sorption processes, the desorption of Fe from PCFe and PCTTFe was verified with atomic absorption spectrometry. The morphology of PC changed after iron modification. The specific area and pzc differed significantly between the iron-modified non-pyrolyzed and pyrolyzed P. crispum. The kinetics of the arsenite and arsenate sorption processes were described with a pseudo-second-order model. The Langmuir-Freundlich model provided the isotherms with the best fit. Less than 0.02% of the Fe was desorbed from the PCFe and PCTTFe after the As(III) and As(V) sorption processes. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Prediction of phycoremediation of As(III) and As(V) from synthetic wastewater by Chlorella pyrenoidosa using artificial neural network

    NASA Astrophysics Data System (ADS)

    Podder, M. S.; Majumder, C. B.

    2017-03-01

    An artificial neural network (ANN) model was developed to predict the phycoremediation efficiency of Chlorella pyrenoidosa for the removal of both As(III) and As(V) from synthetic wastewater based on 49 data-sets obtained from experimental study and increased the data using CSCF technique. The data were divided into training (60%) validation (20%) and testing (20%) sets. The data collected was used for training a three-layer feed-forward back propagation (BP) learning algorithm having 4-5-1 architecture. The model used tangent sigmoid transfer function at input to hidden layer (tansing) while a linear transfer function (purelin) was used at output layer. Comparison between experimental results and model results gave a high correlation coefficient (R{allANN/2} equal to 0.99987 for both ions and exhibited that the model was able to predict the phycoremediation of As(III) and As(V) from wastewater. Experimental parameters influencing phycoremediation process like pH, inoculum size, contact time and initial arsenic concentration [either As(III) or As(V)] were investigated. A contact time of 168 h was mainly required for achieving equilibrium at pH 9.0 with an inoculum size of 10% (v/v). At optimum conditions, metal ion uptake enhanced with increasing initial metal ion concentration.

  20. Design, synthesis, and biological evaluation of chicoric acid analogs as inhibitors of HIV-1 integrase.

    PubMed

    Charvat, Trevor T; Lee, Deborah J; Robinson, W Edward; Chamberlin, A Richard

    2006-07-01

    A series of analogs of the potent HIV-1 integrase (HIV IN) inhibitor chicoric acid (CA) was designed with the intention of ameliorating some of the parent natural product's undesirable properties, in particular its toxicity, instability, and poor membrane permeability. More than 70 analogs were synthesized and assayed for three types of activity: (1) the ability to inhibit 3'-end processing and strand transfer reactions using recombinant HIV IN in vitro, (2) toxicity against the CD4+ lymphoblastoid cell line, MT2, and (3) anti-HIV activity against HIV(LAI). CA analogs lacking one of the carboxyl groups of CA and with 3,4,5-trihydroxycinnamoyl sidechains in place of the caffeoyl group of CA exhibited the most potent inhibition of HIV replication and end-processing activity. Galloyl-substituted derivatives also displayed very potent in vitro and in vivo activities, in most cases exceeding the inhibitory effects of CA itself. Conversely, analogous monocarboxy caffeoyl analogs exhibited only modest inhibition, while the corresponding 3,4-dihydroxybenzoyl-substituted compounds were devoid of activity.

  1. A docking study of L-chicoric acid with HIV-1 integrase.

    PubMed

    Healy, Eamonn F; Sanders, Jonathan; King, Peter J; Robinson, W Edward

    2009-01-01

    Human immunodeficiency virus 1 integrase (HIV-1 IN) is the enzyme responsible for integrating the viral DNA into the host genome, and is essential to the replication of the virus. L-Chicoric acid (L-CA) is a bidentate catechol that has been identified as a potent inhibitor of HIV-1 IN. Using the new Autodock 4.0 free-energy function we have obtained a L-CA binding mode that explains its observed potency and is consistent with available experimental data. Because of the alpha,beta-unsaturated ester functionality of the side arms of L-CA we first performed an extensive conformational analysis of L-CA using semiempirical and ab initio calculations. As a result we have identified two distinct L-CA binding modes, one for the s-cis/s-cis and another for the s-cis/s-trans isomers. The most stable conformer was found to be the structure with the alpha,beta-unsaturated ester in the s-cis conformation for both arms of L-CA. This conformer also gave the top-ranked docking solution. Analysis of the interactions with key IN residues, combined with results using a L-CA tetraacetylated derivative and a Q148A IN mutant, correlate well with the experimental data.

  2. HIV integrase variability and genetic barrier in antiretroviral naïve and experienced patients

    PubMed Central

    2011-01-01

    Background HIV-1 integrase (IN) variability in treatment naïve patients with different HIV-1 subtypes is a major issue. In fact, the effect of previous exposure to antiretrovirals other than IN inhibitors (INI) on IN variability has not been satisfactorily defined. In addition, the genetic barrier for specific INI resistance mutations remains to be calculated. Methods IN variability was analyzed and compared with reverse transcriptase (RT) and protease (PR) variability in 41 treatment naïve and 54 RT inhibitor (RTI) and protease inhibitor (PRI) experienced patients from subjects infected with subtype B and non-B strains. In addition, four HIV-2 strains were analyzed in parallel. Frequency and distribution of IN mutations were compared between HAART-naïve and RTI/PI-experienced patients; the genetic barrier for 27 amino acid positions related to INI susceptibility was calculated as well. Results Primary mutations associated with resistance to INI were not detected in patients not previously treated with this class of drug. However, some secondary mutations which have been shown to contribute to INI resistance were found. Only limited differences in codon usage distribution between patient groups were found. HIV-2 strains from INI naïve patients showed the presence of both primary and secondary resistance mutations. Conclusion Exposure to antivirals other than INI does not seem to significantly influence the emergence of mutations implicated in INI resistance. HIV-2 strain might have reduced susceptibility to INI. PMID:21453487

  3. Synthesis and biological evaluation of geminal disulfones as HIV-1 integrase inhibitors.

    PubMed

    Meadows, D Christopher; Mathews, Timothy B; North, Thomas W; Hadd, Michael J; Kuo, Chih Lin; Neamati, Nouri; Gervay-Hague, Jacquelyn

    2005-07-14

    Integration of HIV-1 viral DNA into the host genome is carried out by HIV-integrase (IN) and is a critical step in viral replication. Although several classes of compounds have been reported to inhibit IN in enzymatic assays, inhibition is not always correlated with antiviral activity. Moreover, potent antiviral IN inhibitors such as the chicoric acids do not act upon the intended enzymatic target but behave as entry inhibitors instead. The charged nature of the chicoric acids contributes to poor cellular uptake, and these compounds are further plagued by rapid ester hydrolysis in vivo. To address these critical deficiencies, we designed neutral, nonhydrolyzable analogues of the chicoric acids. Herein, we report the synthesis, enzyme inhibition studies, and cellular antiviral data for a series of geminal disulfones. Of the 10 compounds evaluated, 8 showed moderate to high inhibition of IN in purified enzyme assays. The purified enzyme data correlated with antiviral assays for all but two compounds, suggesting alternative modes of inhibition. Time-of-addition studies were performed on these analogues, and the results indicate that they inhibit an early stage in the replication process, perhaps entry. In contrast, the most potent member of the correlative group shows behavior consistent with IN being the cellular target.

  4. Methods for the Analyses of Inhibitor-Induced Aberrant Multimerization of HIV-1 Integrase

    PubMed Central

    Kessl, Jacques J.; Sharma, Amit; Kvaratskhelia, Mamuka

    2016-01-01

    HIV-1 integrase (IN) is an important therapeutic target as its function is essential for the viral lifecycle. The discovery of multifunctional allosteric IN inhibitors or ALLINIs, which potently impair viral replication by promoting aberrant, higher order IN multimerization as well as inhibit IN interactions with its cellular cofactor, LEDGF/p75, has opened new venues to exploit IN multimerization as a therapeutic target. Furthermore, the recent discovery of multimerization selective IN inhibitors or MINIs, has provided new investigational probes to study the direct effects of aberrant IN multimerization in vitro and in infected cells. Here we describe three complementary methods designed to detect and quantify the effects of these new classes of inhibitors on IN multimerization. These methods include a homogenous time-resolved fluorescence-based assay which allows for measuring EC50 values for the inhibitor-induced aberrant IN multimerization, a dynamic light scattering-based assay which allows for monitoring the formation and sizes of oligomeric IN particles in a time-dependent manner, and a chemical cross-linking-based assay of interacting IN subunits which allows for the determination of IN oligomers in viral particles. PMID:26714710

  5. Metabolism, excretion, and mass balance of the HIV-1 integrase inhibitor dolutegravir in humans.

    PubMed

    Castellino, Stephen; Moss, Lee; Wagner, David; Borland, Julie; Song, Ivy; Chen, Shuguang; Lou, Yu; Min, Sherene S; Goljer, Igor; Culp, Amanda; Piscitelli, Stephen C; Savina, Paul M

    2013-08-01

    The pharmacokinetics, metabolism, and excretion of dolutegravir, an unboosted, once-daily human immunodeficiency virus type 1 integrase inhibitor, were studied in healthy male subjects following single oral administration of [(14)C]dolutegravir at a dose of 20 mg (80 μCi). Dolutegravir was well tolerated, and absorption of dolutegravir from the suspension formulation was rapid (median time to peak concentration, 0.5 h), declining in a biphasic fashion. Dolutegravir and the radioactivity had similar terminal plasma half-lives (t1/2) (15.6 versus 15.7 h), indicating metabolism was formation rate limited with no long-lived metabolites. Only minimal association with blood cellular components was noted with systemic radioactivity. Recovery was essentially complete (mean, 95.6%), with 64.0% and 31.6% of the dose recovered in feces and urine, respectively. Unchanged dolutegravir was the predominant circulating radioactive component in plasma and was consistent with minimal presystemic clearance. Dolutegravir was extensively metabolized. An inactive ether glucuronide, formed primarily via UGT1A1, was the principal biotransformation product at 18.9% of the dose excreted in urine and the principal metabolite in plasma. Two minor biotransformation pathways were oxidation by CYP3A4 (7.9% of the dose) and an oxidative defluorination and glutathione substitution (1.8% of the dose). No disproportionate human metabolites were observed.

  6. Metabolism, Excretion, and Mass Balance of the HIV-1 Integrase Inhibitor Dolutegravir in Humans

    PubMed Central

    Castellino, Stephen; Moss, Lee; Wagner, David; Borland, Julie; Song, Ivy; Chen, Shuguang; Lou, Yu; Min, Sherene S.; Goljer, Igor; Culp, Amanda; Piscitelli, Stephen C.

    2013-01-01

    The pharmacokinetics, metabolism, and excretion of dolutegravir, an unboosted, once-daily human immunodeficiency virus type 1 integrase inhibitor, were studied in healthy male subjects following single oral administration of [14C]dolutegravir at a dose of 20 mg (80 μCi). Dolutegravir was well tolerated, and absorption of dolutegravir from the suspension formulation was rapid (median time to peak concentration, 0.5 h), declining in a biphasic fashion. Dolutegravir and the radioactivity had similar terminal plasma half-lives (t1/2) (15.6 versus 15.7 h), indicating metabolism was formation rate limited with no long-lived metabolites. Only minimal association with blood cellular components was noted with systemic radioactivity. Recovery was essentially complete (mean, 95.6%), with 64.0% and 31.6% of the dose recovered in feces and urine, respectively. Unchanged dolutegravir was the predominant circulating radioactive component in plasma and was consistent with minimal presystemic clearance. Dolutegravir was extensively metabolized. An inactive ether glucuronide, formed primarily via UGT1A1, was the principal biotransformation product at 18.9% of the dose excreted in urine and the principal metabolite in plasma. Two minor biotransformation pathways were oxidation by CYP3A4 (7.9% of the dose) and an oxidative defluorination and glutathione substitution (1.8% of the dose). No disproportionate human metabolites were observed. PMID:23669385

  7. Dolutegravir: a new integrase strand transfer inhibitor for the treatment of HIV.

    PubMed

    Shah, Bhavik M; Schafer, Jason J; Desimone, Joseph A

    2014-05-01

    The first two integrase strand transfer inhibitors (INSTIs) approved for treatment of patients infected with human immunodeficiency virus (HIV) were raltegravir and elvitegravir. Both raltegravir and elvitegravir are now guideline-preferred agents as part of an antiretroviral regimen for treatment-naive patients. However, raltegravir is dosed twice/day. Elvitegravir is available in a single-tablet regimen and dosed once/day because it is administered with the pharmacokinetic booster cobicistat, a potent CYP3A4 inhibitor that can lead to clinically significant drug-drug interactions. In addition, raltegravir and elvitegravir have a low genetic barrier to resistance and are associated with cross-resistance. Dolutegravir is a new-generation INSTI administered once/day without a pharmacokinetic booster and can be coformulated in a single-tablet regimen. Phase III studies have demonstrated the efficacy and safety of dolutegravir for treatment-naive and treatment-experienced patients. Compared with other INSTIs, dolutegravir has a higher genetic barrier to resistance. Dolutegravir was approved by the U.S. Food and Drug Administration in August 2013 and joins raltegravir and elvitegravir as guideline-preferred agents for the management for HIV-infected treatment-naive patients. © 2013 Pharmacotherapy Publications, Inc.

  8. Cryo-EM reveals a novel octameric integrase structure for β-retroviral intasome function

    PubMed Central

    Ballandras-Colas, Allison; Brown, Monica; Cook, Nicola J.; Dewdney, Tamaria G.; Demeler, Borries; Cherepanov, Peter; Lyumkis, Dmitry; Engelman, Alan N.

    2016-01-01

    Retroviral integrase (IN) catalyzes the integration of viral DNA (vDNA) into host target (tDNA), which is an essential step in the lifecycle of all retroviruses1. Prior structural characterization of IN-vDNA complexes, or intasomes, from the spumavirus prototype foamy virus (PFV) revealed a functional IN tetramer2–5, and it is generally believed that intasomes derived from other retroviral genera will employ tetrameric IN6–9. However, the intasomes of orthoretroviruses, which include all known pathogenic species, have not been characterized structurally. Using single-particle cryo-electron microscopy (cryo-EM) and X-ray crystallography, we determine here an unexpected octameric IN architecture for the β-retrovirus mouse mammary tumor virus (MMTV) intasome. The structure is composed of two core IN dimers, which interact with the vDNA ends and structurally mimic the PFV IN tetramer, and two flanking IN dimers that engage the core structure via their IN C-terminal domains (CTDs). Contrary to the belief that tetrameric IN components are sufficient to catalyze integration, the flanking IN dimers were necessary for MMTV IN activity. The IN octamer solves a conundrum for the β- as well as α-retroviruses by providing critical CTDs to the intasome core that cannot be provided in cis due to evolutionarily restrictive catalytic core domain (CCD)-CTD linker regions. The octameric architecture of the MMTV intasome provides a new paradigm for the structural basis of retroviral DNA integration. PMID:26887496

  9. Integrase-mediated spacer acquisition during CRISPR–Cas adaptive immunity

    PubMed Central

    Nuñez, James K.; Lee, Amy S.Y.; Engelman, Alan; Doudna, Jennifer A.

    2015-01-01

    Bacteria and archaea insert spacer sequences acquired from foreign DNAs into CRISPR loci to generate immunological memory. The Escherichia coli Cas1–Cas2 complex mediates spacer acquisition in vivo, but the molecular mechanism of this process is unknown. Here we show that the purified Cas1–Cas2 complex integrates oligonucleotide DNA substrates into acceptor DNA to yield products similar to those generated by retroviral integrases and transposases. Cas1 is the catalytic subunit, whereas Cas2 substantially increases integration activity. Protospacer DNA with free 3'-OH ends and supercoiled target DNA are required, and integration occurs preferentially at the ends of CRISPR repeats and at sequences adjacent to cruciform structures abutting A-T rich regions, similar to the CRISPR leader sequence. Our results demonstrate the Cas1–Cas2 complex to be the minimal machinery that catalyzes spacer DNA acquisition and explain the significance of CRISPR repeats in providing sequence and structural specificity for Cas1–Cas2-mediated adaptive immunity. PMID:25707795

  10. Inhibition on HIV-1 integrase activity and nitric oxide production of compounds from Ficus glomerata.

    PubMed

    Bunluepuech, Kingkan; Sudsai, Teeratad; Wattanapiromsakul, Chatchai; Tewtrakul, Supinya

    2011-08-01

    An ethanol Ficus glomerata wood extract and its purified components were investigated for their HIV-1 integrase (IN) and nitric oxide (NO) inhibitory activities. From bioassay-guided isolation, five compounds: beta-sitosterol-D-glucoside (1), aloe-emodin (2), genistein (3), 1,3,6-trihydroxy-8-methyl-anthraquinone (4) and 3-(1-C-beta-D-glucopyranosyl)-2,6-dihydroxy-5-methoxybenzoic acid (5) were isolated. Among the tested samples, at concentrations of 100 microM; compound 2 showed 31.9% inhibition of HIV-1 IN, followed by 4 (19.5%), whereas other compounds were inactive. With regard to the inhibitory effect on NO production, 3 possessed the highest activity with an IC50 value of 27.5 microM, followed by 4 (IC50 = 34.7 microM) and 2 (IC50 = 41.8 microM), respectively. This is the first time that compounds 2-5 have been isolated from Ficus glomerata.

  11. The Preserved HTH-Docking Cleft of HIV-1 Integrase Is Functionally Critical.

    PubMed

    Galilee, Meytal; Britan-Rosich, Elena; Griner, Sarah L; Uysal, Serdar; Baumgärtel, Viola; Lamb, Don C; Kossiakoff, Anthony A; Kotler, Moshe; Stroud, Robert M; Marx, Ailie; Alian, Akram

    2016-11-01

    HIV-1 integrase (IN) catalyzes viral DNA integration into the host genome and facilitates multifunctional steps including virus particle maturation. Competency of IN to form multimeric assemblies is functionally critical, presenting an approach for anti-HIV strategies. Multimerization of IN depends on interactions between the distinct subunit domains and among the flanking protomers. Here, we elucidate an overlooked docking cleft of IN core domain that anchors the N-terminal helix-turn-helix (HTH) motif in a highly preserved and functionally critical configuration. Crystallographic structure of IN core domain in complex with Fab specifically targeting this cleft reveals a steric overlap that would inhibit HTH-docking, C-terminal domain contacts, DNA binding, and subsequent multimerization. While Fab inhibits in vitro IN integration activity, in vivo it abolishes virus particle production by specifically associating with preprocessed IN within Gag-Pol and interfering with early cytosolic Gag/Gag-Pol assemblies. The HTH-docking cleft may offer a fresh hotspot for future anti-HIV intervention strategies. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Diketoacid-genre HIV-1 integrase inhibitors containing enantiomeric arylamide functionality.

    PubMed

    Zhao, Xue Zhi; Maddali, Kasthuraiah; Marchand, Christophe; Pommier, Yves; Burke, Terrence R

    2009-07-15

    Using our recently disclosed 2,3-dihydro-6,7-dihydroxy-1H-isoindol-1-one and 4,5-dihydroxy-1H-isoindole-1,3(2H)-dione integrase inhibitors, we report differential effects on inhibitory potency induced by introduction of an alpha-chiral center into a key aryl substituent. We show that introduction of the chiral center is uniformly deleterious to binding, with the (R)-enantiomer being more deleterious than the (S)-enantiomer. A greater enantiomeric difference in potency is shown by inhibitors that have restricted rotation of the aryl ring, with the larger difference being due to poorer potency of the (R)-enantiomer rather than higher potency of the (S)-enantiomer. The potency difference for enantiomers based on the isoindoline-1,3-dione ring system is less than for those derived from the isoindol-1-one ring system. Our findings provide useful information that should aid in understanding molecular binding interactions of DKA-derived IN inhibitors.

