Characterization of the DNA-Binding Activity of HIV-1 Integrase Using a Filter Binding Assay

Haugan, I.R.; Nilsen, Bente M.; Worland, Stephen T.; Olsen, L.R.; Helland, Dag E.

doi:10.1006/bbrc.1995.2843

Cited by 19 publications

(17 citation statements)

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“…The effect of antibodies on IN binding to DNA was assessed by filter‐binding assays. These have been already used to study IN–DNA interactions [41] and to screen for IN inhibitors [41, 46, 47]. Here, IN was preincubated with antibodies at different antibodies/IN molar ratios before addition of oligonucleotide substrates, i.e.…”

Section: Resultsmentioning

confidence: 99%

“…The reaction conditions were described previously [41]. Typically, in a 20‐µL volume, HIV‐1 IN (5.6 pmol) was incubated with antisera at various IN/antibody molar ratios or P159 and K159 at different peptide concentrations in the reaction buffer (20 m m Tris/HCl, pH 7.5, 1 m m β‐mercaptoethanol, 1 m m dithiothreitol, 1 m m MnCl 2 , EDTA 0.3 m m ) for 15 min at 34 °C then the DNA‐labeled substrates (0.6 pmol) were added and incubated to additional 10 min at 34 °C.…”

Section: Methodsmentioning

confidence: 99%

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Conformational aspects of HIV‐1 integrase inhibition by a peptide derived from the enzyme central domain and by antibodies raised against this peptide

Maroun

Krebs

Roshani

et al. 1999

European Journal of Biochemistry

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Monospecific antibodies were raised against a synthetic peptide K159 (SQGVVESMNKELKKIIGQVRDQAEHLKTA) reproducing the segment 147–175 of HIV‐1 integrase (IN). Synthesis of substituted and truncated analogs of K159 led us to identify the functional epitope reacting with antibodies within the C‐terminal portion 163–175 of K159. Conformational studies combining secondary structure predictions, CD and NMR spectroscopy together with ELISA assays, showed that the greater is the propensity of the epitope for helix formation the higher is the recognition by anti‐K159. Both the antibodies and the antigenic peptide K159 exhibited inhibitory activities against IN. In contrast, neither P159, a Pro‐containing analog of K159 that presents a kink around proline but with intact epitope conformation, nor the truncated analogs encompassing the epitope, were inhibitors of IN. While the activity of antibodies is restricted to recognition of the sole epitope portion, that of the antigenic K159 likely requires interactions of the peptide with the whole 147–175 segment in the protein [Sourgen F., Maroun, R.G., Frère, V., Bouziane, A., Auclair, C., Troalen, F. & Fermandjian, S. (1996) Eur. J. Biochem. 240, 765–773 ]. Actually, of all tested peptides only K159 was found to fulfill conditions of minimal number of helical heptads to achieve the formation of a stable coiled‐coil structure with the IN 147–175 segment. The binding of antibodies and of the antigenic peptide to this segment of IN hampers the binding of IN to its DNA substrates in filter‐binding assays. This appears to be the main effect leading to inhibition of integration. Quantitative analysis of filter‐binding assay curves indicates that two antibody molecules react with IN implying that the enzyme is dimeric within these experimental conditions. Together, present data provide an insight into the structure–function relationship for the 147–175 peptide domain of the enzyme. They also strongly suggest that the functional enzyme is dimeric. Results could help to assess models for binding of peptide fragments to IN and to develop stronger inhibitors. Moreover, K159 antibodies when expressed in vivo might exhibit useful inhibitory properties.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Conformational aspects of HIV‐1 integrase inhibition by a peptide derived from the enzyme central domain and by antibodies raised against this peptide

Maroun

Krebs

Roshani

et al. 1999

European Journal of Biochemistry

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“…Functions attributed to the IN CTD include enhancement of IN multimerization (43), nonspecific and, presumptively, specific DNA binding capabilities (19,28,29,38,41,44,55,56,74), and facilitation of host factor binding (2,10,35,54,63,75). Reports also highlight a direct and apparently functional interaction between IN and reverse transcriptase (RT) (40,69,77,79,82), with recent evidence suggesting that this association is mediated through the CTD (40,77).…”

mentioning

confidence: 99%

Sequential Deletion of the Integrase (Gag-Pol) Carboxyl Terminus Reveals Distinct Phenotypic Classes of Defective HIV-1

Mohammed

Topper

Muesing

2011

J Virol

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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.The defining hallmarks of retroviruses are reverse transcription of the viral genomic information as encoded in polyadenylated RNA and the subsequent integration of the copied DNA genome into that of a host cell. The latter is an essential and irreversible event which is mediated by the catalytic activities of the viral integrase protein (IN), the recent target of successful chemotherapeutic intervention against HIV-1 infection (1). HIV-1 IN is a 288-amino-acid, 32-kDa protein that is cleaved from the C terminus of the Gag-Pol polyprotein (Pr160 Gag-Pol ) via viral proteolytic activity. The biochemical mechanisms that lead to retroviral integration, which have been extensively studied in vitro, are defined by two catalytically related and sequentially dependent steps (18) which may be distinguished by their respective sensitivities to current inhibitors of IN function (39). Following the completion of reverse transcription of the viral RNA into its DNA copy, IN removes two nucleotides from the 3Ј end of each strand of the viral DNA. This step, termed 3Ј processing, generates a chemically reactive 3Ј-hydroxyl group (CA OH -3Ј) at the 3Ј ends of the DNA molecule, effectively activating the termini for the subsequent reaction (strand transfer). This enzymatic step is the target of all I...

