HIV-1 Integrase Activity Research Articles

Nucleotides

The new (2′ – 5′)oligoadenylate trimers 26 – 34 containing 5′-amino-5′-deoxyadenosine or 5′-amino-3′,5′-dideoxyadenosine or their lipophilic 5′-deoxy-5′-(hexadecanoylamino) derivatives at the 5′-terminus, and adenosine or 3′-deoxyadenosine (=cordycepin) at the penultimate and 2′-end position of the trimers were synthesized by the phosphoramidite method. The newly synthesized trimers 26 – 34 inhibited, at 100 μM concentration, HIV-1-induced syncytia formation (SYN) by 19 – 96% and reverse-transcriptase activity (RT) by 27 – 100% (see Table). The two hexadecanoylamino derivatives 27 and 30 which were found to be potent inhibitors of SYN and RT showed also a 73 and 49% inhibition, respectively, of expression of HIV-1 p24 antigen (p24-EX). The same compounds 27 and 30 inhibited also, with a 100% efficacy, an amplification of HIV-1 partial reverse transcripts (PCR) and HIV-1 integrase activity (INT), respectively.

Selective and controlled hydrolysis of chloropeptin I. HIV-1 integrase activity of fragments

Selective amide bond cleavages of chloropeptin I were accomplished using trifluoroacetic acid (TFA), and a mixture of acetic acid, hydrochloric acid and thioglycolic acid. The hydrolysis products maintaining the bicyclic core retained HIV-1 integrase inhibitory activity similar to that of the chloropeptin I. The hydrolysis products and their HIV-1 integrase activities are described.

Cobalamin Inhibition of HIV-1 Integrase and Integration of HIV-1 DNA into Cellular DNA

Our prior studies showed that certain cobalamins inhibit productive HIV-1 infection of primary cultures of blood lymphocytes and monocytes. We demonstrate here that this antiviral activity may be mediated by an inhibition of HIV-1 integrase, an enzyme required for productive infection. Purified recombinant HIV-1 integrase activity was inhibitedin vitroby hydroxocobalamin (OH-Cbl), methylcobalamin (Me-Cbl), adenosylcobalamin (Ado-Cbl), and dicyanocobinamide (CN2-Cbi) with IC50values of approximately 17, 17, 17, and 4 μM, respectively. The agents inhibited HIV-1 infection of cultured monocytes (IC50values for OH-Cbl, Me-Cbl, Ado-Cbl, and CN2-Cbi of 6, 7, 4, and 1 μM, respectively) and of cultured lymphocytes (IC50values of 60, 50, 60, and 11 μM, respectively). Experiments using cultured monocytes or lymphocytes demonstrated that OH-Cbl inhibited integration of HIV-1 DNA into cellular DNA. Thus, cobalamins and cobinamides represent novel inhibitors of HIV-1 integrase. These or related agents may be useful as anti-viral treatments that target HIV-1 integrase.

Structure-function analysis of integrase interactor 1/hSNF5L1 reveals differential properties of two repeat motifs present in the highly conserved region.

Retroviral integrase (IN) catalyzes the integration of retroviral cDNA into host chromosome. Ini1 (integrase interactor 1) is a host protein that specifically binds and stimulates in vitro joining activity of HIV-1 IN. Ini1 has homology to yeast transcription factor SNF5 and is a component of the analogous mammalian SWI/SNF complex that can remodel chromatin. Little is known about the function of Ini1 in mammalian cells. To gain insight into the functional domains of Ini1, and to understand the details of protein-protein interactions of IN and Ini1, a structure-function analysis of Ini1 was initiated. By means of the yeast two-hybrid system, the minimal IN binding domain of Ini1 was characterized. One of the two repeat motifs present in the highly conserved regions of Ini1 was found necessary and sufficient to bind to IN in yeast as well as in vitro. Because IN binds to only one of the two repeat motifs in this conserved region of Ini1, it appears that the IN-Ini1 interaction is very specific and functionally significant. Characterization of DNA-binding properties of Ini1 revealed that Ini1 can bind to plasmid DNA, binding more readily to supercoiled DNA than to the relaxed circular DNA. The minimal domain for DNA binding was localized to a region upstream of repeat 1. The DNA binding activity of Ini1 is not required for its ability to interact with IN. The finding that the two repeat motifs of Ini1 display differential binding to HIV-1 IN and that this discrete component of mammalian SWI/SNF complex binds to DNA will help understand the role of Ini1 in HIV-1 integration and in cellular process.