  13. Comparison of DNA binding and integration half-site selection by avian myeloblastosis virus integrase.

    PubMed Central

    Grandgenett, D P; Inman, R B; Vora, A C; Fitzgerald, M L

    1993-01-01

    Insertion of the linear retrovirus DNA genome into the host DNA by the virus-encoded integrase (IN) is essential for efficient replication. We devised an efficient virus-like DNA plasmid integration assay which mimics the standard oligonucleotide assay for integration. It permitted us to study, by electron microscopy and sequence analysis, insertion of a single long terminal repeat terminus (LTR half-site) of one plasmid into another linearized plasmid. The reaction was catalyzed by purified avian myeloblastosis virus IN in the presence of Mg2+. The recombinant molecules were easily visualized and quantitated by agarose gel electrophoresis. Agarose gel-purified recombinants could be genetically selected by transformation of ligated recombinants into Escherichia coli HB101 cells. Electron microscopy also permitted the identification and localization of IN-DNA complexes on the virus-like substrate in the absence of the joining reaction. Intramolecular and intermolecular DNA looping by IN was visualized. Although IN preferentially bound to AT-rich regions in the absence of the joining reaction, there was a bias towards GC-rich regions for the joining reaction. Alignment of 70 target site sequences 5' of the LTR half-site insertions with 68 target sites previously identified for the concerted insertion of both LTR termini (LTR full-site reaction) indicated similar GC inflection patterns with both insertional events. Comparison of the data suggested that IN recognized only half of the target sequences necessary for integration with the LTR half-site reaction. Images PMID:8474165

  14. Class 1 integrase, sulfonamide and tetracycline resistance genes in wastewater treatment plant and surface water.

    PubMed

    Makowska, Nicoletta; Koczura, Ryszard; Mokracka, Joanna

    2016-02-01

    Wastewater treatment plants are considered hot spots for multiplication and dissemination of antibiotic-resistant bacteria and resistance genes. In this study, we determined the presence of class 1 integron integrase and genes conferring resistance to tetracyclines and sulfonamides in the genomes of culturable bacteria isolated from a wastewater treatment plant and the river that receives the treated wastewater. Moreover, using PCR-based metagenomic approach, we quantified intI1, tet and sul genes. Wastewater treatment caused the decrease in the total number of culturable heterotrophs and bacteria resistant to tetracycline and sulfonamides, along with the decrease in the number of intI1, sul and tet gene copies per ml, with significant reduction of tet(B). On the other hand, the treatment process increased both the frequency of tetracycline- and sulfonamide-resistant bacteria and intI1-positive strains, and the relative abundance of all quantified antibiotic resistance genes (ARGs) and intI1 gene; in the case of tet(A) and sul2 significantly. The discharge of treated wastewater increased the number of intI1, tet and sul genes in the receiving river water both in terms of copy number per ml and relative abundance. Hence, despite the reduction of the number of ARGs and ARBs, wastewater treatment selects for bacteria with ARGs in effluent.

  15. Using the class 1 integron-integrase gene as a proxy for anthropogenic pollution

    PubMed Central

    Gillings, Michael R; Gaze, William H; Pruden, Amy; Smalla, Kornelia; Tiedje, James M; Zhu, Yong-Guan

    2015-01-01

    Around all human activity, there are zones of pollution with pesticides, heavy metals, pharmaceuticals, personal care products and the microorganisms associated with human waste streams and agriculture. This diversity of pollutants, whose concentration varies spatially and temporally, is a major challenge for monitoring. Here, we suggest that the relative abundance of the clinical class 1 integron-integrase gene, intI1, is a good proxy for pollution because: (1) intI1 is linked to genes conferring resistance to antibiotics, disinfectants and heavy metals; (2) it is found in a wide variety of pathogenic and nonpathogenic bacteria; (3) its abundance can change rapidly because its host cells can have rapid generation times and it can move between bacteria by horizontal gene transfer; and (4) a single DNA sequence variant of intI1 is now found on a wide diversity of xenogenetic elements, these being complex mosaic DNA elements fixed through the agency of human selection. Here we review the literature examining the relationship between anthropogenic impacts and the abundance of intI1, and outline an approach by which intI1 could serve as a proxy for anthropogenic pollution. PMID:25500508

  16. Using the class 1 integron-integrase gene as a proxy for anthropogenic pollution.

    PubMed

    Gillings, Michael R; Gaze, William H; Pruden, Amy; Smalla, Kornelia; Tiedje, James M; Zhu, Yong-Guan

    2015-06-01

    Around all human activity, there are zones of pollution with pesticides, heavy metals, pharmaceuticals, personal care products and the microorganisms associated with human waste streams and agriculture. This diversity of pollutants, whose concentration varies spatially and temporally, is a major challenge for monitoring. Here, we suggest that the relative abundance of the clinical class 1 integron-integrase gene, intI1, is a good proxy for pollution because: (1) intI1 is linked to genes conferring resistance to antibiotics, disinfectants and heavy metals; (2) it is found in a wide variety of pathogenic and nonpathogenic bacteria; (3) its abundance can change rapidly because its host cells can have rapid generation times and it can move between bacteria by horizontal gene transfer; and (4) a single DNA sequence variant of intI1 is now found on a wide diversity of xenogenetic elements, these being complex mosaic DNA elements fixed through the agency of human selection. Here we review the literature examining the relationship between anthropogenic impacts and the abundance of intI1, and outline an approach by which intI1 could serve as a proxy for anthropogenic pollution.

  17. Initial Characterization of Integrase-Defective Lentiviral Vectors for Pancreatic Cancer Gene Therapy.

    PubMed

    Hanoun, Naima; Gayral, Marion; Pointreau, Adeline; Buscail, Louis; Cordelier, Pierre

    2016-02-01

    The vast majority (85%) of pancreatic ductal adenocarcinomas (PDACs) are discovered at too of a late stage to allow curative surgery. In addition, PDAC is highly resistant to conventional methods of chemotherapy and radiotherapy, which only offer a marginal clinical benefit. Consequently, the prognosis of this cancer is devastating, with a 5-year survival rate of less than 5%. In this dismal context, we recently demonstrated that PDAC gene therapy using nonviral vectors is safe and feasible, with early signs of efficacy in selected patients. Our next step is to transfer to the clinic HIV-1-based lentiviral vectors (LVs) that outshine other therapeutic vectors to treat experimental models of PDAC. However, a primary safety issue presented by LVs that may delay their use in patients is the risk of oncogenesis after vector integration in the host's cell DNA. Thus, we developed a novel anticancerous approach based on integrase-defective lentiviral vectors (IDLVs) and demonstrated that IDLVs can be successfully engineered to transiently deliver therapeutic genes to inhibit pancreatic cancer cells proliferation. This work stems for the use of therapeutic IDLVs for the management of PDAC, in forthcoming early phase gene therapy clinical trial for this disease with no cure.

  18. Speciation analysis of inorganic arsenic in river water by Amberlite IRA 910 resin immobilized in a polyacrylamide gel as a selective binding agent for As(V) in diffusive gradient thin film technique.

    PubMed

    Rolisola, Ana M C M; Suárez, Carlos A; Menegário, Amauri A; Gastmans, Didier; Kiang, Chang H; Colaço, Camila D; Garcez, Daniel L; Santelli, Ricardo E

    2014-09-07

    In this study, a method is proposed for the selective retention of As(V) using diffusive gradient in thin film (DGT) samplers containing a strongly basic anion exchange resin (Amberlite IRA 910) supported on a polyacrylamide gel. In addition, the total arsenic content is determined by ferrihydrite gel discs. Subsequently, the concentration of As(III) was obtained by determining the difference between the total As and As(V). DGT experiments showed linear accumulation of As(V) (up to 280 ng) until a deployment time of 8 h deployment (R(2) > 0.99). The retention of As(V) was appropriate (97.9-112.3%) between pH 5 and 9. For a solution with an ionic strength ranging from 0.001 to 0.05 mol L(-1), the As(V) uptake ranged from 90-120%. The proposed method was applied for the speciation of arsenic in river water. For the analysis of spiked samples collected at the Furnas stream, the recoveries of total arsenic content ranged between 103.9% and 118.8%. However, the recoveries of As(III) and As(V) were 43.3-75.2% and 147.3-153.4%, respectively. These differences were probably because of the oxidation of As(III) to As(V) during deployments. For spiked samples collected at the Ribeirão Claro, the recoveries of dissolved As(III), As(V) and As(T) were 103.1%, 108.0% and 106.3%, respectively. Thus, the DGT technique with Amberlite IRA 910 resin as the binding phase can be employed for the in situ redox speciation of inorganic arsenic.

  19. X-ray crystal structure of the N-terminal region of Moloney murine leukemia virus integrase and its implications for viral DNA recognition.

    PubMed

    Guan, Rongjin; Aiyer, Sriram; Cote, Marie L; Xiao, Rong; Jiang, Mei; Acton, Thomas B; Roth, Monica J; Montelione, Gaetano T

    2017-04-01

    The retroviral integrase (IN) carries out the integration of a dsDNA copy of the viral genome into the host DNA, an essential step for viral replication. All IN proteins have three general domains, the N-terminal domain (NTD), the catalytic core domain, and the C-terminal domain. The NTD includes an HHCC zinc finger-like motif, which is conserved in all retroviral IN proteins. Two crystal structures of Moloney murine leukemia virus (M-MuLV) IN N-terminal region (NTR) constructs that both include an N-terminal extension domain (NED, residues 1-44) and an HHCC zinc-finger NTD (residues 45-105), in two crystal forms are reported. The structures of IN NTR constructs encoding residues 1-105 (NTR1-105 ) and 8-105 (NTR8-105 ) were determined at 2.7 and 2.15 Å resolution, respectively and belong to different space groups. While both crystal forms have similar protomer structures, NTR1-105 packs as a dimer and NTR8-105 packs as a tetramer in the asymmetric unit. The structure of the NED consists of three anti-parallel β-strands and an α-helix, similar to the NED of prototype foamy virus (PFV) IN. These three β-strands form an extended β-sheet with another β-strand in the HHCC Zn(2+) binding domain, which is a unique structural feature for the M-MuLV IN. The HHCC Zn(2+) binding domain structure is similar to that in HIV and PFV INs, with variations within the loop regions. Differences between the PFV and MLV IN NEDs localize at regions identified to interact with the PFV LTR and are compared with established biochemical and virological data for M-MuLV. Proteins 2017; 85:647-656. © 2016 Wiley Periodicals, Inc.

  20. Sequential Deletion of the Integrase (Gag-Pol) Carboxyl Terminus Reveals Distinct Phenotypic Classes of Defective HIV-1 ▿ † §

    PubMed Central

    Mohammed, Kevin D.; Topper, Michael B.; Muesing, Mark A.

    2011-01-01

    A requisite step in the life cycle of human immunodeficiency virus type 1 (HIV-1) is the insertion of the viral genome into that of the host cell, a process catalyzed by the 288-amino-acid (32-kDa) viral integrase (IN). IN recognizes and cleaves the ends of reverse-transcribed viral DNA and directs its insertion into the chromosomal DNA of the target cell. IN function, however, is not limited to integration, as the protein is required for other aspects of viral replication, including assembly, virion maturation, and reverse transcription. Previous studies demonstrated that IN is comprised of three domains: the N-terminal domain (NTD), catalytic core domain (CCD), and C-terminal domain (CTD). Whereas the CCD is mainly responsible for providing the structural framework for catalysis, the roles of the other two domains remain enigmatic. This study aimed to elucidate the primary and subsidiary roles that the CTD has in protein function. To this end, we generated and tested a nested set of IN C-terminal deletion mutants in measurable assays of virologic function. We discovered that removal of up to 15 residues (IN 273) resulted in incremental diminution of enzymatic function and infectivity and that removal of the next three residues resulted in a loss of infectivity. However, replication competency was surprisingly reestablished with one further truncation, corresponding to IN 269 and coinciding with partial restoration of integration activity, but it was lost permanently for all truncations extending N terminal to this position. Our analyses of these replication-competent and -incompetent truncation mutants suggest potential roles for the IN CTD in precursor protein processing, reverse transcription, integration, and IN multimerization. PMID:21367893

  1. A new perovskite-type NdFeO3 adsorbent: synthesis, characterization, and As(V) adsorption

    NASA Astrophysics Data System (ADS)

    Luu, Minh Dai; Nhiem Dao, Ngoc; Van Nguyen, Duc; Chuc Pham, Ngoc; Ninh Vu, The; Dung Doan, Trung

    2016-06-01

    Nanocrystalline NdFeO3 perovskite oxide was prepared by the combustion method using polyvinyl alcohol (PVA) and corresponding metal nitrate precursors under optimum process conditions, using a solution with a pH of 2, a metal/PVA molar ratio of 1:3, and a calcination temperature of 600 °C, and was characterized by x-ray diffraction, scanning electron microscopy, transmission electron microscopy, Brunauer-Emmett-Teller nitrogen adsorption and desorption, energy dispersive x-ray spectroscopy, Fourier transform infrared spectroscopy, laser Raman spectroscopy, and thermogravimetric and differential thermal analysis. The synthesized perovskite NdFeO3, which had an average size of 20 nm and a relatively high surface area of 20 m2 g-1, was investigated for adsorbing hazardous arsenate from aqueous solution. Arsenate adsorption by perovskite NdFeO3 is a pH-dependent process with a high adsorption capacity at pH levels between 4 and 7 and a maximum As(V) adsorption capacity of 126.58 mg g-1, higher than most arsenate adsorbents reported in the literature. Kinetic and equilibrium data of reaction under the experimental conditions are best described by a pseudo-second-order and the Langmuir isotherm equation. The values of enthalpy, Gibbs free energy and entropy changes (ΔH0 = +63.916 kJ mol-1, ΔG0 = -6.551 kJ mol-1 to -14.021 kJ mol-1 at T = 283-313 K, ΔS0 = +0.249 kJ mol-1 K-1) suggested that the reaction was endothermic, spontaneous, and took place with increasing entropy.

  2. Determination of traces of Sb(III) using ASV in Sb-rich water samples affected by mining.

    PubMed

    Cidu, Rosa; Biddau, Riccardo; Dore, Elisabetta

    2015-01-07

    Chemical speciation [Sb(V) and Sb(III)] affects the mobility, bioavailability and toxicity of antimony. In oxygenated environments Sb(V) dominates whereas thermodynamically unstable Sb(III) may occur. In this study, a simple method for the determination of Sb(III) in non acidic, oxygenated water contaminated with antimony is proposed. The determination of Sb(III) was performed by anodic stripping voltammetry (ASV, 1-20 μg L(-1) working range), the total antimony, Sb(tot), was determined either by inductively coupled plasma mass spectrometry (ICP-MS, 1-100μgL(-1) working range) or inductively coupled plasma optical emission spectrometry (ICP-OES, 100-10,000 μg L(-1) working range) depending on concentration. Water samples were filtered on site through 0.45 μm pore size filters. The aliquot for determination of Sb(tot) was acidified with 1% (v/v) HNO3. Different preservatives, namely HCl, L(+) ascorbic acid or L(+) tartaric acid plus HNO3, were used to assess the stability of Sb(III) in synthetic solutions. The method was tested on groundwater and surface water draining the abandoned mine of Su Suergiu (Sardinia, Italy), an area heavily contaminated with Sb. The waters interacting with Sb-rich mining residues were non acidic, oxygenated, and showed extreme concentrations of Sb(tot) (up to 13,000 μg L(-1)), with Sb(III) <10% of total antimony. The stabilization with L(+) tartaric acid plus HNO3 appears useful for the determination of Sb(III) in oxygenated, Sb-rich waters. Due to the instability of Sb(III), analyses should be carried out within 7 days upon the water collection. The main advantage of the proposed method is that it does not require time-consuming preparation steps prior to analysis of Sb(III).

  3. Discovery of Novel HIV-1 Integrase Inhibitors Using QSAR-Based Virtual Screening of the NCI Open Database.

    PubMed

    Ko, Gene M; Garg, Rajni; Bailey, Barbara A; Kumar, Sunil

    2016-01-01

    Quantitative structure-activity relationship (QSAR) models can be used as a predictive tool for virtual screening of chemical libraries to identify novel drug candidates. The aims of this paper were to report the results of a study performed for descriptor selection, QSAR model development, and virtual screening for identifying novel HIV-1 integrase inhibitor drug candidates. First, three evolutionary algorithms were compared for descriptor selection: differential evolution-binary particle swarm optimization (DE-BPSO), binary particle swarm optimization, and genetic algorithms. Next, three QSAR models were developed from an ensemble of multiple linear regression, partial least squares, and extremely randomized trees models. A comparison of the performances of three evolutionary algorithms showed that DE-BPSO has a significant improvement over the other two algorithms. QSAR models developed in this study were used in consensus as a predictive tool for virtual screening of the NCI Open Database containing 265,242 compounds to identify potential novel HIV-1 integrase inhibitors. Six compounds were predicted to be highly active (plC50 > 6) by each of the three models. The use of a hybrid evolutionary algorithm (DE-BPSO) for descriptor selection and QSAR model development in drug design is a novel approach. Consensus modeling may provide better predictivity by taking into account a broader range of chemical properties within the data set conducive for inhibition that may be missed by an individual model. The six compounds identified provide novel drug candidate leads in the design of next generation HIV- 1 integrase inhibitors targeting drug resistant mutant viruses.

  4. The activity of the integrase inhibitor dolutegravir against HIV-1 variants isolated from raltegravir-treated adults.

    PubMed

    Underwood, Mark R; Johns, Brian A; Sato, Akihiko; Martin, Jeffrey N; Deeks, Steven G; Fujiwara, Tamio

    2012-11-01

    Dolutegravir (DTG, S/GSK1349572) is an integrase inhibitor with low nanomolar potency. Susceptibility to dolutegravir and raltegravir was determined for raltegravir-resistant clinical isolates. Genotypic and phenotypic susceptibility to integrase inhibitors was examined using 39 clinical isolate samples obtained from 18 adults who had exhibited incomplete viral suppression on a raltegravir-based regimen. Of 39 samples evaluated, 30 had genotypic and phenotypic resistance to raltegravir. All samples lacking raltegravir resistance retained complete susceptibility to dolutegravir. Of the 30 samples with genotypic evidence of raltegravir resistance, the median level of phenotypic resistance to raltegravir was high (median fold change in inhibitory concentration at 50%, >81; range, 3.7 to >87), while the level of resistance to dolutegravir was close to that of wild-type variants (median fold change, 1.5; range, 0.9-19.0). Longitudinal samples from 5 subjects collected during long-term failure of raltegravir revealed time-dependent general decreases in phenotypic susceptibility to raltegravir, with minimal changes in phenotypic susceptibility to dolutegravir. The median fold change to dolutegravir for isolates containing changes at G140S + Q148H, G140S + Q148R, T97A + Y143R, and N155H (thus including raltegravir signature resistance codons) were 3.75, 13.3, 1.05, and 1.37, respectively. Dolutegravir retained in vitro activity against clinical isolates obtained from subjects who failed raltegravir-based therapy at near wild-type levels for variants containing the Y143 and N155 resistance mutations. Isolates with Q148 plus additional integrase mutations possessed a broader range of and more reduced susceptibility to dolutegravir.