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“…To investigate the binding characteristics of HIV-1 integrase, we have developed an FCS-based method to determine its binding constants. The advantage of this method is the possibility of performing measurements on a tiny volume without UV cross-linking (16), filtering (17), or the fixing of any component to a solid carrier (18 The oligonucleotides INT1-TMR3, INT1-TMR5, INT1, and INT2 are 20-mers that correspond to the U5 LTR ends of the HIV-1 genome (19); (INT1, 5Ј-TGTGGAAAATCTCTAGCAGT-3Ј; INT2, 5Ј-ACTGCTAGAGATTTTCCACA-3Ј). They were synthesized and purified by Eurogentec (Seraing, Belgium).…”

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confidence: 99%

DNA-induced Polymerization of HIV-1 Integrase Analyzed with Fluorescence Fluctuation Spectroscopy

Vercammen¹,

Maertens²,

Gérard³

et al. 2002

Journal of Biological Chemistry

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Human immunodeficiency virus type 1 (HIV-1) integrase is essential for viral replication. Integrase inserts the viral DNA into the host DNA. We studied the association of integrase to fluorescently labeled oligonucleotides using fluorescence correlation spectroscopy. The binding of integrase to the fluorescent oligonucleotides resulted in the appearance of bright spikes during fluorescence correlation spectroscopy measurements. These spikes arise from the formation of high molecular mass protein-DNA complexes. The fluorescence of the free DNA was separated from the spikes with a statistical method. From the decrease of the concentration of free oligonucleotides, a site association constant was determined. The DNA-protein complexes were formed rapidly in a salt-dependent manner with site association constants ranging between 5 and 40 M ؊1 under different conditions. We also analyzed the kinetics of the DNA-protein complex assembly and the effect of different buffer components. The formation of the fluorescent protein-DNA complex was inhibited by guanosine quartets, and the inhibition constant was determined at 1.8 ؎ 0.6 ؋ 10 8 M ؊1 . Displacement of bound DNA with G-quartets allowed the determination of the dissociation rate constant and proves the reversibility of the association process.Integration of the viral DNA of the human immunodeficiency virus type 1 (HIV-1) 1 into the genome of a human cell is an essential step in the retroviral replication cycle. Genetic studies have shown that retrovirus integration requires two viral components, integrase and DNA sequences at the ends of the viral long terminal repeats (LTRs) (1). In the cytoplasm, integrase forms a complex with the viral DNA. This is followed by two enzymatic reactions. In the 3Ј-processing reaction, integrase removes a dinucleotide from each 3Ј-end of the viral DNA, resulting in a terminating CA sequence. The processed DNA is transported into the nucleus and inserted in the host chromosome, producing a gapped intermediate. Gap repair is probably carried out by cellular DNA repair enzymes (2).Integrase is a 32-kDa protein that is composed of three distinct domains. The N terminus contains the "HHCC" zinc finger-like motif. Evidence suggests that the binding of zinc has a prominent role in the self-assembly of IN into an active multimeric complex (3, 4). The N terminus has been suggested to be involved in protein-protein interactions and substrate specificity (5). The central core and the C-terminal domain are DNA binding domains. The central core contains the catalytic triad, the D,D(35)E motif, which is responsible for the specific binding of the substrate and the enzymatic activity of integrase. The C-terminal domain has a nonspecific DNA binding activity. In solution, integrase has been shown to form monomers, dimers, and tetramers (6 -8). However, the active complex present in the preintegration complex is a multimer as required for the enzymatic reactions of integrase (4, 10).Fluorescence correlation spectroscopy (FCS) is based on the statistic...

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Characterization of the DNA-Binding Activity of HIV-1 Integrase Using a Filter Binding Assay

Cited by 19 publications

References 0 publications

Conformational aspects of HIV‐1 integrase inhibition by a peptide derived from the enzyme central domain and by antibodies raised against this peptide

Conformational aspects of HIV‐1 integrase inhibition by a peptide derived from the enzyme central domain and by antibodies raised against this peptide

Sequential Deletion of the Integrase (Gag-Pol) Carboxyl Terminus Reveals Distinct Phenotypic Classes of Defective HIV-1

DNA-induced Polymerization of HIV-1 Integrase Analyzed with Fluorescence Fluctuation Spectroscopy

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