  5. The Activity of the Integrase Inhibitor Dolutegravir Against HIV-1 Variants Isolated From Raltegravir-Treated Adults

    PubMed Central

    Underwood, Mark R.; Johns, Brian A.; Sato, Akihiko; Martin, Jeffrey N.; Deeks, Steven G.; Fujiwara, Tamio

    2013-01-01

    Background Dolutegravir (DTG, S/GSK1349572) is an integrase inhibitor with low nanomolar potency. Susceptibility to dolutegravir and raltegravir was determined for raltegravir-resistant clinical isolates. Methods Genotypic and phenotypic susceptibility to integrase inhibitors was examined using 39 clinical isolate samples obtained from 18 adults who had exhibited incomplete viral suppression on a raltegravir-based regimen. Results Of 39 samples evaluated, 30 had genotypic and phenotypic resistance to raltegravir. All samples lacking raltegravir resistance retained complete susceptibility to dolutegravir. Of the 30 samples with genotypic evidence of raltegravir resistance, the median level of phenotypic resistance to raltegravir was high (median fold change in inhibitory concentration at 50%, >81; range, 3.7 to >87) while the level of resistance to dolutegravir was close to that of wild-type variants (median fold change, 1.5; range, 0.9–19.0). Longitudinal samples from 5 subjects collected during long-term failure of raltegravir revealed time-dependent general decreases in phenotypic susceptibility to raltegravir, with minimal changes in phenotypic susceptibility to dolutegravir. The median fold change to dolutegravir for isolates containing changes at G140S + Q148H; G140S + Q148R; T97A + Y143R; and N155H (thus including raltegravir signature resistance codons) were 3.75, 13.3, 1.05, and 1.37, respectively. Conclusions Dolutegravir retained in vitro activity against clinical isolates obtained from subjects who failed raltegravir-based therapy at near wild-type levels for variants containing the Y143 and N155 resistance mutations. Isolates with Q148 plus additional integrase mutations possessed a broader range of and more reduced susceptibility to dolutegravir. PMID:22878423

  6. Rapid activity prediction of HIV-1 integrase inhibitors: harnessing docking energetic components for empirical scoring by chemometric and artificial neural network approaches

    NASA Astrophysics Data System (ADS)

    Thangsunan, Patcharapong; Kittiwachana, Sila; Meepowpan, Puttinan; Kungwan, Nawee; Prangkio, Panchika; Hannongbua, Supa; Suree, Nuttee

    2016-06-01

    Improving performance of scoring functions for drug docking simulations is a challenging task in the modern discovery pipeline. Among various ways to enhance the efficiency of scoring function, tuning of energetic component approach is an attractive option that provides better predictions. Herein we present the first development of rapid and simple tuning models for predicting and scoring inhibitory activity of investigated ligands docked into catalytic core domain structures of HIV-1 integrase (IN) enzyme. We developed the models using all energetic terms obtained from flexible ligand-rigid receptor dockings by AutoDock4, followed by a data analysis using either partial least squares (PLS) or self-organizing maps (SOMs). The models were established using 66 and 64 ligands of mercaptobenzenesulfonamides for the PLS-based and the SOMs-based inhibitory activity predictions, respectively. The models were then evaluated for their predictability quality using closely related test compounds, as well as five different unrelated inhibitor test sets. Weighting constants for each energy term were also optimized, thus customizing the scoring function for this specific target protein. Root-mean-square error (RMSE) values between the predicted and the experimental inhibitory activities were determined to be <1 (i.e. within a magnitude of a single log scale of actual IC50 values). Hence, we propose that, as a pre-functional assay screening step, AutoDock4 docking in combination with these subsequent rapid weighted energy tuning methods via PLS and SOMs analyses is a viable approach to predict the potential inhibitory activity and to discriminate among small drug-like molecules to target a specific protein of interest.

  7. A quantum mechanic/molecular mechanic study of the wild-type and N155S mutant HIV-1 integrase complexed with diketo acid.

    PubMed

    Alves, Cláudio Nahum; Martí, Sergio; Castillo, Raquel; Andrés, Juan; Moliner, Vicent; Tuñón, Iñaki; Silla, Estanislao

    2008-04-01

    Integrase (IN) is one of the three human immunodeficiency virus type 1 (HIV-1) enzymes essential for effective viral replication. Recently, mutation studies have been reported that have shown that a certain degree of viral resistance to diketo acids (DKAs) appears when some amino acid residues of the IN active site are mutated. Mutations represent a fascinating experimental challenge, and we invite theoretical simulations for the disclosure of still unexplored features of enzyme reactions. The aim of this work is to understand the molecular mechanisms of HIV-1 IN drug resistance, which will be useful for designing anti-HIV inhibitors with unique resistance profiles. In this study, we use molecular dynamics simulations, within the hybrid quantum mechanics/molecular mechanics (QM/MM) approach, to determine the protein-ligand interaction energy for wild-type and N155S mutant HIV-1 IN, both complexed with a DKA. This hybrid methodology has the advantage of the inclusion of quantum effects such as ligand polarization upon binding, which can be very important when highly polarizable groups are embedded in anisotropic environments, for example in metal-containing active sites. Furthermore, an energy terms decomposition analysis was performed to determine contributions of individual residues to the enzyme-inhibitor interactions. The results reveal that there is a strong interaction between the Lys-159, Lys-156, and Asn-155 residues and Mg(2+) cation and the DKA inhibitor. Our calculations show that the binding energy is higher in wild-type than in the N155S mutant, in accordance with the experimental results. The role of the mutated residue has thus been checked as maintaining the structure of the ternary complex formed by the protein, the Mg(2+) cation, and the inhibitor. These results might be useful to design compounds with more interesting anti-HIV-1 IN activity on the basis of its three-dimensional structure.

  8. A Quantum Mechanic/Molecular Mechanic Study of the Wild-Type and N155S Mutant HIV-1 Integrase Complexed with Diketo Acid

    PubMed Central

    Alves, Cláudio Nahum; Martí, Sergio; Castillo, Raquel; Andrés, Juan; Moliner, Vicent; Tuñón, Iñaki; Silla, Estanislao

    2008-01-01

    Integrase (IN) is one of the three human immunodeficiency virus type 1 (HIV-1) enzymes essential for effective viral replication. Recently, mutation studies have been reported that have shown that a certain degree of viral resistance to diketo acids (DKAs) appears when some amino acid residues of the IN active site are mutated. Mutations represent a fascinating experimental challenge, and we invite theoretical simulations for the disclosure of still unexplored features of enzyme reactions. The aim of this work is to understand the molecular mechanisms of HIV-1 IN drug resistance, which will be useful for designing anti-HIV inhibitors with unique resistance profiles. In this study, we use molecular dynamics simulations, within the hybrid quantum mechanics/molecular mechanics (QM/MM) approach, to determine the protein-ligand interaction energy for wild-type and N155S mutant HIV-1 IN, both complexed with a DKA. This hybrid methodology has the advantage of the inclusion of quantum effects such as ligand polarization upon binding, which can be very important when highly polarizable groups are embedded in anisotropic environments, for example in metal-containing active sites. Furthermore, an energy terms decomposition analysis was performed to determine contributions of individual residues to the enzyme-inhibitor interactions. The results reveal that there is a strong interaction between the Lys-159, Lys-156, and Asn-155 residues and Mg2+ cation and the DKA inhibitor. Our calculations show that the binding energy is higher in wild-type than in the N155S mutant, in accordance with the experimental results. The role of the mutated residue has thus been checked as maintaining the structure of the ternary complex formed by the protein, the Mg2+ cation, and the inhibitor. These results might be useful to design compounds with more interesting anti-HIV-1 IN activity on the basis of its three-dimensional structure. PMID:17981909

  9. Mechanism of inhibition of HIV-1 integrase by G-tetrad-forming oligonucleotides in Vitro.

    PubMed

    Jing, N; Marchand, C; Liu, J; Mitra, R; Hogan, M E; Pommier, Y

    2000-07-14

    The G-tetrad-forming oligonucleotides and have been identified as potent inhibitors of human immunodeficiency virus type 1 integrase (HIV-1 IN) activity (Rando, R. F., Ojwang, J., Elbaggari, A., Reyes, G. R., Tinder, R., McGrath, M. S., and Hogan, M. E. (1995) J. Biol. Chem. 270, 1754-1760; Mazumder, A., Neamati, N., Ojwang, J. O., Sunder, S., Rando, R. F., and Pommier, Y. (1996) Biochemistry 35, 13762-13771; Jing, N., and Hogan, M. E. (1998) J. Biol. Chem. 273, 34992-34999). To understand the inhibition of HIV-1 IN activity by the G-quartet inhibitors, we have designed the oligonucleotides and, composed of three and four G-quartets with stem lengths of 19 and 24 A, respectively. The fact that increasing the G-quartet stem length from 15 to 24 A kept inhibition of HIV-1 IN activity unchanged suggests that the binding interaction occurs between a GTGT loop domain of the G-quartet inhibitors and a catalytic site of HIV-1 IN, referred to as a face-to-face interaction. Docking the NMR structure of (Jing and Hogan (1998)) into the x-ray structure of the core domain of HIV-1 IN, HIV-1 IN-(51-209) (Maignan, S., Guilloteau, J.-P. , Qing, Z.-L., Clement-Mella, C., and Mikol, V. (1998) J. Mol. Biol. 282, 359-368), was performed using the GRAMM program. The statistical distributions of hydrogen bonding between HIV-1 IN and were obtained from the analyses of 1000 random docking structures. The docking results show a high probability of interaction between the GTGT loop residues of the G-quartet inhibitors and the catalytic site of HIV-1 IN, in agreement with the experimental observation.

  10. Placental transfer of the HIV integrase inhibitor dolutegravir in an ex vivo human cotyledon perfusion model.

    PubMed

    Schalkwijk, Stein; Greupink, Rick; Colbers, Angela P; Wouterse, Alfons C; Verweij, Vivienne G M; van Drongelen, Joris; Teulen, Marga; van den Oetelaar, Daphne; Burger, David M; Russel, Frans G M

    2016-02-01

    Data on fetal exposure to antiretroviral agents during pregnancy are important to estimate their potential for prevention of mother-to-child transmission (PMTCT) and possible toxicity. For the recently developed HIV integrase inhibitor dolutegravir, clinical data on fetal disposition are not yet available. Dual perfusion of a single placental lobule (cotyledon) provides a useful ex vivo model to predict the in vivo maternal-to-fetal transfer of this drug. The aim of this study was to estimate the transfer of dolutegravir across the human term placenta, using a dual-perfusion cotyledon model. After cannulation of the cotyledons (n = 6), a fetal circulation of 6 mL/min and maternal circulation of 12 mL/min were initiated. The perfusion medium consisted of Krebs-Henseleit buffer (pH = 7.2-7.4) supplemented with 10.1 mM glucose, 30 g/L human serum albumin and 0.5 mL/L heparin 5000IE. Dolutegravir was administered to the maternal circulation (∼ 4.2 mg/L) and analysed by UPLC-MS/MS. After 3 h of perfusion, the mean ± SD fetal-to-maternal (FTM) concentration ratio of dolutegravir was 0.6 ± 0.2 and the mean ± SD concentrations in the maternal and fetal compartments were 2.3 ± 0.4 and 1.3 ± 0.3 mg/L, respectively. Dolutegravir crosses the blood-placental barrier with a mean FTM concentration ratio of 0.6. Compared with other antiretroviral agents, placental transfer of dolutegravir is moderate to high. These data suggest that dolutegravir holds clinical potential for pre-exposure prophylaxis and consequently PMTCT, but also risk of fetal toxicity. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  11. Impact of resistance mutations on inhibitor binding to HIV-1 integrase

    SciTech Connect

    Chen, Qi; Buolamwini, John K.; Smith, Jeremy C.; Li, Aixiu; Xu, Qin; Cheng, Xiaolin; Wei, Dongqing

    2013-11-08

    Here, HIV-1 integrase (IN) is essential for HIV-1 replication, catalyzing two key reaction steps termed 3' processing and strand transfer. Therefore, IN has become an important target for antiviral drug discovery. However, mutants have emerged, such as E92Q/N155H and G140S/Q148H, which confer resistance to raltegravir (RAL), the first IN strand transfer inhibitor (INSTI) approved by the FDA, and to the recently approved elvitegravir (EVG). To gain insights into the molecular mechanisms of ligand binding and drug resistance, we performed molecular dynamics (MD) simulations of homology models of the HIV-1 IN and four relevant mutants complexed with viral DNA and RAL. The results show that the structure and dynamics of the 140s loop, comprising residues 140 to 149, are strongly influenced by the IN mutations. In the simulation of the G140S/Q148H double mutant, we observe spontaneous dissociation of RAL from the active site, followed by an intrahelical swing-back of the 3' -OH group of nucleotide A17, consistent with the experimental observation that the G140S/Q148H mutant exhibits the highest resistance to RAL compared to other IN mutants. An important hydrogen bond between residues 145 and 148 is present in the wild-type IN but not in the G140S/Q148H mutant, accounting for the structural and dynamical differences of the 140s' loop and ultimately impairing RAL binding in the double mutant. End-point free energy calculations that broadly capture the experimentally known RAL binding profiles elucidate the contributions of the 140s' loop to RAL binding free energies and suggest possible approaches to overcoming drug resistance.

  12. Prevalence of Integrase Strand Transfer Inhibitors (INSTI) Resistance Mutations in Taiwan

    PubMed Central

    Chang, Sui-Yuan; Lin, Pi-Han; Cheng, Chien-Lin; Chen, Mao-Yuan; Sun, Hsin-Yun; Hsieh, Szu-Min; Sheng, Wang-Huei; Su, Yi-Ching; Su, Li-Hsin; Chang, Shu-Fang; Liu, Wen-Chun; Hung, Chien-Ching; Chang, Shan-Chwen

    2016-01-01

    Antiretroviral therapy containing an integrase strand transfer inhibitor (INSTI) plus two NRTIs has become the recommended treatment for antiretroviral-naive HIV-1-infected patients in the updated guidelines. We aimed to determine the prevalence of INSTI-related mutations in Taiwan. Genotypic resistance assays were performed on plasma from ARV-naïve patients (N = 948), ARV-experienced but INSTI-naive patients (N = 359), and raltegravir-experienced patients (N = 63) from 2006 to 2015. Major INSTI mutations were defined according to the IAS-USA list and other substitutions with a Stanford HIVdb score ≧ 10 to at least one INSTI were defined as minor mutations. Of 1307 HIV-1 samples from patients never exposed to INSTIs, the overall prevalence of major resistance mutations to INSTIs was 0.9% (n = 12), with an increase to 1.2% in 2013. Of these 12 sequences, 11 harboured Q148H/K/R, one Y143R, and none N155H. Of 30 sequences (47.6%) with INSTI-resistant mutations from raltegravir-experienced patients, 17 harboured Q148H/K/R, 8 N155H, and 6 Y143C/R. Other than these major mutations, the prevalence of minor mutations were 5.3% and 38.1%, respectively, in ARV-naive and raltegravir-experienced patients. The overall prevalence of INSTI mutations remains low in Taiwan. Surveillance of INSTI resistance is warranted due to circulation of polymorphisms contributing to INSTI resistance and expected increasing use of INSTIs. PMID:27779200

  13. Dolutegravir: clinical and laboratory safety in integrase inhibitor-naive patients.

    PubMed

    Curtis, L; Nichols, G; Stainsby, C; Lim, J; Aylott, A; Wynne, B; Clark, A; Bloch, M; Maechler, G; Martin-Carpenter, L; Raffi, F; Min, S

    2014-01-01

    The efficacy of dolutegravir (DTG) has been demonstrated in 5 randomized studies in integrase inhibitor (INI)-naive adult populations. To date, a detailed safety review of DTG has not been provided in the literature. To describe the safety and tolerability profile of DTG in adults based on 5 randomized, controlled trials and comparison with drugs in 3 major antiretroviral (ARV) classes. Safety data from phase IIb/III/IIIb trials in ART-naive and ART-experienced, INI-naive adults were integrated. In 4 ART-naive (SPRING-1, SPRING-2, SINGLE, FLAMINGO) and 1 ART-experienced, INI-naive study (SAILING), 1,579 individuals received a DTG-containing regimen. The proportion of individuals from DTG treatment arms who withdrew due to adverse events (AEs) was low (≤2%) compared to raltegravir (RAL; 2% SPRING-2, 4% SAILING), efavirenz (EFV)-containing comparator arm (10% SINGLE), and darunavir + ritonavir (DRV/r; 4% FLAMINGO). The most frequently observed AEs (diarrhea, nausea, headache), typically grade 1 or 2 in severity, did not lead to study discontinuation. Psychiatric and nervous system disorders with DTG were comparable to RAL- and DRV/r-containing regimens and favorable to EFV-containing regimens. In hepatitis B and/or C coinfected ART-naive individuals, the incidence of transaminase elevations was lower with DTG versus RAL and EFV comparators, but was similar to DRV/r. In SAILING, transaminase elevations were more commonly observed with DTG, particularly in the setting of inadequate hepatitis B therapy or immune reconstitution. On DTG treatment, mild creatinine elevations occurred and stabilized early. Few cases of hypersensitivity reaction and/or severe rash were seen. Rates of these events were comparable to or lower than with RAL-, EFV-, and DRV/r-containing regimens. The safety profile for DTG 50 mg once daily in INI-naive individuals was comparable to RAL- and DRV/r-containing regimens and generally favorable compared with EFV-containing regimens.

  14. Recent Advances in the Development of Small-Molecular Inhibitors Target HIV Integrase-LEDGF/p75 Interaction.

    PubMed

    Zhao, Yu; Luo, Zaigang

    2015-01-01

    Lens epithelium-derived growth factor (LEDGF/p75) plays an essential role in the HIV-1 replication. It acts by tethering integrase (IN) into the host cellular chromatin. Due to its significance of the IN-LEDGF/p75 interaction affords a novel therapeutic approach for the design of new classes of antiretroviral agents. To date, many small molecules have been found to be the inhibitors of INLEDGF/ p75 interaction. This review summarizes recent advances in the development of potential structure-based IN-LEDGF/p75 interaction inhibitors. The work will be helpful to shed light on the antiretroviral drug development pipeline in the next future.

  15. Dolutegravir Resistance Mutation R263K Cannot Coexist in Combination with Many Classical Integrase Inhibitor Resistance Substitutions

    PubMed Central

    Anstett, Kaitlin; Mesplede, Thibault; Oliveira, Maureen; Cutillas, Vincent

    2015-01-01

    The new integrase strand transfer inhibitor (INSTI) dolutegravir (DTG) displays limited cross-resistance with older drugs of this class and selects for the R263K substitution in treatment-experienced patients. We performed tissue culture selections with DTG, using viruses resistant to older INSTIs and infectivity and resistance assays, and showed that the presence of the E92Q or N155H substitution was compatible with the emergence of R263K, whereas the G140S Q148R, E92Q N155H, G140S, Y143R, and Q148R substitutions were not. PMID:25653436

  16. Metal-dependent inhibition of HIV-1 integrase by 5CITEP inhibitor: A theoretical QM/MM approach

    NASA Astrophysics Data System (ADS)

    do Nascimento, Josenaide P.; Araújo Silva, José Rogério; Lameira, Jerônimo; Alves, Cláudio N.

    2013-09-01

    HIV-1 integrase (IN) is a potential target for developing drugs against AIDS. In this letter, QM/MM approach was used to study the inhibition of IN by 5CITEP inhibitor in presence of divalent cations (Mg2+ or Mn2+). In addition, the main interactions occurring in 5CITEP-IN complex and the influence of divalent cations (Mg2+ or Mn2+) in enzymatic inhibition were investigated using B3LYP/6-31+G(d,p)/MM. The results suggest that the Asp64, Asp116 and four crystal water molecules plays a crucial role in cation (Mg2+ or Mn2+) coordination sphere.

  17. Optimization of Streptomyces bacteriophage φC31 integrase system to prevent post integrative gene silencing in pulmonary type II cells

    PubMed Central

    Aneja, Manish Kumar; Geiger, Johannes; Imker, Rabea; Üzgün, Senta; Kormann, Michael; Hasenpusch, Guenther; Maucksch, Christof

    2009-01-01

    φC31 integrase has emerged as a potent tool for achieving long-term gene expression in different tissues. The present study aimed at optimizing elements of φC31 integrase system for alveolar type II cells. Luciferase and β-galactosidase activities were measured at different time points post transfection. 5-Aza-2'deoxycytidine (AZA) and trichostatin A (TSA) were used to inhibit DNA methyltransferase and histone deacetylase complex (HDAC) respectively. In A549 cells, expression of the integrase using a CMV promoter resulted in highest integrase activity, whereas in MLE12 cells, both CAG and CMV promoter were equally effective. Effect of polyA site was observed only in A549 cells, where replacement of SV40 polyA by bovine growth hormone (BGH) polyA site resulted in an enhancement of integrase activity. Addition of a C-terminal SV40 nuclear localization signal (NLS) did not result in any significant increase in integrase activity. Long-term expression studies with AZA and TSA, provided evidence for post-integrative gene silencing. In MLE12 cells, both DNA methylases and HDACs played a significant role in silencing, whereas in A549 cells, it could be attributed majorly to HDAC activity. Donor plasmids comprising cellular promoters ubiquitin B (UBB), ubiquitin C (UCC) and elongation factor 1α (EF1α) in an improved backbone prevented post-integrative gene silencing. In contrast to A549 and MLE12 cells, no silencing could be observed in human bronchial epithelial cells, BEAS-2B. Donor plasmid coding for murine erythropoietin under the EF1α promoter when combined with φC31 integrase resulted in higher long-term erythropoietin expression and subsequently higher hematocrit levels in mice after intravenous delivery to the lungs. These results provide evidence for cell specific post integrative gene silencing with φC31 integrase and demonstrate the pivotal role of donor plasmid in long-term expression attained with this system. PMID:19745601

  18. The influence of particle size and structure on the sorption and oxidation behavior of birnessite: I. Adsorption of As(V) and oxidation of As(III)

    NASA Astrophysics Data System (ADS)

    Villalobos, Mario; Escobar-Quiroz, Ingrid N.; Salazar-Camacho, Carlos

    2014-01-01

    Sorption and oxidation reactions in the environment may affect substantially the mobility of redox-sensitive toxic trace elements and compounds. Investigating the environmental factors that influence these reactions is crucial in understanding and predicting the geochemical fate of these environmental species, as well as to design appropriate engineered remediation schemes. Arsenic is a widespread contaminant of concern, especially in its oxidized forms, and Mn oxide minerals are some of the major contributors to its oxidation. The goal of this work was to investigate the influence of particle size and structural differences of environmentally-relevant Mn(IV) birnessites on the adsorption of As(V) and on the oxidation of As(III). An acid birnessite of 39 m2/g and a δ-MnO2 of 114 m2/g were used. Both birnessites sorbed a maximum Pb(II) of 0.3 Pb/Mn, indicating a significantly larger layer cationic vacancy content for acid birnessite, and a density of reactive edge sites for both of 12 sites/nm2. As(V) forms a bidentate bridging complex on singly-coordinated surface sites at the birnessite particle edges regardless of loading, pH, birnessite type, and presence of pre-sorbed metals(II). Maximum As(V) adsorption, under repulsive electrostatic pH conditions did not yield adsorption congruency behavior between both birnessites at constant pH, presumably because the increase in internal vacancy content causes negative electrostatic repulsion towards external As(V) oxyanion binding. At pH 4.5 As(III) oxidation on birnessites was fast and quantitative at As/Mn ratios of 0.3-0.33, the reaction being largely driven by the proton concentration. At pH 6 δ-MnO2 oxidized As(III) faster and to a higher extent than acid birnessite, at equal masses; but the reverse at equal total surface areas. The oxidation driving force (independently from protons) was higher at pH 6 than at pH 4.5 because of Mn(II) product removal by sorption to interlayer vacancies, which overcomes reactive

  19. Thermodynamic and kinetic studies of As(V) removal from water by zirconium oxide-coated marine sand.

    PubMed

    Khan, Tabrez Alam; Chaudhry, Saif Ali; Ali, Imran

    2013-08-01

    Arsenic contamination of groundwater is a major threat to human beings globally. Among various methods available for arsenic removal, adsorption is fast, inexpensive, selective, accurate, reproducible and eco-friendly in nature. The present paper describes removal of arsenate from water on zirconium oxide-coated sand (novel adsorbent). In the present work, zirconium oxide-coated sand was prepared and characterised by infrared and X-ray diffraction techniques. Batch experiments were performed to optimise different adsorption parameters such as initial arsenate concentration (100-1,000 μg/L), dose (1-8 g/L), pH of the solution (2-14), contact time (15-150 min.), and temperature (20, 30, 35 and 40 °C). The experimental data were analysed by Langmuir, Freundlich, Temkin and Dubinin-Radushkevich isotherm models. Furthermore, thermodynamic and kinetic parameters were evaluated to know the mode of adsorption between ZrOCMS and As(V). The maximum removal of arsenic, 97 %, was achieved at initial arsenic concentration of 200 μg/L, after 75 min at dosage of 5.0 g/L, pH 7.0 and 27 ± 2 °C. For 600 μg/L concentration, the maximum Langmuir monolayer adsorption capacity was found to be 270 μg/g at 35 °C. Kinetic modelling data indicated that adsorption process followed pseudo-second-order kinetics. The mechanism is controlled by liquid film diffusion model. Thermodynamic parameter, ΔH°, was -57.782, while the values of ΔG° were -9.460, -12.183, -13.343 and -13.905 kJ/mol at 20, 30, 35 and 40 °C, respectively, suggesting exothermic and spontaneous nature of the process. The change in entropy, ΔS°= -0.23 kJ/mol indicated that the entropy decreased due to adsorption of arsenate ion onto the solid adsorbent. The results indicated that the reported zirconium oxide-coated marine sand (ZrOCMS) was good adsorbent with 97 % removal capacity at 200 μg/L concentration. It is interesting to note that the permissible limit of arsenic as per World Health Organization is 10

  20. Identification of redox-regulated components of arsenate (As(V)) tolerance through thiourea supplementation in rice.

    PubMed

    Srivastava, A K; Srivastava, S; Mishra, S; D'Souza, S F; Suprasanna, P

    2014-09-01

    Arsenic (As) is a ubiquitously present environmental carcinogen that enters into the human food chain through rice grains. In our previous research, the application of thiourea (TU; a non-physiological thiol based ROS scavenger) has been demonstrated to enhance salt and UV stress tolerance as well as the crop yield under field conditions. These effects were associated with the ability of TU to maintain plant redox homeostasis. Since As stress also induces redox imbalance, the present research was initiated to evaluate the efficiency of TU in regulating As tolerance/accumulation in rice. The supplementation of TU (75 μM) to As(V) (25 μM) improved the root growth and also reduced the As concentration by 56% in the aerial parts, which could be attributed to significant downregulation of the Lsi2 transporter responsible for the translocation of As from root to shoot. The fact that these effects were not due to direct interaction between As and TU was confirmed from complexation studies using HPLC-(ICP-MS)-(ESI-MS). Short-term kinetic studies of GSH levels and the GSH/GSSG ratio confirmed the establishment of differential redox states in As and As + TU treated seedlings. The real-time RT-PCR based comparative expression profiling under As with/without TU treatment identified Sultr1;1 and Sultr1;2 as major redox-regulated sulfate transporters. Their specific induction in shoots coupled with enhanced root-to-shoot sulfate translocation (analyzed using (35)S-sulfate as a radiotracer) was observed under TU supplementation. Furthermore, the level of thiolic metabolites (PC2 in roots and GSH and PC3 in shoots) and activities of sulfur metabolism enzymes (ATP sulfurylase and cysteine synthase in roots and 5'-adenylylsulfate reductase in shoot) were also increased with As + TU as compared to As treatment. Thus, this study utilizes the interaction between As and TU to identify the critical redox regulated components of As tolerance in rice.

  1. Study of As(III) and As(V) Oxoanion Adsorption onto Single and Mixed Ferrite and Hausmannite Nanomaterials.

    PubMed

    Garcia, Sandra; Sardar, Saima; Maldonado, Stephanie; Garcia, Velia; Tamez, C; Parsons, J G

    2014-11-01

    The removal of arsenic(III) and arsenic(V) from an aqueous solution through adsorption on to Fe3O4, MnFe2O4, 50% Mn substituted Fe3O4, 75% Mn substituted Fe3O4, and Mn3O4 nanomaterials was investigated. Characterization of the nanomaterials using XRD showed only pure phases for Mn3O4, MnFe2O4, and Fe3O4. The 50% and 75% substituted nanomaterials were found to be mixtures of Mn3O4 and Fe3O4. From batch studies the optimum binding pH of arsenic(III) and arsenic(V) to the nanomaterials was determined to be pH 3. The binding capacity for As(III) and As(VI) to the various nanomaterials was determined using Isotherm studies. The binding capacity of Fe3O4 was determined to be 17.1 mg/g for arsenic(III) and 7.0 mg/g for arsenic(V). The substitution of 25% Mn into the Fe3O4 lattice showed a slight increase in the binding capacity for As(III) and As(VI) to 23.8 mg/g and 7.9 mg/g, respectively. The 50% substituted showed the maximum binding capacity of 41.5 mg/g and 13.9 mg/g for arsenic(III) and arsenic(V). The 75% Mn substituted Fe3O4 capacities were 16.7 mg/g for arsenic(III) and 8.2 mg/g for arsenic(V). The binding capacity of the Mn3O4 was determined to be 13.5 mg/g for arsenic(III) and 7.5 mg/g for arsenic(V). In addition, interference studies on the effects of SO(2-)4, PO(3-)4, Cl(-), and NO(-)3 investigated. All the interferences had very minimal effects on the As(III) and As(V) binding never fell below 20% even in the presence of 1000 ppm interfering ions.

  2. Study of As(III) and As(V) Oxoanion Adsorption onto Single and Mixed Ferrite and Hausmannite Nanomaterials

    PubMed Central

    Garcia, Sandra; Sardar, Saima; Maldonado, Stephanie; Garcia, Velia; Tamez, C.; Parsons, J. G.

    2014-01-01

    The removal of arsenic(III) and arsenic(V) from an aqueous solution through adsorption on to Fe3O4, MnFe2O4, 50% Mn substituted Fe3O4, 75% Mn substituted Fe3O4, and Mn3O4 nanomaterials was investigated. Characterization of the nanomaterials using XRD showed only pure phases for Mn3O4, MnFe2O4, and Fe3O4. The 50% and 75% substituted nanomaterials were found to be mixtures of Mn3O4 and Fe3O4. From batch studies the optimum binding pH of arsenic(III) and arsenic(V) to the nanomaterials was determined to be pH 3. The binding capacity for As(III) and As(VI) to the various nanomaterials was determined using Isotherm studies. The binding capacity of Fe3O4 was determined to be 17.1 mg/g for arsenic(III) and 7.0 mg/g for arsenic(V). The substitution of 25% Mn into the Fe3O4 lattice showed a slight increase in the binding capacity for As(III) and As(VI) to 23.8 mg/g and 7.9 mg/g, respectively. The 50% substituted showed the maximum binding capacity of 41.5 mg/g and 13.9 mg/g for arsenic(III) and arsenic(V). The 75% Mn substituted Fe3O4 capacities were 16.7 mg/g for arsenic(III) and 8.2 mg/g for arsenic(V). The binding capacity of the Mn3O4 was determined to be 13.5 mg/g for arsenic(III) and 7.5 mg/g for arsenic(V). In addition, interference studies on the effects of SO2−4, PO3−4, Cl−, and NO−3 investigated. All the interferences had very minimal effects on the As(III) and As(V) binding never fell below 20% even in the presence of 1000 ppm interfering ions. PMID:25097269

  3. Superparamagnetic nanomaterial Fe3O4-TiO2 for the removal of As(V) and As(III) from aqueous solutions.

    PubMed

    Beduk, Fatma

    2016-01-01

    A magnetically separable nanomaterial Fe3O4-TiO2 was synthesized and characterized which was subsequently used for the removal of arsenic (V) from aqueous solutions. The surface morphology, magnetic properties, crystalline structure, thermal stability and Brunauer-Emmet-Teller surface area of the synthesized Fe3O4-TiO2 nanoparticles (NPs) are characterized by scanning electron microscope and high-resolution transmission electron microscope, vibrating sample magnetometry, X-ray diffractometer, thermogravimetric analysis and multi point function surface area analyzer. The saturation magnetization of Fe3O4-TiO2 NPs was determined to be 50.97 emu/g, which makes them superparamagnetic. The surface area of Fe3O4-TiO2 NPs was as much as 94.9 m(2)/g. The main factors affecting adsorption efficiency, such as solution pH, reaction time, initial As(V) concentration and adsorbent concentration are investigated. When the adsorption isotherms were analyzed by the Langmuir, Freundlich and Dubinin-Radushkevich models, equilibrium data were found to be well represented by Freundlich isotherm, and adsorption on Fe3O4-TiO2 NPs fitted well with pseudo-second-order kinetic model. The maximum adsorption capacity of As(V) on Fe3O4-TiO2 NPs, calculated by the Freundlich model was determined at 11.434 µg/g. 1.0 g/L of Fe3O4-TiO2 NPs was efficient for complete removal of 100 µg/L As(V) in 1 h. Fe3O4-TiO2 NPs was also effective for 93% removal of 100 µg/L As(III). Matrix effect was determined using As(V)-contaminated well water. Successfull results were obtained for purification of real well water containing 137.12 µg/L As(V). Results show that Fe3O4-TiO2 NPs are promising adsorbents with an advantage of magnetic separation.

  4. Temporospatial evolution and removal mechanisms of As(V) and Se(VI) in ZVI column with H2O2 as corrosion accelerator.

    PubMed

    Yang, Zhe; Shan, Chao; Zhang, Weiming; Jiang, Zhao; Guan, Xiaohong; Pan, Bingcai

    2016-12-01

    Enhanced removal of As(V) and Se(VI) by zero valent iron (ZVI) has been recently revealed by using H2O2 as the corrosion accelerator, however, the detailed performance of such enhanced removal in ZVI column as well as the underlying mechanism is still unclear. In this study, the temporospatial evolution of As(V) and Se(VI) along a self-designed ZVI/H2O2 column in down-flow mode was systematically investigated. The variations of concerned aqueous parameters (pH, ORP, H2O2, Fe(2+), As, and Se) were monitored at different positions along the column throughout the experiments. Results showed the corrosion degree of ZVI decreased with the depth of the column, as confirmed by SEM and XRD analyses of the solid samples from different layers. The retention of As and Se also decreased along the column, suggesting the uptake of As(V) and Se(VI) was highly dependent upon the ZVI corrosion evolution. In the initial stage, the influent H2O2 was mostly consumed by ZVI in the top layer. With the continuous corrosion of ZVI, the breakthrough of H2O2 would activate the ZVI at lower positions, resulting in the reactive zone continuously shifting downward along the column. The reduction of As(V) and Se(VI) to aqueous As(III) and Se(IV) was significantly inhibited at the positions in the presence of H2O2, whereas favorably enhanced in the presence of abundant Fe(2+). The retention of As(III) in the lower part of the column was observed while that of Se(IV) was negligible, as related to the different effects of pH on the adsorption of As(III) and Se(IV). In addition, the evolution of different oxidation states of As and Se retained in the column were identified by XPS, further demonstrating the comprehensive mechanisms of As(V)/Se(VI) removal involving reduction and adsorption in the ZVI/H2O2 column. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. LEDGIN-mediated Inhibition of Integrase-LEDGF/p75 Interaction Reduces Reactivation of Residual Latent HIV.

    PubMed

    Vranckx, Lenard S; Demeulemeester, Jonas; Saleh, Suha; Boll, Annegret; Vansant, Gerlinde; Schrijvers, Rik; Weydert, Caroline; Battivelli, Emilie; Verdin, Eric; Cereseto, Anna; Christ, Frauke; Gijsbers, Rik; Debyser, Zeger

    2016-06-01

    Persistence of latent, replication-competent Human Immunodeficiency Virus type 1 (HIV-1) provirus is the main impediment towards a cure for HIV/AIDS (Acquired Immune Deficiency Syndrome). Therefore, different therapeutic strategies to eliminate the viral reservoirs are currently being explored. We here propose a novel strategy to reduce the replicating HIV reservoir during primary HIV infection by means of drug-induced retargeting of HIV integration. A novel class of integration inhibitors, referred to as LEDGINs, inhibit the interaction between HIV integrase and the LEDGF/p75 host cofactor, the main determinant of lentiviral integration site selection. We show for the first time that LEDGF/p75 depletion hampers HIV-1 reactivation in cell culture. Next we demonstrate that LEDGINs relocate and retarget HIV integration resulting in a HIV reservoir that is refractory to reactivation by different latency-reversing agents. Taken together, these results support the potential of integrase inhibitors that modulate integration site targeting to reduce the likeliness of viral rebound.

  6. Discovery of a Potent HIV Integrase Inhibitor That Leads to a Prodrug with Significant anti-HIV Activity

    PubMed Central

    2011-01-01

    Worldwide research efforts in drug discovery involving HIV integrase have produced only one compound, raltegravir, that has been approved for clinical use in HIV/AIDS. As resistance, toxicity, and drug–drug interactions are recurring issues with all classes of anti-HIV drugs, the discovery of novel integrase inhibitors remains a significant scientific challenge. We have designed a lead HIV-1 strand transfer (ST) inhibitor (IC50 70 nM), strategically assembled on a pyridinone scaffold. A focused structure–activity investigation of this parent compound led to a significantly more potent ST inhibitor, 2 (IC50 6 ± 3 nM). Compound 2 exhibits good stability in pooled human liver microsomes. It also displays a notably favorable profile with respect to key human cytochrome P450 (CYP) isozymes and human UDP glucuronosyl transferases (UGTs). The prodrug of inhibitor 2, i.e., compound 10, was found to possess remarkable anti-HIV-1 activity in cell culture (EC50 9 ± 4 nM, CC50 135 ± 7 μM, therapeutic index = 15 000). PMID:22328963

  7. Neurological Response to cART vs. cART plus Integrase Inhibitor and CCR5 Antagonist Initiated during Acute HIV

    PubMed Central

    Valcour, Victor G.; Spudich, Serena S.; Sailasuta, Napapon; Phanuphak, Nittaya; Lerdlum, Sukalaya; Fletcher, James L. K.; Kroon, Eugene D. M. B.; Jagodzinski, Linda L.; Allen, Isabel E.; Adams, Collin L.; Prueksakaew, Peeriya; Slike, Bonnie M.; Hellmuth, Joanna M.; Kim, Jerome H.; Ananworanich, Jintanat

    2015-01-01

    Objective To compare central nervous system (CNS) outcomes in participants treated during acute HIV infection with standard combination antiretroviral therapy (cART) vs. cART plus integrase inhibitor and CCR5 antagonist (cART+). Design 24-week randomized open-label prospective evaluation. Method Participants were evaluated then randomized to initiate cART (efavirenz, tenofovir, and either emtricitabine or lamivudine) vs. cART+ (cART plus raltegravir and maraviroc) during acute HIV and re-evaluated at 4, 12 and 24 weeks. We examined plasma and CSF cytokines, HIV RNA levels, neurological and neuropsychological findings, and brain MRS across groups and compared to healthy controls. Results At baseline, 62 participants were in Fiebig stages I-V. Randomized groups were similar for mean age (27 vs. 25, p = 0.137), gender (each 94% male), plasma log10 HIV RNA (5.4 vs. 5.6, p = 0.382), CSF log10 HIV RNA (2.35 vs. 3.31, p = 0.561), and estimated duration of HIV (18 vs. 17 days, p = 0.546). Randomized arms did not differ at 24 weeks by any CNS outcome. Combining arms, all measures concurrent with antiretroviral treatment improved, for example, neuropsychological testing (mean NPZ-4 of -0.408 vs. 0.245, p<0.001) and inflammatory markers by MRS (e.g. mean frontal white matter (FWM) choline of 2.92 vs. 2.84, p = 0.045) at baseline and week 24, respectively. Plasma neopterin (p<0.001) and interferon gamma-induced protein 10 (IP-10) (p = 0.007) remained elevated in participants compared to controls but no statistically significant differences were seen in CSF cytokines compared to controls, despite individual variability among the HIV-infected group. Conclusions A 24-week course of cART+ improved CNS related outcomes, but was not associated with measurable differences compared to standard cART. PMID:26555069

  8. Polymorphic substitution E157Q in HIV-1 integrase increases R263K-mediated dolutegravir resistance and decreases DNA binding activity.

    PubMed

    Anstett, Kaitlin; Cutillas, Vincent; Fusco, Robert; Mesplède, Thibault; Wainberg, Mark A

    2016-08-01

    The E157Q substitution in HIV-1 integrase (IN) is a relatively common natural polymorphism associated with HIV resistance to IN strand transfer inhibitors (INSTIs). Although R263K is the most common resistance substitution for the INSTI dolutegravir, an INSTI treatment-experienced individual recently failed dolutegravir-based therapy, with E157Q being the only resistance-associated change reported. Given that different resistance pathways can sometimes synergize to confer high levels of resistance to antiretroviral drugs, we studied the effects of E157Q in association with R263K. Because Glu157 is thought to lie within the binding site of HIV IN DNA binding inhibitors such as FZ41, we also evaluated DNA binding activity and resistance to IN inhibitors in the presence of E157Q. Purified recombinant IN proteins were assessed in cell-free assays for their strand transfer and DNA binding activities. NL4.3 viral stocks harbouring IN mutations were generated and characterized in the presence and absence of IN inhibitors in tissue culture. E157Q alone had little if any effect on the biochemical activity of IN, and partially restored the activity of R263K-containing IN. The E157Q/R263K double viral mutant displayed infectiousness in culture equivalent to WT, while increasing resistance to dolutegravir by 10-fold compared with lower-level resistance associated with R263K alone. None of the mutations tested showed significant resistance to either raltegravir or FZ41. This study shows that E157Q may act as a compensatory mutation for R263K. Since E157Q is a natural polymorphism present in 1%-10% of HIV-positive individuals, it may be of particular importance for patients receiving INSTI therapy. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  9. Polymorphic substitution E157Q in HIV-1 integrase increases R263K-mediated dolutegravir resistance and decreases DNA binding activity

    PubMed Central

    Anstett, Kaitlin; Cutillas, Vincent; Fusco, Robert; Mesplède, Thibault; Wainberg, Mark A.

    2016-01-01

    Objectives The E157Q substitution in HIV-1 integrase (IN) is a relatively common natural polymorphism associated with HIV resistance to IN strand transfer inhibitors (INSTIs). Although R263K is the most common resistance substitution for the INSTI dolutegravir, an INSTI treatment-experienced individual recently failed dolutegravir-based therapy, with E157Q being the only resistance-associated change reported. Given that different resistance pathways can sometimes synergize to confer high levels of resistance to antiretroviral drugs, we studied the effects of E157Q in association with R263K. Because Glu157 is thought to lie within the binding site of HIV IN DNA binding inhibitors such as FZ41, we also evaluated DNA binding activity and resistance to IN inhibitors in the presence of E157Q. Methods Purified recombinant IN proteins were assessed in cell-free assays for their strand transfer and DNA binding activities. NL4.3 viral stocks harbouring IN mutations were generated and characterized in the presence and absence of IN inhibitors in tissue culture. Results E157Q alone had little if any effect on the biochemical activity of IN, and partially restored the activity of R263K-containing IN. The E157Q/R263K double viral mutant displayed infectiousness in culture equivalent to WT, while increasing resistance to dolutegravir by 10-fold compared with lower-level resistance associated with R263K alone. None of the mutations tested showed significant resistance to either raltegravir or FZ41. Conclusions This study shows that E157Q may act as a compensatory mutation for R263K. Since E157Q is a natural polymorphism present in 1%–10% of HIV-positive individuals, it may be of particular importance for patients receiving INSTI therapy. PMID:27084918

  10. Neurological Response to cART vs. cART plus Integrase Inhibitor and CCR5 Antagonist Initiated during Acute HIV.

    PubMed

    Valcour, Victor G; Spudich, Serena S; Sailasuta, Napapon; Phanuphak, Nittaya; Lerdlum, Sukalaya; Fletcher, James L K; Kroon, Eugene D M B; Jagodzinski, Linda L; Allen, Isabel E; Adams, Collin L; Prueksakaew, Peeriya; Slike, Bonnie M; Hellmuth, Joanna M; Kim, Jerome H; Ananworanich, Jintanat

    2015-01-01

    To compare central nervous system (CNS) outcomes in participants treated during acute HIV infection with standard combination antiretroviral therapy (cART) vs. cART plus integrase inhibitor and CCR5 antagonist (cART+). 24-week randomized open-label prospective evaluation. Participants were evaluated then randomized to initiate cART (efavirenz, tenofovir, and either emtricitabine or lamivudine) vs. cART+ (cART plus raltegravir and maraviroc) during acute HIV and re-evaluated at 4, 12 and 24 weeks. We examined plasma and CSF cytokines, HIV RNA levels, neurological and neuropsychological findings, and brain MRS across groups and compared to healthy controls. At baseline, 62 participants were in Fiebig stages I-V. Randomized groups were similar for mean age (27 vs. 25, p = 0.137), gender (each 94% male), plasma log10 HIV RNA (5.4 vs. 5.6, p = 0.382), CSF log10 HIV RNA (2.35 vs. 3.31, p = 0.561), and estimated duration of HIV (18 vs. 17 days, p = 0.546). Randomized arms did not differ at 24 weeks by any CNS outcome. Combining arms, all measures concurrent with antiretroviral treatment improved, for example, neuropsychological testing (mean NPZ-4 of -0.408 vs. 0.245, p<0.001) and inflammatory markers by MRS (e.g. mean frontal white matter (FWM) choline of 2.92 vs. 2.84, p = 0.045) at baseline and week 24, respectively. Plasma neopterin (p<0.001) and interferon gamma-induced protein 10 (IP-10) (p = 0.007) remained elevated in participants compared to controls but no statistically significant differences were seen in CSF cytokines compared to controls, despite individual variability among the HIV-infected group. A 24-week course of cART+ improved CNS related outcomes, but was not associated with measurable differences compared to standard cART.

  11. Corynebacterium glutamicum MTCC 2745 immobilized on granular activated carbon/MnFe2O4 composite: A novel biosorbent for removal of As(III) and As(V) ions.

    PubMed

    Podder, M S; Majumder, C B

    2016-11-05

    The optimization of biosorption/bioaccumulation process of both As(III) and As(V) has been investigated by using the biosorbent; biofilm of Corynebacterium glutamicum MTCC 2745 supported on granular activated carbon/MnFe2O4 composite (MGAC). The presence of functional groups on the cell wall surface of the biomass that may interact with the metal ions was proved by FT-IR. To determine the most appropriate correlation for the equilibrium curves employing the procedure of the non-linear regression for curve fitting analysis, isotherm studies were performed for As(III) and As(V) using 30 isotherm models. The pattern of biosorption/bioaccumulation fitted well with Vieth-Sladek isotherm model for As(III) and Brouers-Sotolongo and Fritz-Schlunder-V isotherm models for As(V). The maximum biosorption/bioaccumulation capacity estimated using Langmuir model were 2584.668mg/g for As(III) and 2651.675mg/g for As(V) at 30°C temperature and 220min contact time. The results showed that As(III) and As(V) removal was strongly pH-dependent with an optimum pH value of 7.0. D-R isotherm studies specified that ion exchange might play a prominent role.

  12. Corynebacterium glutamicum MTCC 2745 immobilized on granular activated carbon/MnFe2O4 composite: A novel biosorbent for removal of As(III) and As(V) ions

    NASA Astrophysics Data System (ADS)

    Podder, M. S.; Majumder, C. B.

    2016-11-01

    The optimization of biosorption/bioaccumulation process of both As(III) and As(V) has been investigated by using the biosorbent; biofilm of Corynebacterium glutamicum MTCC 2745 supported on granular activated carbon/MnFe2O4 composite (MGAC). The presence of functional groups on the cell wall surface of the biomass that may interact with the metal ions was proved by FT-IR. To determine the most appropriate correlation for the equilibrium curves employing the procedure of the non-linear regression for curve fitting analysis, isotherm studies were performed for As(III) and As(V) using 30 isotherm models. The pattern of biosorption/bioaccumulation fitted well with Vieth-Sladek isotherm model for As(III) and Brouers-Sotolongo and Fritz-Schlunder-V isotherm models for As(V). The maximum biosorption/bioaccumulation capacity estimated using Langmuir model were 2584.668 mg/g for As(III) and 2651.675 mg/g for As(V) at 30 °C temperature and 220 min contact time. The results showed that As(III) and As(V) removal was strongly pH-dependent with an optimum pH value of 7.0. D-R isotherm studies specified that ion exchange might play a prominent role.

  13. Anti-HIV-1 integrase compounds from Dioscorea bulbifera and molecular docking study.

    PubMed

    Chaniad, Prapaporn; Wattanapiromsakul, Chatchai; Pianwanit, Somsak; Tewtrakul, Supinya

    2016-01-01

    Dioscorea bulbifera L. (Dioscoreaceae) has been used in a traditional Thai longevity medicine preparation. Isolation of inhibitors from natural products is a potential source for continuous development of new HIV-1 integrase (IN) inhibitors. The objective of this study is to isolate the compounds and evaluate their anti-HIV-1 IN activity, as well as to predict the potential interactions of the compounds with an IN. The ethyl acetate and water fractions (1-100 μg/mL) of Dioscorea bulbifera bulbils were isolated and tested for their anti-HIV-1 IN activity using the multiplate integration assay (MIA). The interactions of the active compounds with IN were investigated using a molecular docking method. The ethyl acetate and water fractions of Dioscorea bulbifera bulbils afforded seven compounds. Among these, allantoin (1), 2,4,3',5'-tetrahydroxybibenzyl (2), and 5,7,4'-trihydroxy-2-styrylchromone (5) were isolated for the first time from this plant. Myricetin (4) exhibited the most potent activity with an IC50 value of 3.15 μM, followed by 2,4,6,7-tetrahydroxy-9,10-dihydrophenanthrene (3, IC50 value= 14.20 μM), quercetin-3-O-β-D-glucopyranoside (6, IC50 value = 19.39 μM) and quercetin-3-O-β-D-galactopyranoside (7, IC50 value = 21.80 μM). Potential interactions of the active compounds (3, 4, 6, and 7) with the IN active site were additionally investigated. Compound 4 showed the best binding affinity to IN and formed strong interactions with various amino acid residues. These compounds interacted with Asp64, Thr66, His67, Glu92, Asp116, Gln148, Glu152, Asn155, and Lys159, which are involved in both the 3'-processing and strand transfer reactions of IN. In particular, galloyl, catechol, and sugar moieties were successful inhibitors for HIV-1 IN.

  14. Lack of Pharmacokinetic Interaction between Rilpivirine and Integrase Inhibitors Dolutegravir and GSK1265744

    PubMed Central

    Ford, Susan L.; Gould, Elizabeth; Chen, Shuguang; Margolis, David; Spreen, William; Crauwels, Herta

    2013-01-01

    Dolutegravir (DTG) and GSK1265744 are HIV integrase inhibitors (INIs) in clinical development. The oral formulation of rilpivirine (RPV), a nonnucleoside reverse transcriptase inhibitor (NNRTI), has been approved for treatment-naive HIV infection. Long-acting depot injections of GSK1265744 and RPV are also being developed. This study evaluated the potential for drug interactions between RPV and these INIs. This phase 1, open-label, two-cohort, three-period, single-sequence crossover study evaluated oral coadministration of RPV with DTG or GSK1265744. Healthy subjects received DTG (50 mg every 24 h for 5 days) or GSK1265744 (30 mg every 24 h for 12 days) in period 1 followed by a washout, RPV (25 mg every 24 h for 11 or 12 days) in period 2, immediately followed by RPV (25 mg every 24 h) plus DTG (50 mg every 24 h) for 5 days or GSK1265744 (30 mg every 24 h) for 12 days in period 3. Steady-state pharmacokinetic (PK) parameters were estimated using noncompartmental analysis of data collected on the last day of each period. The combinations of RPV and DTG (n = 16) and of RPV and GSK1265744 (n = 11) were well tolerated; no grade 3 or 4 adverse events (AEs) or AE-related discontinuations were observed. The 90% confidence intervals for the area under the curve from time zero until the end of the dosage interval [AUC0–τ] and maximum concentration of drug in serum (Cmax) geometric mean ratios were within 0.8 to 1.25. Following administration of DTG + RPV, DTG and RPV Cτ increased by 22% and 21%, respectively. Following administration of GSK1265744 + RPV, RPV Cτ decreased 8%. DTG and GSK1265744 can be administered with RPV without dosage adjustment for either agent. These results support coadministration of RPV with DTG or GSK1265744 as either oral or long-acting depot injection regimens. (This study has been registered at ClinicalTrials.gov under registration no. NCT01467531.) PMID:23979733

  15. Retrovirus DNA termini bound by integrase communicate in trans for full-site integration in vitro.

    PubMed

    McCord, M; Chiu, R; Vora, A C; Grandgenett, D P

    1999-07-05

    Integration of linear retrovirus DNA involves the concerted insertion of the viral termini (full-site integration) into the host chromosome. We investigated the interactions that occur between long terminal repeat (LTR) termini bound by avian retrovirus integrase (IN) for full-site integration in vitro. Wild-type (wt) or mutant LTR donors that possess gain-of-function ("G") or loss-of-function ("L") for full-site integration activity were used. G LTR termini are characterized as having significantly higher strand transfer activity than the wt and the L LTR termini. L LTR mutations are classified as partially or extremely defective for strand transfer activity. The L mutations were further classified by their ability to either permit or block the assembly of G or wt LTR termini into nucleoprotein complexes capable of full-site strand transfer. We demonstrated that avian myeloblastosis virus IN bound to G LTR termini increased the incorporation of partially defective L LTR termini into nucleoprotein complexes that were capable of full-site integration. The observed full-site integration activity of these assembled nucleoprotein complexes appeared to be influenced by each individual IN-LTR complex in trans. In contrast, extremely defective L LTR termini exhibited the ability to effectively block the assembly of wt LTR termini into nucleoprotein complexes capable of full-site strand transfer. Data from nonspecific DNA competition experiments suggested that IN had an apparent higher affinity for G LTR donor termini than for partially defective L LTR donor termini as measured by full-site integration activity. However, assembled nucleoprotein complexes containing either two G or two L LTR donors were stable, having a similar half-life of approximately 2 h on ice. The results suggest that LTR termini bound by IN exhibit an allosteric effect to modulate full-site integration in vitro. Similar regulatory controls also appear to exist in vivo between the wt U3 and wt U5 LTR

  16. Integrase inhibitors in late pregnancy and rapid HIV viral load reduction.

    PubMed

    Rahangdale, Lisa; Cates, Jordan; Potter, JoNell; Badell, Martina L; Seidman, Dominika; Miller, Emilly S; Coleman, Jenell S; Lazenby, Gweneth B; Levison, Judy; Short, William R; Yawetz, Sigal; Ciaranello, Andrea; Livingston, Elizabeth; Duthely, Lunthita; Rimawi, Bassam H; Anderson, Jean R; Stringer, Elizabeth M

    2016-03-01

    Minimizing time to HIV viral suppression is critical in pregnancy. Integrase strand transfer inhibitors (INSTIs), like raltegravir, are known to rapidly suppress plasma HIV RNA in nonpregnant adults. There are limited data in pregnant women. We describe time to clinically relevant reduction in HIV RNA in pregnant women using INSTI-containing and non-INSTI-containing antiretroviral therapy (ART) options. We conducted a retrospective cohort study of pregnant HIV-infected women in the United States from 2009 through 2015. We included women who initiated ART, intensified their regimen, or switched to a new regimen due to detectable viremia (HIV RNA >40 copies/mL) at ≥20 weeks gestation. Among women with a baseline HIV RNA permitting 1-log reduction, we estimated time to 1-log RNA reduction using the Kaplan-Meier estimator comparing women starting/adding an INSTI in their regimen vs other ART. To compare groups with similar follow-up time, we also conducted a subgroup analysis limited to women with ≤14 days between baseline and follow-up RNA data. This study describes 101 HIV-infected pregnant women from 11 US clinics. In all, 75% (76/101) of women were not taking ART at baseline; 24 were taking non-INSTI containing ART, and 1 received zidovudine monotherapy. In all, 39% (39/101) of women started an INSTI-containing regimen or added an INSTI to their ART regimen. Among 90 women with a baseline HIV RNA permitting 1-log reduction, the median time to 1-log RNA reduction was 8 days (interquartile range [IQR], 7-14) in the INSTI group vs 35 days (IQR, 20-53) in the non-INSTI ART group (P < .01). In a subgroup of 39 women with first and last RNA measurements ≤14 days apart, median time to 1-log reduction was 7 days (IQR, 6-10) in the INSTI group vs 11 days (IQR, 10-14) in the non-INSTI group (P < .01). ART that includes INSTIs appears to induce more rapid viral suppression than other ART regimens in pregnancy. Inclusion of an INSTI may play a role in optimal reduction

  17. Combinations of reverse transcriptase, protease, and integrase inhibitors can be synergistic in vitro against drug-sensitive and RT inhibitor-resistant molecular clones of HIV-1.

    PubMed

    Beale, K K; Robinson, W E

    2000-06-01

    Combinations of anti-HIV agents including one or two reverse transcriptase inhibitors with a protease inhibitor are potent and effective. However, toxicities, costs and the emergence of drug-resistant organisms have compromised their long-term efficacy in people. A next, likely, target for anti-HIV therapy is HIV-1 integrase. Viral integration, catalyzed by integrase, is absolutely required for HIV replication. L-chicoric acid is a potent and selective inhibitor of HIV-1 integrase that also inhibits HIV-1 replication in cell culture. As a first step in understanding the potential role for integrase inhibitors in clinical medicine, the activities of L-chicoric acid alone and in combination with 2', 3'-dideoxycytidine, zidovudine, and a protease inhibitor, nelfinavir, were tested in vitro against molecular clones of HIV-1 resistant to reverse transcriptase inhibitors. L-chicoric acid was equally effective against a wild-type clone of HIV-1, HIV(NL4-3), or against HIV-1 resistant to either zidovudine or dideoxycytidine. L-chicoric acid was largely synergistic with zidovudine and synergistic with both dideoxycytidine and nelfinavir.

  18. Fragment-Based Design of Ligands Targeting a Novel Site on the Integrase Enzyme of Human Immunodeficiency Virus;#8197;1

    SciTech Connect

    Wielens, Jerome; Headey, Stephen J.; Deadman, John J.; Rhodes, David I.; Parker, Michael W.; Chalmers, David K.; Scanlon, Martin J.

    2011-08-17

    Fragment-based screening has been used to identify a novel ligand binding site on HIV-1 integrase. Crystal structures of fragments bound at this site (shown) have been used to design elaborated second-generation compounds that bind with higher affinity and good ligand efficiency.

  19. Evaluation and modelling of dissolved organic matter reactivity toward As(III) and As(V) – implication in environmental arsenic speciation.

    PubMed

    Lenoble, V; Dang, D H; Loustau Cazalet, M; Mounier, S; Pfeifer, H-R; Garnier, C

    2015-03-01

    Many studies have been carried out to identify dissolved organic matter-trace metals interactions, as organic matter (OM) was demonstrated to be a governing parameter of metals speciation. Concerning arsenic (As), such OM-As studies are scarce and concluded that, when As binding occurred, it was probably through cationic bridges or, in some cases, directly. Yet, analytical proofs remained complex to obtain. In this work, As binding with Suwanee River Humic Acid (SRHA), as an example of dissolved organic matter, was studied, considering both As(III) and As(V), at various pH and in absence/presence of Na and Ca. Dialysis, fluorescence measurements and PHREEQC modelling were performed to identify and characterize the mechanisms at work for the various performed experiments. It was observed that As(III) binding on SRHA occurred through direct SRHA-As(III) binding and that neither Na nor Ca presence modify this mechanism. As(V) appeared to be also bound by SRHA through direct interaction, but suffered from the competition of Na for the SRHA binding sites. Oppositely, in presence of Ca, the overall As(V)-SRHA binding was significantly enhanced, Ca acting as an efficient cationic bridge through the formation of an SRHA-Ca-As(V) ternary complex. All the obtained data were satisfactorily simulated using a unique set of binding parameters which can therefore be implemented in any speciation code to better address As behaviour in environmental conditions.

  20. Facile synthesis of yeast cross-linked Fe3O4 nanoadsorbents for efficient removal of aquatic environment contaminated with As(V).

    PubMed

    Rajesh Kumar, S; Jayavignesh, V; Selvakumar, R; Swaminathan, K; Ponpandian, N

    2016-12-15

    A facile solvothermal method was adopted to prepare monodispersed surface functionalized Fe3O4 nanoparticles via self assembly process. The pure yeast, diethylamine functionalized Fe3O4 nanoparticles (DMNPs) and yeast cross-linked Fe3O4 nanoparticles (YcMNPs) were used for the efficient removal of arsenate from aqueous solution. The crystal structure, morphology and magnetic properties of these nanoparticles were characterized by using X-ray diffraction, field emission scanning electron microscopy and vibrating sample magnetometer. The observed physico-chemical properties confirms the metal binding nature of prepared samples. The adsorption of As(V) on the functionalized magnetite nanoparticles was tested under different operating conditions like contact time, adsorbate dosage, adsorbate concentration and pH. The faster removal of As(V) was obtained using YcMNPs (99%) than DMNPs and pure yeast. The adsorption equilibrium data obeys Langmuir isotherm than Freundlich model and the kinetics data well depicts the pseudo-second-order model. The batch column experiment confirms the adequate desorption as well as reusability without significant loss of efficiency. The results reveal the technical feasibility of the prepared nanoparticles for their easy synthesis, recovery, cost effective, eco-friendly and a promising advanced adsorbent for environmental pollution. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Enhancing As(V) adsorption and passivation using biologically formed nano-sized FeS coatings on limestone: Implications for acid mine drainage treatment and neutralization.

    PubMed

    Liu, Jing; Zhou, Lei; Dong, Faqin; Hudson-Edwards, Karen A

    2017-02-01

    The iron-reducing bacterium Acidiphilium cryputum JF-5 and a sulfate reducing bacterium (SRB) collected and purified from the mine drainage of a copper mine in the northwest of Sichuan Province, China, were used to biologically synthesize nano-sized FeS-coated limestone to remove As(V) from solution. The adsorption efficiency of As(V) is improved from 6.64 μg/g with limestone alone to 187 μg/g with the FeS coated limestone in both batch and column experiments. The hydraulic conductivity of the columns are also improved by the presence of the nano-sized FeS coatings, but the solution neutralization performance of the limestone can be reduced by passivation by gypsum and Fe(III) precipitates. Calculations for FeS-coated limestone dissolution experiments show that the process can be described as nCa.sol = At(1/2) - nCa,gyp. The results suggest that FeS-coated limestone may be an effective medium for remediating As(V)-bearing solutions such as acid mine drainage in systems such as Permeable Reactive Barriers. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Removal of As(III) and As(V) using iron-rich sludge produced from coal mine drainage treatment plant.

    PubMed

    Yang, Jung-Seok; Kim, Young-Soo; Park, Sang-Min; Baek, Kitae

    2014-09-01

    To test the feasibility of the reuse of iron-rich sludge (IRS) produced from a coal mine drainage treatment plant for removing As(III) and As(V) from aqueous solutions, we investigated various parameters, such as contact time, pH, initial As concentration, and competing ions, based on the IRS characterization. The IRS consisted of goethite and calcite, and had large surface area and small particles. According to energy dispersive X-ray spectroscopy mapping results, As was mainly removed by adsorption onto iron oxides. The adsorption kinetic studies showed that nearly 70 % adsorption of As was achieved within 1 h, and the pseudo-second-order model well explained As sorption on the IRS. The adsorption isotherm results agreed with the Freundlich isotherm model, and the maximum adsorption capacities for As(III) and As(V) were 66.9 and 21.5 mg/g, respectively, at 293 K. In addition, the adsorption showed the endothermic character. At high pH or in the presence of phosphate, the adsorption of As was decreased. When the desorption experiment was conducted to reuse the IRS, 85 % As was desorbed with 1.0 N NaOH. In the column experiment, adsorbed As in real acid mine drainage was 43 % of the maximum adsorbed amount of As in the batch test. These results suggested that the IRS is an effective adsorbent for As and can be effectively applied for the removal of As in water and wastewater.

  3. Facile and economical synthesis of large hollow ferrites and their applications in adsorption for As(V) and Cr(VI).

    PubMed

    Dui, Jingna; Zhu, Gongyu; Zhou, Shaomin

    2013-10-23

    Unlike the previous ferrites (MFe2O4; M=Fe, Co, Zn, and Mn) solid nanospheres/nanoparticles, which were prepared by polluted solvothermal (glycol) approaches, here controllable monodisperse porous ferrites hollow nanospheres are promptly synthesized by a nontemplate hydrothermal method which has introduced an addition agent, polyacrylamide. The hollow nanospheres with different size can be prepared by varying the synthetic compositions. Scanning/transmission micros-graphs show the outside diameters of ferrite nanospheres are 180-380 nm and the shell thicknesses of that are only 20-45 nm, which could be adjusted by controlling CH3COONa concentration. X-ray diffraction (XRD) and X-ray photoelectron (XPS) spectroscopy, scanning electron (SEM) and transmission electron (TEM) microscopy, energy-dispersive spectrometer (EDS), the measurement of N2 adsorption-desorption isotherms and Brunauer-Emmett-Teller (BET) surface area, and superconducting quantum interference device (SQID) magnetometer were adopted to analyze their phase composition, morphology, porosity, and magnetic properties, respectively. The results of controlled experiments show that citrate and polyacrylamide are vital for the phase purities and morphology of ferrites. In particular, the as-obtained samples exhibit a large adsorption capacity for the toxic solution containing As(V) and Cr(VI) ions, and the calculated result of the maximum adsorption capacity is 340 mg/g based on Langmuir model, which shows excellent As(V) and Cr(VI) ions uptake capacity in contrast to other solid nanosphere materials.

  4. Identification of Phe187 as a crucial dimerization determinant facilitates crystallization of a monomeric retroviral integrase core domain.

    PubMed

    Galilee, Meytal; Alian, Akram

    2014-10-07

    Retroviral DNA integration into the host genome is mediated by nucleoprotein assemblies containing tetramers of viral integrase (IN). Whereas the fully active form of IN comprises a dimer of dimers, the molecular basis of IN multimerization has not been fully characterized. IN has consistently been crystallized in an analogous dimeric form in all crystallographic structures and experimental evidence as to the level of similarity between IN monomeric and dimeric conformations is missing because of the lack of IN monomeric structures. Here we identify Phe187 as a critical dimerization determinant of IN from feline immunodeficiency virus (FIV), a nonprimate lentivirus that causes AIDS in the natural host, and report, in addition to a canonical dimeric structure of the FIV IN core-domain, a monomeric structure revealing the preservation of the backbone structure between the two multimeric forms and suggest a role for Phe187 in "hinging" the flexible IN dimer. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. N-3 Hydroxylation of Pyrimidine-2,4-diones Yields Dual Inhibitors of HIV Reverse Transcriptase and Integrase

    PubMed Central

    2010-01-01

    A new molecular scaffold featuring an N-hydroxyimide functionality and capable of inhibiting both reverse transcriptase (RT) and integrase (IN) of human immunodeficiency virus (HIV) was rationally designed based on 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) non-nucleoside RT inhibitors (NNRTIs). The design involves a minimal 3-N hydroxylation of the pyrimidine ring of HEPT compound to yield a chelating triad which, along with the existing benzyl group, appeared to satisfy major structural requirements for IN binding. In the mean time, this chemical modification did not severely compromise the compound’s ability to inhibit RT. A preliminary structure−activity relationship (SAR) study reveals that this N-3 OH is essential for IN inhibition and that the benzyl group on N-1 side chain is more important for IN binding than the one on C-6. PMID:21499541

  6. The dynamics of interconverting D- and E-forms of the HIV-1 integrase N-terminal domain.

    PubMed

    Sangeetha, Balasubramanian; Muthukumaran, Rajagopalan; Amutha, Ramaswamy

    2014-11-01

    The N-terminal domain (NTD) of HIV-1 integrase adopts two inter-converting forms (D- and E-) due to their specific coordination of a Zn(2+) ion by an HHCC motif. Mutational studies on NTD have suggested the importance of conformational transition in regulating the functions of tetramers and dimers of HIV-1 integrase. This study explores the stability and dynamics of native NTD forms and the conformational transition between D- and E-forms using molecular dynamics simulations elucidating their role in regulation of viral and host DNA integration. Simulation of native forms of NTD revealed stable dynamics. Transition studies between D- and E-forms using conventional molecular dynamics simulations for 50 ns partially revealed conformational change towards the target during D- to -E simulation (the extension of α1-helix), which failed in the E- to -D simulation. This could be attributed to the existence of the D-form (-1,945.907 kCal/mol) in higher energy than the E-form (-2,002.383 kCal/mol). The conformational transition pathway between these two states was explored using targeted molecular dynamics simulations. Analysis of the targeted molecular dynamics trajectories revealed conformations closer to the experimentally-reported intermediate form of an NTD during the transition phase. The role of Met22 in stabilizing the E-form was studied by simulating the E-form with Met22Ala mutation, revealing a highly dynamic α1-helix as compared to the native form. The present study reveals the significant role of the Zn(2+) ion-coordinated HHCC motif and its interaction with Met22 as the basis for understanding the biological implications of D- and E-forms of the NTD in regulating integration reaction.

  7. Molecular Dynamics Approaches Estimate the Binding Energy of HIV-1 Integrase Inhibitors and Correlate with In Vitro Activity

    PubMed Central

    Johnson, Barry C.; Métifiot, Mathieu; Pommier, Yves

    2012-01-01

    The design of novel integrase (IN) inhibitors has been aided by recent crystal structures revealing the binding mode of these compounds with a full-length prototype foamy virus (PFV) IN and synthetic viral DNA ends. Earlier docking studies relied on incomplete structures and did not include the contribution of the viral DNA to inhibitor binding. Using the structure of PFV IN as the starting point, we generated a model of the corresponding HIV-1 complex and developed a molecular dynamics (MD)-based approach that correlates with the in vitro activities of novel compounds. Four well-characterized compounds (raltegravir, elvitegravir, MK-0536, and dolutegravir) were used as a training set, and the data for their in vitro activity against the Y143R, N155H, and G140S/Q148H mutants were used in addition to the wild-type (WT) IN data. Three additional compounds were docked into the IN-DNA complex model and subjected to MD simulations. All three gave interaction potentials within 1 standard deviation of values estimated from the training set, and the most active compound was identified. Additional MD analysis of the raltegravir- and dolutegravir-bound complexes gave internal and interaction energy values that closely match the experimental binding energy of a compound related to raltegravir that has similar activity. These approaches can be used to gain a deeper understanding of the interactions of the inhibitors with the HIV-1 intasome and to identify promising scaffolds for novel integrase inhibitors, in particular, compounds that retain activity against a range of drug-resistant mutants, making it possible to streamline synthesis and testing. PMID:22037850

  8. Lack of resistance to integrase inhibitors among antiretroviral-naive subjects with primary HIV-1 infection, 2007–2013

    PubMed Central

    Stekler, Joanne D; McKernan, Jennifer; Milne, Ross; Tapia, Kenneth A; Mykhalchenko, Kateryna; Holte, Sarah; Maenza, Janine; Stevens, Claire E; Buskin, Susan E; Mullins, James I; Frenkel, Lisa M; Collier, Ann C

    2014-01-01

    Background U.S. guidelines recommend genotyping for persons newly diagnosed with HIV infection to identify transmitted drug resistance mutations associated with decreased susceptibility to NRTIs, NNRTIs, and PIs. To date, testing for integrase strand transfer inhibitor (INSTI) mutations has not been routinely recommended. We aimed to evaluate the prevalence of transmitted INSTI mutations among persons with primary HIV-1 infection in Seattle, WA. Methods Persons with primary HIV-1 infection have enrolled in an observational cohort at the University of Washington Primary Infection Clinic since 1992. We performed a retrospective analysis of plasma specimens collected prospectively from the 82 antiretroviral-naive subjects who were enrolled from 2007–13, after FDA-approval of the first INSTI. Resistance testing was performed by consensus sequencing. Results Specimens for analysis had been obtained a median of 24 (lQR 18–41, range 8–108) days after the estimated date of HIV-1 infection. All subjects were infected with HIV-1 subtype B except for one subject infected with subtype C. Consensus sequencing identified no subjects with major INSTI mutations (T66I, E92Q, G140S, Y143C/H/R, S147G, Q148H/K/R, N155H). Using exact binomial confidence intervals, the upper bound of the 95% CI was 4.4%. Conclusions Although our sample size was small, this study does not support the need at this time to evaluate integrase mutations as part of routine consensus sequencing among persons newly diagnosed with HIV-1 infection. However, it is likely that the prevalence of transmitted INSTI mutations may increase with the recent commercial introduction of additional INSTIs and presumably greater INST1 use among persons living with HIV-1. PMID:24831260

  9. Pseudo attP sites in favor of transgene integration and expression in cultured porcine cells identified by streptomyces phage phiC31 integrase

    PubMed Central

    2013-01-01

    Phage PhiC31 integrase integrates attB-containing plasmid into pseudo attP site in eukaryotic genomes in a unidirectional site-specific manner and maintains robust transgene expression. Few studies, however, explore its potential in livestock. This study aims to discover the molecular basis of PhiC31 integrase-mediated site-specific recombination in pig cells. We show that PhiC31 integrase can mediate site-specific transgene integration into the genome of pig kidney PK15 cells. Intramolecular recombination in pig PK15 cell line occurred at maximum frequency of 82% with transiently transfected attB- and attP-containing plasmids. An optimal molar ratio of pCMV-Int to pEGFP-N1-attB at 5:1 was observed for maximum number of cell clones under drug selection. Four candidate pseudo attP sites were identified by TAIL-PCR from those cell clones with single-copy transgene integration. Two of them gave rise to higher integration frequency occurred at 33%. 5′ and 3′ junction PCR showed that transgene integration mediated by PhiC31 integrase was mono-allelic. Micro- deletion and insertion were observed by sequencing the integration border, indicating that double strand break was induced by the recombination. We then constructed rescue reporter plasmids by ABI-REC cloning of the four pseudo attP sites into pBCPB + plasmid. Transfection of these rescue plasmids and pCMV-Int resulted in expected intramolecular recombination between attB and pseudo attP sites. This proved that the endogenous pseudo attP sites were functional substrates for PhiC31 integrase-mediated site-specific recombination. Two pseudo attP sites maintained robust extracellular and intracellular EGFP expression. Alamar blue assay showed that transgene integration into these specific sites had little effect on cell proliferation. This is the first report to document the potential use of PhiC31 integrase to mediate site-specific recombination in pig cells. Our work established an ideal model to study the

  10. Is the effect of silicon on rice uptake of arsenate (AsV) related to internal silicon concentrations, iron plaque and phosphate nutrition?

    PubMed

    Guo, W; Zhu, Y-G; Liu, W-J; Liang, Y-C; Geng, C-N; Wang, S-G

    2007-07-01

    Solution culture experiments were conducted to investigate the effects of silicon (Si) on arsenate (As(V)) uptake by rice. The addition of Si to the pretreatment or uptake solution significantly decreased shoot and root As concentrations (P<0.001 and P<0.05). The presence of Si in the pretreatment or uptake solution also significantly decreased shoot P concentrations (P<0.001). The data demonstrated that both internal and external Si inhibited the uptake of As and P. Results of As uptake kinetics showed that the mechanism of the effect of Si on arsenate uptake is not caused by direct competition for active sites of transporters with As. The effect of Si on As uptake was not entirely mediated through the effect of Si on P uptake. Although the addition of Si to pretreatment solutions still significantly decreased shoot and root As concentrations, the extent of reduction became smaller when rice roots were coated with iron plaque.

  11. Synthesis and Characterization of Iron-impregnated Pre-oxidized Activated Carbon Prepared by Microwave Radiation for As(V) Removal from Water

    NASA Astrophysics Data System (ADS)

    Yurum, Yuda; Yurum, Alp; Ozlem Kocabas, Zuleyha; Semiat, Raphael

    2013-04-01

    One of the most efficient ways to treat water is probably by adsorption and catalytic oxidation. Surely, for such a process to be economical, the catalyst and the adsorber should have a high catalytic activity and adsorption capacity, and be inexpensive. One of these materials is iron oxide, which is studied and used in areas like catalysis and environmental applications. It is known that synthesizing iron oxides in nano size enhances the catalytic activity. Pre-oxidized activated carbons impregnated with iron-based nanoparticles are prepared in a single step under hydrothermal conditions with microwave radiation. The hydrothermal treatment provides an important advantage by forming fine particles that can easily impregnate deep in to the porous support by the help of water. Their efficiency for the removal of As(V) from water was compared with the pure pre-oxidized activated carbon and iron oxide nanoparticles impregnated without microwave radiation. The synthesized nanomaterials with different iron oxide loadings were characterized by x-ray diffraction (XRD), scanning electron microscopy (SEM), and Brunauer-Emmett-Teller (BET) surface area analyzer. Iron loadings were calculated using flame atomic absorbance. Microwave radiation provided much faster iron impregnation on the active carbon surface. At the first stage of microwave radiation iron oxide impregnation is low but after 6 minutes, iron oxide nanoparticles of 100 nm size started to cover the surface homogeneously. Further treatment with microwave increased the size of particles and the amount of surface coverage. Additionally, with microwave hydrothermal treatment, relatively higher iron oxide loadings were achieved within 10 minutes. From the XRD characterization it was seen that at the first stage of radiation, iron deposited in the form of β-FeOOH, but after the first stage the structure became Fe2O3. While radiation increased the surface area of the material during the first stages, at the last stage

  12. Size effects of nanocrystalline TiO2 on As(V) and As(III) adsorption and As(III) photooxidation.

    PubMed

    Xu, Zhonghou; Meng, Xiaoguang

    2009-09-15

    The physicochemical properties of TiO(2) particles in the diameter range between 6.6 and 30.1 nm and the effect of the crystalline size on arsenic adsorption and photocatalytical oxidation were investigated. TiO(2) nanoparticles of different sizes were single-phase anatase. The adsorption capacity of the TiO(2) for As(III) and As(V) increased linearly with the N(2) Brunauer-Emmett-Teller surface area (S(BET)) of the particles. There was not much difference in the rate of As(III) photooxidation when the diameter of the TiO(2) nanoparticles was between 6.6 and 14.8 nm. However, the As(III) photooxidation rate clearly decreased when the particle size increased to 30.1 nm. Arsenite photooxidation data could be fitted with a first-order kinetics equation.

  13. Ecotoxicity assessment of ionic As(III), As(V), In(III) and Ga(III) species potentially released from novel III-V semiconductor materials.

    PubMed

    Zeng, Chao; Gonzalez-Alvarez, Adrian; Orenstein, Emily; Field, Jim A; Shadman, Farhang; Sierra-Alvarez, Reyes

    2017-06-01

    III-V materials such as indium arsenide (InAs) and gallium arsenide (GaAs) are increasingly used in electronic and photovoltaic devices. The extensive application of these materials may lead to release of III-V ionic species during semiconductor manufacturing or disposal of decommissioned devices into the environment. Although arsenic is recognized as an important contaminant due to its high toxicity, there is a lack of information about the toxic effects of indium and gallium ions. In this study, acute toxicity of As(III), As(V), In(III) and Ga(III) species was evaluated using two microbial assays testing for methanogenic activity and O2 uptake, as well as two bioassays targeting aquatic organisms, including the marine bacterium Aliivibrio fischeri (bioluminescence inhibition) and the crustacean Daphnia magna (mortality). The most noteworthy finding was that the toxicity is mostly impacted by the element tested. Secondarily, the toxicity of these species also depended on the bioassay target. In(III) and Ga(III) were not or only mildly toxic in the experiments. D. magna was the most sensitive organism for In(III) and Ga(III) with 50% lethal concentrations of 0.5 and 3.4mM, respectively. On the other hand, As(III) and As(V) caused clear inhibitory effects, particularly in the methanogenic toxicity bioassay. The 50% inhibitory concentrations of both arsenic species towards methanogens were about 0.02mM, which is lower than the regulated maximum allowable daily effluent discharge concentration (2.09mg/L or 0.03mM) for facilities manufacturing electronic components in the US. Overall, the results indicate that the ecotoxicity of In(III) and Ga(III) is much lower than that of the As species tested. This finding is important in filling the knowledge gap regarding the ecotoxicology of In and Ga. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Spectroscopic characterization and solubility investigation on the effects of As(V) on mineral structure tooeleite (Fe6(AsO3)4SO4(OH)4·H2O)

    NASA Astrophysics Data System (ADS)

    Liu, Jing; Deng, Shiming; Zhao, Fenghua; Cheng, Hongfei; Frost, Ray L.

    2015-01-01

    Tooeleite is an unique ferric arsenite sulfate mineral, which has the potential significance of directly fixing As(III) as mineral trap. The tooeleite and various precipitates were hydrothermally synthesized under the different of initial As(III)/As(V) molar ratios and characterized by XRD, FTIR, XPS and SEM. The crystallinity of tooeleite decreases with the amount of As(V). The precipitate is free of any crystalline tooeleite at the level of that XRD could detect when the ratio of As(III)/As(V) of 7:3 and more. The characteristic bands of tooeleite are observed in 772, 340, 696 and 304 cm-1, which are assigned to the ν1, ν2, ν3 and ν4 vibrations of AsO33-. These intensities of bands gradually decreases with the presence of As(V) and its increasing. An obviously wide band is observed in 830 cm-1, which is the ν1 vibration of AsO4. The result of XPS reveals that the binding energies of As3d increase from 44.0 eV to 45.5 eV, which indicates that the amount of As(V) in the precipitates increases. The concentrations of arsenic released of these precipitates are 350-650 mg/L. The stability of tooeleite decreases by comparison when the presence of coexisting As(V) ions.

  15. The effect of adaptive servo ventilation (ASV) on objective and subjective outcomes in Cheyne-Stokes respiration (CSR) with central sleep apnea (CSA) in heart failure (HF): A systematic review.

    PubMed

    Yang, Hyunju; Sawyer, Amy M

    2016-01-01

    To summarize the current evidence for adaptive servo ventilation (ASV) in Cheyne-Stokes respiration (CSR) with central sleep apnea (CSA) in heart failure (HF) and advance a research agenda and clinical considerations for ASV-treated CSR-CSA in HF. CSR-CSA in HF is associated with higher overall mortality, worse outcomes and lower quality of life (QOL) than HF without CSR-CSA. Five databases were searched using key words (n = 234). Randomized controlled trials assessed objective sleep quality, cardiac, and self-reported outcomes in adults (≥18 years) with HF (n = 10). ASV has a beneficial effect on the reduction of central sleep apnea in adult patients with CSR-CSA in HF, but it is not be superior to CPAP, bilevel PPV, or supplemental oxygen in terms of sleep quality defined by polysomnography, cardiovascular outcomes, subjective daytime sleepiness, and quality of life. ASV is not recommended for CSR-CSA in HF. It is important to continue to refer HF patients for sleep evaluation to clearly discern OSA from CSR-CSA. Symptom management research, inclusive of objective and subjective outcomes, in CSR-CSA in HF adults is needed.

  16. Creating transgenic Drosophila by microinjecting the site-specific phiC31 integrase mRNA and a transgene-containing donor plasmid.

    PubMed

    Fish, Matthew P; Groth, Amy C; Calos, Michele P; Nusse, Roel

    2007-01-01

    We describe a microinjection-based phiC31 integrase mRNA-mediated method for creating transgenic Drosophila strains. This approach is more efficient than traditional methods and ensures that the transgene is targeted to a precise genomic position. The method involves targeting integration of an exogenous plasmid (containing the transgene and sequences to facilitate integration) to a preplaced recipient site in the Drosophila genome. The plasmid is coinjected into embryos with mRNA encoding the phiC31 integrase, the enzyme that catalyzes the integration reaction. Using the protocol described here, transgenic lines can be established from, on average, 46% of fertile adults obtained after injection, and all integrations should be targeted to the chosen genomic insertion site. The whole procedure, from injection to established transgenic stocks, can be completed in three generations (approximately 1 month) and can be adapted for other types of transgenesis and mRNA injections in Drosophila.

  17. Study of Genotypic and Phenotypic HIV-1 Dynamics of Integrase Mutations During Raltegravir Treatment: A Refined Analysis by Ultra-Deep 454 Pyrosequencing

    PubMed Central

    Armenia, Daniele; Vandenbroucke, Ina; Fabeni, Lavinia; Van Marck, Herwig; Cento, Valeria; D’Arrigo, Roberta; Van Wesenbeeck, Liesbeth; Scopelliti, Fernanda; Micheli, Valeria; Bruzzone, Bianca; Lo Caputo, Sergio; Aerssens, Jeroen; Rizzardini, Giuliano; Tozzi, Valerio; Narciso, Pasquale; Antinori, Andrea; Stuyver, Lieven; Perno, Carlo Federico

    2012-01-01

    Background. The dynamics of raltegravir-resistant variants and their impact on virologic response in 23 HIV-1–infected patients, who started a salvage raltegravir-containing regimen, were investigated. Methods. Integrase population sequencing and Ultra-Deep-454 Pyrosequencing (UDPS) were performed on plasma samples at baseline and at raltegravir failure. All integrase mutations detected at a frequency ≥1% were considered to be reliable for the UDPS analyses. Phylogenetic and phenotypic resistance analyses were also performed. Results. At baseline, primary resistance mutations were not detected by both population and UDPS genotypic assays; few secondary mutations (T97A-V151I-G163R) were rarely detected and did not show any statistically association either with virologic response at 24-weeks or with the development of resistant variants at failure. At UDPS, not all resistant variants appearing early during treatment evolved as major populations during failure; only specific resistance pathways (Y143R-Q148H/R-N155H) associated with an increased rate of fitness and phenotypic resistance were selected. Conclusions. Resistance to raltegravir in integrase strand transfer inhibitor–naive patients remains today a rare event, which might be changed by future extensive use of such drugs. In our study, pathways of resistance at failure were not predicted by baseline mutations, suggesting that evolution plus stochastic selection plays a major role in the appearance of integrase-resistance mutations, whereas fitness and resistance are dominant factors acting for the late selection of resistant quasispecies. PMID:22238474

  18. Synthesis and evaluation of substituted 4-(N-benzylamino)cinnamate esters as potential anti-cancer agents and HIV-1 integrase inhibitors.

    PubMed

    Faridoon; Edkins, Adrienne L; Isaacs, Michelle; Mnkandhla, Dumisani; Hoppe, Heinrich C; Kaye, Perry T

    2016-08-01

    Encouraging selectivity and low micromolar activity against HeLa cervical carcinoma (IC50⩾3.0μM) and the aggressive MDA-MB-231 triple negative breast carcinoma (IC50⩾9.6μM) cell lines has been exhibited by a number of readily accessible 4-(N-benzylamino)cinnamate esters. The potential of the ligands as HIV-1 integrase inhibitors has also been examined. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Genetic analysis of human immunodeficiency virus type 1 integrase and the U3 att site: unusual phenotype of mutants in the zinc finger-like domain.

    PubMed Central

    Masuda, T; Planelles, V; Krogstad, P; Chen, I S

    1995-01-01

    Retroviral integration is the step which leads to establishment of the provirus, cis- and trans-acting regions of the human immunodeficiency type 1 (HIV-1) retrovirus genome, including the attachment site (att) at the ends of the unintegrated viral DNA and the conserved domains within the integrase (IN) protein, have been identified as being important for integration. We investigated the role of each of these regions in the context of an infectious HIV-1 molecular clone through point mutagenesis of the att site and the zinc finger-like and catalytic domains of IN. The effect of each mutation on integration activity was examined by using a single-step infection system with envelope-pseudotype virus. The relative integration efficiency was estimated by monitoring the levels of viral DNA over time in the infected cells. The integration activities of catalytic domain point mutants and att site deletion mutants were estimated to be 0.5 and 5% of wild-type activity, respectively. However, in contrast with previous in vitro cell-free integration studies, alteration of the highly conserved CA dinucleotide resulted in a mutant which still retained 40% of wild-type integration activity. The relative levels of expression of each mutant, as measured by a luciferase reporter gene, correlated with levels of integration. This observation is consistent with those of previous studies indicating that integration is an obligatory step for retroviral gene expression. Interestingly, we found that three different HIV-1 constructs bearing point mutations in the zinc finger-like domain synthesized much lower levels of viral DNA after infection, suggesting impairment of these mutants before or at the initiation of reverse transcription. Western blot (immunoblot) analysis demonstrated wild-type levels of reverse transcriptase within the mutant virions. In vitro endogenous reverse transcription assays indicated that all three mutants in the zinc finger-like domain had wild-type levels of

  20. Critical Contribution of Tyr15 in the HIV-1 Integrase (IN) in Facilitating IN Assembly and Nonenzymatic Function through the IN Precursor Form with Reverse Transcriptase.

    PubMed

    Takahata, Tatsuro; Takeda, Eri; Tobiume, Minoru; Tokunaga, Kenzo; Yokoyama, Masaru; Huang, Yu-Lun; Hasegawa, Atsuhiko; Shioda, Tatsuo; Sato, Hironori; Kannagi, Mari; Masuda, Takao

    2017-01-01

    Nonenzymatic roles for HIV-1 integrase (IN) at steps prior to the enzymatic integration step have been reported. To obtain structural and functional insights into the nonenzymatic roles of IN, we performed genetic analyses of HIV-1 IN, focusing on a highly conserved Tyr15 in the N-terminal domain (NTD), which has previously been shown to regulate an equilibrium state between two NTD dimer conformations. Replacement of Tyr15 with alanine, histidine, or tryptophan prevented HIV-1 infection and caused severe impairment of reverse transcription without apparent defects in reverse transcriptase (RT) or in capsid disassembly kinetics after entry into cells. Cross-link analyses of recombinant IN proteins demonstrated that lethal mutations of Tyr15 severely impaired IN structure for assembly. Notably, replacement of Tyr15 with phenylalanine was tolerated for all IN functions, demonstrating that a benzene ring of the aromatic side chain is a key moiety for IN assembly and functions. Additional mutagenic analyses based on previously proposed tetramer models for IN assembly suggested a key role of Tyr15 in facilitating the hydrophobic interaction among IN subunits, together with other proximal residues within the subunit interface. A rescue experiment with a mutated HIV-1 with RT and IN deleted (ΔRT ΔIN) and IN and RT supplied in trans revealed that the nonenzymatic IN function might be exerted through the IN precursor conjugated with RT (RT-IN). Importantly, the lethal mutations of Tyr15 significantly reduced the RT-IN function and assembly. Taken together, Tyr15 seems to play a key role in facilitating the proper assembly of IN and RT on viral RNA through the RT-IN precursor form. Inhibitors of the IN enzymatic strand transfer function (INSTI) have been applied in combination antiretroviral therapies to treat HIV-1-infected patients. Recently, allosteric IN inhibitors (ALLINIs) that interact with HIV-1 IN residues, the locations of which are distinct from the catalytic

  1. Enzymology of a carbonyl reduction clearance pathway for the HIV integrase inhibitor, S-1360: role of human liver cytosolic aldo-keto reductases.

    PubMed

    Rosemond, M Jane Cox; St John-Williams, Lisa; Yamaguchi, Toshiro; Fujishita, Toshio; Walsh, John S

    2004-03-15

    S-1360, a 1,3-diketone derivative, was the first HIV integrase inhibitor to enter human trials. Clinical data suggested involvement of non-cytochrome P450 clearance pathways, including reduction and glucuronidation. Reduction of S-1360 generates a key metabolite in humans, designated HP1, and constitutes a major clearance pathway. For characterization of subcellular location and cofactor dependence of HP1 formation, [(14)C]-S-1360 was incubated with commercially available pooled human liver fractions, including microsomes, cytosol, and mitochondria, followed by HPLC analysis with radiochemical detection. Incubations were performed in the presence and absence of the cofactors NADH or NADPH. Results showed that the enzyme system responsible for generation of HP1 in vitro is cytosolic and NADPH-dependent, implicating aldo-keto reductases (AKRs) and/or short-chain dehydrogenases/reductases (SDRs). A validated LC/MS/MS method was developed for investigating the reduction of S-1360 in detail. The reduction reaction exhibited sigmoidal kinetics with a K(m,app) of 2 microM and a Hill coefficient of 2. The ratio of V(max)/K(m) was approximately 1 ml/(min mg cytosolic protein). The S-1360 kinetic data were consistent with positive cooperativity and a single enzyme system. The relative contributions of AKRs and SDRs were examined through the use of chemical inhibitors. For these experiments, non-radiolabeled S-1360 was incubated with pooled human liver cytosol and NADPH in the presence of inhibitors, followed by quantitation of HP1 by LC/MS/MS. Quercetin and menadione produced approximately 30% inhibition at a concentration of 100 microM. Enzymes sensitive to these inhibitors include the carbonyl reductases (CRs), a subset of the SDR enzyme family predominantly located in the cytosol. Flufenamic acid and phenolphthalein were the most potent inhibitors, with > 67% inhibition at a concentration of 20 microM, implicating the AKR enzyme family. The cofactor dependence

  2. Molecular dynamics simulation studies of the wild type and E92Q/N155H mutant of Elvitegravir-resistance HIV-1 integrase

    SciTech Connect

    Chen, Qi; Cheng, Xiaolin; Wei, Dongqing; Xu, Qin

    2014-11-06

    Although Elvitegravir (EVG) is a newly developed antiretrovirals drug to treat the acquired immunodeficiency syndrome (AIDS), drug resistance has already been found in clinic, such as E92Q/N155H and Q148H/G140S. Several structural investigations have already been reported to reveal the molecular mechanism of the drug resistance. As full length crystal structure for HIV-1 integrase is still unsolved, we use in this paper the crystal structure of the full length prototype foamy virus (PFV) in complex with virus DNA and inhibitor Elvitegravir as a template to construct the wild type and E92Q/N155H mutant system of HIV-1 integrase. Molecular dynamic simulations was used to revel the binding mode and the drug resistance of the EVG ligand in E92Q/N155H. Several important interactions were discovered between the mutated residues and the residues in the active site of the E92Q/N155H double mutant pattern, and cross correlation and clustering methods were used for detailed analysis. The results from the MD simulation studies will be used to guide the experimental efforts of developing novel inhibitors against drug-resistant HIV integrase mutants.

  3. Construction of a stepwise gene integration system by transient expression of actinophage R4 integrase in cyanobacterium Synechocystis sp. PCC 6803.

    PubMed

    Miura, Takamasa; Nishizawa, Akito; Nishizawa, Tomoyasu; Asayama, Munehiko; Takahashi, Hideo; Shirai, Makoto

    2014-08-01

    The integrase of actinophage R4, which belongs to the large serine-recombinase family, catalyzes site-specific recombination between two distinct attachment site sequences of the phage (attP) and actinomycete Streptomyces parvulus 2297 chromosome (attB). We previously reported that R4 integrase (Sre) catalyzed site-specific recombination both in vivo and in vitro. In the present study, a Sre-based system was developed for the stepwise site-specific integration of multiple genes into the chromosome of cyanobacterium Synechocystis sp. PCC 6803 (hereafter PCC 6803). A transgene-integrated plasmid with two attP sites and a non-replicative sre-containing plasmid were co-introduced into attB-inserted PCC 6803 cells. The transiently expressed Sre catalyzed highly efficient site-specific integration between one of the two attP sites on the integration plasmid and the attB site on the chromosome of PCC 6803. A second transgene-integrated plasmid with an attB site was integrated into the residual attP site on the chromosome by repeating site-specific recombination. The transformation frequencies (%) of the first and second integrations were approximately 5.1 × 10(-5) and 8.2 × 10(-5), respectively. Furthermore, the expression of two transgenes was detected. This study is the first to apply the multiple gene site-specific integration system based on R4 integrase to cyanobacteria.

  4. Antiviral Activity of Bictegravir (GS-9883), a Novel Potent HIV-1 Integrase Strand Transfer Inhibitor with an Improved Resistance Profile

    PubMed Central

    Tsiang, Manuel; Jones, Gregg S.; Goldsmith, Joshua; Mulato, Andrew; Hansen, Derek; Kan, Elaine; Tsai, Luong; Bam, Rujuta A.; Stepan, George; Stray, Kirsten M.; Niedziela-Majka, Anita; Yant, Stephen R.; Yu, Helen; Kukolj, George; Cihlar, Tomas; Lazerwith, Scott E.; Jin, Haolun

    2016-01-01

    Bictegravir (BIC; GS-9883), a novel, potent, once-daily, unboosted inhibitor of HIV-1 integrase (IN), specifically targets IN strand transfer activity (50% inhibitory concentration [IC50] of 7.5 ± 0.3 nM) and HIV-1 integration in cells. BIC exhibits potent and selective in vitro antiretroviral activity in both T-cell lines and primary human T lymphocytes, with 50% effective concentrations ranging from 1.5 to 2.4 nM and selectivity indices up to 8,700 relative to cytotoxicity. BIC exhibits synergistic in vitro antiviral effects in pairwise combinations with tenofovir alafenamide, emtricitabine, or darunavir and maintains potent antiviral activity against HIV-1 variants resistant to other classes of antiretrovirals. BIC displayed an in vitro resistance profile that was markedly improved compared to the integrase strand transfer inhibitors (INSTIs) raltegravir (RAL) and elvitegravir (EVG), and comparable to that of dolutegravir (DTG), against nine INSTI-resistant site-directed HIV-1 mutants. BIC displayed statistically improved antiviral activity relative to EVG, RAL, and DTG against a panel of 47 patient-derived HIV-1 isolates with high-level INSTI resistance; 13 of 47 tested isolates exhibited >2-fold lower resistance to BIC than DTG. In dose-escalation experiments conducted in vitro, BIC and DTG exhibited higher barriers to resistance than EVG, selecting for HIV-1 variants with reduced phenotypic susceptibility at days 71, 87, and 20, respectively. A recombinant virus with the BIC-selected M50I/R263K dual mutations in IN exhibited only 2.8-fold reduced susceptibility to BIC compared to wild-type virus. All BIC-selected variants exhibited low to intermediate levels of cross-resistance to RAL, DTG, and EVG (<8-fold) but remained susceptible to other classes of antiretrovirals. A high barrier to in vitro resistance emergence for both BIC and DTG was also observed in viral breakthrough studies in the presence of constant clinically relevant drug concentrations. The

  5. Production of transgenic cattle highly expressing human serum albumin in milk by phiC31 integrase-mediated gene delivery.

    PubMed

    Luo, Yan; Wang, Yongsheng; Liu, Jun; Lan, Hui; Shao, Minghao; Yu, Yuan; Quan, Fusheng; Zhang, Yong

    2015-10-01

    Transgenic cattle expressing high levels of recombinant human serum albumin (HSA) in their milk may as an alternative source for commercial production. Our objective was to produce transgenic cattle highly expressing HSA in milk by using phiC31 integrase system and somatic cell nuclear transfer (SCNT). The mammary-specific expression plasmid pIACH(-), containing the attB recognition site for phiC31 integrase, were co-transfected with integrase expression plasmid pCMVInt into bovine fetal fibroblast cells (BFFs). PhiC31 integrase-mediated integrations in genome of BFFs were screened by nested inverse PCR. After analysis of sequence of the PCR products, 46.0% (23/50) of the both attB-genome junction sites (attL and attR) were confirmed, and four pseudo attP sites were identified. The integration rates in BF3, BF11, BF19 and BF4 sites were 4.0% (2/50), 6.0% (3/50), 16.0% (8/50) and 20.0% (10/50), respectively. BF3 is located in the bovine chromosome 3 collagen alpha-3 (VI) chain isomer 2 gene, while the other three sites are located in the non-coding region. The transgenic cell lines from BF11, BF19 and BF4 sites were used as donors for SCNT. Two calves from transgenic cells BF19 were born, one died within a few hours after birth, and another calf survived healthy. PCR and Southern blot analysis revealed integration of the transgene in the genome of cloned calves. The nested reverse PCR confirmed that the integration site in cloned calves was identical to the donor cells. The western blotting assessment indicated that recombinant HSA was expressed in the milk of transgenic cattle and the expression level was about 4-8 mg/mL. The present study demonstrated that phiC31 integrase system was an efficient and safety gene delivery tool for producing HSA transgenic cattle. The production of recombinant HSA in the milk of cattle may provide a large-scale and cost-effective resource.

  6. Human immunodeficiency virus type 1 (HIV-1) integrase: resistance to diketo acid integrase inhibitors impairs HIV-1 replication and integration and confers cross-resistance to L-chicoric acid.

    PubMed

    Lee, Deborah J; Robinson, W E

    2004-06-01

    The diketo acids are potent inhibitors of human immunodeficiency virus (HIV) integrase (IN). Mutations in IN, T66I, S153Y, and M154I, as well as T66I-S153Y and T66I-M154I double mutations, confer resistance to diketo acids (D. J. Hazuda et al., Science 287:646-650, 2000). The effects of these IN mutations on viral replication, enzymatic activity, and susceptibility to other HIV inhibitors are reported herein. By immunofluorescence assay and real-time PCR, all mutant viruses demonstrated a modest delay in viral spread compared to that of reference HIV. These viruses also showed a statistically significant defect in integration without defects in reverse transcription. Recombinant IN containing S153Y, T66I, and M154I-T66I mutations had an approximately twofold decrease in both disintegration and 3'-end-processing-strand transfer activities in vitro. In contrast, IN containing M154I demonstrated a greater than twofold increase in specific activity in both reactions. All mutant HIVs were resistant to l-chicoric acid, a dicaffeoyltartaric acid IN inhibitor, both in tissue culture and in biochemical assays, yet remained susceptible to the reverse transcriptase inhibitors zidovudine and nevirapine. Thus, IN mutations conferring resistance to the diketo acids can yield integration defects, attenuated catalysis in vitro, and cross-resistance to l-chicoric acid.

  7. Q148N, a Novel Integrase Inhibitor Resistance Mutation Associated with Low-Level Reduction in Elvitegravir Susceptibility

    PubMed Central

    Pinsky, Benjamin A.; Smith, Darvin S.; Klein, Daniel; Shafer, Robert W.

    2016-01-01

    Abstract The integrase strand transfer inhibitor (INSTI)-resistance mutations Q148H/K/R are arguably the most important INSTI-resistance mutations as they represented the first step to high-level dolutegravir cross-resistance. We describe an individual with transmitted four-class drug resistance whose virus sequence had the previously uncharacterized mutation Q148N. Infectious molecular HIV-1 clones containing Q148N alone and in combination with G140S demonstrated ∼2.4–4.5 reduced elvitegravir susceptibility depending on the virus's genetic context but retained susceptibility to raltegravir and dolutegravir. This level of reduced elvitegravir susceptibility is lower than that observed with Q148H/K/R and in fact the infected individual responded to an initial treatment regimen containing tenofovir/emtricitabine/elvitegravir/cobicistat. Q148N was associated with a higher replication capacity than Q148H, suggesting that this mutation may be more fit in the absence of selective INSTI therapy. PMID:27009474

  8. Q148N, a Novel Integrase Inhibitor Resistance Mutation Associated with Low-Level Reduction in Elvitegravir Susceptibility.

    PubMed

    Varghese, Vici; Pinsky, Benjamin A; Smith, Darvin S; Klein, Daniel; Shafer, Robert W

    2016-07-01

    The integrase strand transfer inhibitor (INSTI)-resistance mutations Q148H/K/R are arguably the most important INSTI-resistance mutations as they represented the first step to high-level dolutegravir cross-resistance. We describe an individual with transmitted four-class drug resistance whose virus sequence had the previously uncharacterized mutation Q148N. Infectious molecular HIV-1 clones containing Q148N alone and in combination with G140S demonstrated ∼2.4-4.5 reduced elvitegravir susceptibility depending on the virus's genetic context but retained susceptibility to raltegravir and dolutegravir. This level of reduced elvitegravir susceptibility is lower than that observed with Q148H/K/R and in fact the infected individual responded to an initial treatment regimen containing tenofovir/emtricitabine/elvitegravir/cobicistat. Q148N was associated with a higher replication capacity than Q148H, suggesting that this mutation may be more fit in the absence of selective INSTI therapy.

  9. Resistance Analyses of Integrase Strand Transfer Inhibitors within Phase 3 Clinical Trials of Treatment-Naive Patients

    PubMed Central

    White, Kirsten L.; Raffi, Francois; Miller, Michael D.

    2014-01-01

    The integrase (IN) strand transfer inhibitors (INSTIs), raltegravir (RAL), elvitegravir (EVG) and dolutegravir (DTG), comprise the newest drug class approved for the treatment of HIV-1 infection, which joins the existing classes of reverse transcriptase, protease and binding/entry inhibitors. The efficacy of first-line regimens has attained remarkably high levels, reaching undetectable viral loads in 90% of patients by Week 48; however, there remain patients who require a change in regimen due to adverse events, virologic failure with emergent resistance or other issues of patient management. Large, randomized clinical trials conducted in antiretroviral treatment-naive individuals are required for drug approval in this population in the US, EU and other countries, with the primary endpoint for virologic success at Week 48. However, there are differences in the definition of virologic failure and the evaluation of drug resistance among the trials. This review focuses on the methodology and tabulation of resistance to INSTIs in phase 3 clinical trials of first-line regimens and discusses case studies of resistance. PMID:25054884

  10. Characterization of Streptococcus gordonii prophage PH15: complete genome sequence and functional analysis of phage-encoded integrase and endolysin.

    PubMed

    van der Ploeg, Jan R

    2008-10-01

    Streptococcus gordonii OMZ1039, isolated from supragingival dental plaque, was found to harbour a prophage, PH15, whose excision could be induced by mitomycin treatment. Phage PH15 belongs to the Siphoviridae. The complete genome sequence of PH15 was determined. The genome was 39 136 bp in size and contained 61 ORFs. The genome of PH15 was most similar in the structural module to the temperate bacteriophages MM1 and phiNIH1.1 from Streptococcus pneumoniae and Streptococcus pyogenes, respectively. In strain OMZ1039, PH15 was found to reside as a prophage in the cysteinyl-tRNA gene. A plasmid, harbouring the attP site and the integrase gene downstream of a constitutive promoter, was capable of site-specific integration into the genomes of different oral streptococcal species. The phage endolysin was purified after expression in Escherichia coli and found to inhibit growth of all S. gordonii strains tested and several different streptococcal species, including the pathogens Streptococcus mutans, S. pyogenes and Streptococcus agalactiae.

  11. Molecular dynamics study of carbon nanotube as a potential dual-functional inhibitor of HIV-1 integrase.

    PubMed

    Zhang, Zhishun; Wang, Bingqiang; Wan, Bo; Yu, Long; Huang, Qiang

    2013-07-12

    HIV-1 integrase (IN) plays an important role in integrating viral DNA into human genome, which has been considered as the drug target for anti-AIDS therapy. The appearance of drug-resistance mutants urgently requires novel inhibitors that act on non-active site of HIV-1 IN. Nanoparticles have such unique geometrical and chemical properties, which inspires us that nanoparticles like nanotubes may serve as better HIV-1 IN inhibitors than the conventional inhibitors. To test this hypothesis, we performed molecular dynamics (MD) simulation to study the binding of a carbon nanotube (CNT) to a full-length HIV-1 IN. The results showed that the CNT could stably bind to the C-terminal domain (CTD) of HIV-1 IN. The CNT also induced a domain-shift which disrupted the binding channel for viral DNA. Further MD simulation showed that a HIV-1 IN inhibitor, 5ClTEP was successfully sealed inside the uncapped CNT. These results indicate that the CNT may serve as a potential dual-functional HIV-1 IN inhibitor, not only inducing conformation change as an allosteric inhibitor but also carrying small-molecular inhibitors as a drug delivery system. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. A QSAR study of integrase strand transfer inhibitors based on a large set of pyrimidine, pyrimidone, and pyridopyrazine carboxamide derivatives

    NASA Astrophysics Data System (ADS)

    de Campos, Luana Janaína; de Melo, Eduardo Borges

    2017-08-01

    In the present study, 199 compounds derived from pyrimidine, pyrimidone and pyridopyrazine carboxamides with inhibitory activity against HIV-1 integrase were modeled. Subsequently, a multivariate QSAR study was conducted with 54 molecules employed by Ordered Predictors Selection (OPS) and Partial Least Squares (PLS) for the selection of variables and model construction, respectively. Topological, electrotopological, geometric, and molecular descriptors were used. The selected real model was robust and free from chance correlation; in addition, it demonstrated favorable internal and external statistical quality. Once statistically validated, the training model was used to predict the activity of a second data set (n = 145). The root mean square deviation (RMSD) between observed and predicted values was 0.698. Although it is a value outside of the standards, only 15 (10.34%) of the samples exhibited higher residual values than 1 log unit, a result considered acceptable. Results of Williams and Euclidean applicability domains relative to the prediction showed that the predictions did not occur by extrapolation and that the model is representative of the chemical space of test compounds.

  13. The Need for Development of New HIV-1 Reverse Transcriptase and Integrase Inhibitors in the Aftermath of Antiviral Drug Resistance

    PubMed Central

    Wainberg, Mark A.

    2012-01-01

    The use of highly active antiretroviral therapy (HAART) involves combinations of drugs to achieve maximal virological response and reduce the potential for the emergence of antiviral resistance. There are two broad classes of reverse transcriptase inhibitors, the nucleoside reverse transcriptase inhibitors (NRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs). Since the first classes of such compounds were developed, viral resistance against them has necessitated the continuous development of novel compounds within each class. This paper considers the NRTIs and NNRTIs currently in both preclinical and clinical development or approved for second line therapy and describes the patterns of resistance associated with their use, as well as the underlying mechanisms that have been described. Due to reasons of both affordability and availability, some reverse transcriptase inhibitors with low genetic barrier are more commonly used in resource-limited settings. Their use results to the emergence of specific patterns of antiviral resistance and so may require specific actions to preserve therapeutic options for patients in such settings. More recently, the advent of integrase strand transfer inhibitors represents another major step forward toward control of HIV infection, but these compounds are also susceptible to problems of HIV drug resistance. PMID:24278679

  14. A novel assay for screening inhibitors targeting HIV-1 integrase dimerization based on Ni-NTA magnetic agarose beads

    PubMed Central

    Zhang, Dawei; He, Hongqiu; Liu, Mengmeng; Meng, Zhixia; Guo, Shunxing

    2016-01-01

    Human immunodeficiency virus (HIV)-1 integrase (IN), which mediates integration of viral cDNA into the cellular chromosome, is a validated antiviral drug target. Three IN inhibitors, raltegravir, elvitegravir and dolutegravir, have been clinically approved since 2008. However, drug resistance have emerged in infected patients receiving treatment using these drugs which share the same mechanism of action and have a low genetic barrier for resistance. Therefore, there is an urgent need to develop drugs with novel mechanism. IN requires a precise and dynamic equilibrium between several oligomeric species for its activities. The modulation of the process which is termed as IN oligomerization, presents an interesting allosteric target for drug development. In this research, we developed a magnetic beads based approach to assay the IN dimerization. Then, using the assay we screened a library of 1000 Food and Drug Administration (FDA)-approved drugs for IN dimerization inhibitors and identified dexlansoprazole as a potential IN dimerization inhibitor. In conclusion, the assay presented here has been proven to be sensitive and specific for the detection of IN dimerization as well as for the identification of antiviral drugs targeting IN dimerization. Moreover, a FDA-approved proton-pump inhibitors, dexlansoprazole, was identified as a potential inhibitor for IN dimerization. PMID:27137477

  15. Selectivity for strand-transfer over 3′-processing and susceptibility to clinical resistance of HIV-1 integrase inhibitors are driven by key enzyme–DNA interactions in the active site

    PubMed Central

    Métifiot, Mathieu; Johnson, Barry C.; Kiselev, Evgeny; Marler, Laura; Zhao, Xue Zhi; Burke, Terrence R.; Marchand, Christophe; Hughes, Stephen H.; Pommier, Yves

    2016-01-01

    Integrase strand transfer inhibitors (INSTIs) are highly effective against HIV infections. Co-crystal structures of the prototype foamy virus intasome have shown that all three FDA-approved drugs, raltegravir (RAL), elvitegravir and dolutegravir (DTG), act as interfacial inhibitors during the strand transfer (ST) integration step. However, these structures give only a partial sense for the limited inhibition of the 3′-processing reaction by INSTIs and how INSTIs can be modified to overcome drug resistance, notably against the G140S-Q148H double mutation. Based on biochemical experiments with modified oligonucleotides, we demonstrate that both the viral DNA +1 and −1 bases, which flank the 3′-processing site, play a critical role for 3′-processing efficiency and inhibition by RAL and DTG. In addition, the G140S-Q148H (SH) mutant integrase, which has a reduced 3′-processing activity, becomes more active and more resistant to inhibition of 3′-processing by RAL and DTG in the absence of the −1 and +1 bases. Molecular modeling of HIV-1 integrase, together with biochemical data, indicate that the conserved residue Q146 in the flexible loop of HIV-1 integrase is critical for productive viral DNA binding through specific contacts with the virus DNA ends in the 3′-processing and ST reactions. The potency of integrase inhibitors against 3′-processing and their ability to overcome resistance is discussed. PMID:27369381

  16. Synthesis and Characterization of β-Cyclodextrin Functionalized Ionic Liquid Polymer as a Macroporous Material for the Removal of Phenols and As(V)

    PubMed Central

    Raoov, Muggundha; Mohamad, Sharifah; Abas, Mhd Radzi

    2014-01-01

    β-Cyclodextrin-ionic liquid polymer (CD-ILP) was first synthesized by functionalized β-cyclodextrin (CD) with 1-benzylimidazole (BIM) to form monofunctionalized CD (βCD-BIMOTs) and was further polymerized using a toluene diisocyanate (TDI) linker to form insoluble CD-ILP (βCD-BIMOTs-TDI). The βCD-BIMOTs-TDI polymer was characterized using various tools and the results obtained were compared with those derived from the native β-cyclodextrin polymer (βCD-TDI). The SEM result shows that the presence of ionic liquid (IL) increases the pore size, while the thermo gravimetric analysis (TGA) result shows that the presence of IL increases the stability of the polymer. Meanwhile, Brunauer-Emmett-Teller (BET) results show that βCD-BIMOTs-TDI polymer has 1.254 m2/g surface areas and the Barret-Joyner-Halenda (BJH) pore size distribution result reveals that the polymer exhibits macropores with a pore size of 77.66 nm. Preliminary sorption experiments were carried out and the βCD-BIMOTs-TDI polymer shows enhanced sorption capacity and high removal towards phenols and As(V). PMID:24366065

  17. Synthesis of green nano iron particles (GnIP) and their application in adsorptive removal of As(III) and As(V) from aqueous solution

    NASA Astrophysics Data System (ADS)

    Prasad, Kumar Suranjit; Gandhi, Pooja; Selvaraj, Kaliaperumal

    2014-10-01

    The present study reports a new approach to synthesise nano iron particles using leaf extract of Mint (Mentha spicata L.) plant. The synthesised GnIPs were subjected to detailed adsorption studies for removal of arsenite and arsenate from aqueous solution of defined concentration. Iron nanoparticles synthesised using leaf extract showed UV-vis absorption peaks at 360 and 430 nm. TEM result showed the formation of polydispersed nanoparticles of size ranging from 20 to 45 nm. Nanoparticles were found to have core-shell structure. The planer reflection of selected area electron diffraction (SAED) and XRD analysis suggested that iron particles were crystalline and belonged to fcc (face centred cubic) type. Energy-dispersive X-ray analysis (EDAX) shows that Fe was an integral component of synthesised nanoparticles. The content of Fe in nanoparticles was found to be 40%, in addition to other elements like C (16%), O (19%) and Cl (23%). FT-IR study suggested that functional groups like sbnd NH, sbnd Cdbnd O, sbnd Cdbnd N and sbnd Cdbnd C were involved in particle formation. The removal efficiency of GnIP-chitosan composite for As(III) and As(V) was found to be 98.79 and 99.65%. Regeneration of adsorbent suggested that synthesised green GnIP may work as an effective tool for removal of arsenic from contaminated water.

  18. Synthesis and characterization of β-cyclodextrin functionalized ionic liquid polymer as a macroporous material for the removal of phenols and As(V).

    PubMed

    Raoov, Muggundha; Mohamad, Sharifah; Abas, Mhd Radzi

    2013-12-23

    β-Cyclodextrin-ionic liquid polymer (CD-ILP) was first synthesized by functionalized β-cyclodextrin (CD) with 1-benzylimidazole (BIM) to form monofunctionalized CD (βCD-BIMOTs) and was further polymerized using a toluene diisocyanate (TDI) linker to form insoluble CD-ILP (βCD-BIMOTs-TDI). The βCD-BIMOTs-TDI polymer was characterized using various tools and the results obtained were compared with those derived from the native β-cyclodextrin polymer (βCD-TDI). The SEM result shows that the presence of ionic liquid (IL) increases the pore size, while the thermo gravimetric analysis (TGA) result shows that