Enzymatic Capability of HIS-Tagged HIV-1 Integrase Using Oligonucleotide Disintegration Substrates.

Disintegration, wherein a half-site integration substrate is resolved into separate viral and host DNA components via DNA strand transfer, is one of three well-established in vitro activities of HIV-1 integrase. The role of disintegration in the HIV-1 replicative cycle, however, remains a mystery. In this report, we describe the expression in Escherichia coli and purification of HIV-1 integrase as a fusion protein containing a 6xHis tag at its amino terminus. Integrase resolved dumbbell and Y-substrates optimally at pH 6.8-7.2 in the presence of 2 mM MnCl(2). Substrate requirements for intramolecular disintegration included a 10 base pair viral U5 LTR arm and a CA dinucleotide located at the 3' end of the LTR. Disintegration was not sensitive to changes in the host DNA portion of the substrate. A dumbbell substrate with a 5' oligo-dA tail also underwent disintegration. The released LTR arm with an oligo-dA tail was utilized as a template primer by several DNA polymerases indicating that disintegration occurred via nucleophilic attack on the phosphodiester bond located immediately adjacent to the CA dinucleotide at the 3' end of the LTR. Coupled disintegration-DNA polymerase reactions provided a highly efficient and sensitive means of detecting disintegration activity. Integrase also catalyzed an apparently concerted disintegration-5'-end joining reaction in which an LTR arm was transferred from one dumbbell substrate molecule to another. Copyright 1996 S. Karger AG, Basel

Assaying the activity of HIV-1 integrase with DNA-coated plates.

Integration of reverse transcribed viral DNA of HIV into host chromosomes is mediated by the viral enzyme, integrase. This enzymatic activity can be monitored in vitro by integration of a small labeled DNA (donor) into a second unlabeled DNA (target). The methodology usually involves isotope labeling and gel electrophoresis. To simplify the measurement, a method mimicking enzyme-linked immunosorbent assay (ELISA) procedures was developed. Fragments of DNA were adsorbed directly on 96-well plates and used as the target DNA. The donor was a synthetic 21-bp DNA duplex of HIV-1 U5 LTR; biotin was incorporated into the 5' end of one strand whose two nucleotides at the 3' end were specifically removed during the integration. As a result of integration, the biotin-labeled donor DNA was joined with the target DNA and became immobilized on plates. These integration products were then measured by binding of avidin-alkaline phosphatase on plates. The method is simple and straightforward and can easily be adapted for high throughput screening of integrase inhibitors.

Effects of tyrphostins, protein kinase inhibitors, on human immunodeficiency virus type 1 integrase.

Efficient replication of HIV-1 requires establishment of the proviral state, i.e., the integration of a DNA copy of the viral genome, synthesized by reverse transcriptase, into a chromosome of the host cell. Integration is catalyzed by the viral integrase protein. We have previously reported that phenolic moieties in compounds such as napthoquinones, flavones, caffeic acid phenethyl ester (CAPE), and curcumin confer inhibitory activity against HIV-1 integrase. We have extended these findings by examining the effects of tryphostins, tyrosine kinase inhibitors. The catalytic activities of HIV-1 integrase and the formation of enzyme-DNA complexes using photocross-linking were examined. Both steps of the integration reaction, 3'-processing and strand transfer, were inhibited by tyrphostins at micromolar concentrations. The DNA binding activity of integrase was inhibited at higher concentrations of tryphostins. Disintegration, an apparent reversal of the strand transfer reaction, catalyzed by an integrase mutant lacking the N-terminal zinc finger and C-terminal DNA binding domains is also inhibited by tyrphostins, indicating that the binding site for these compounds resides in the central catalytic core of HIV-1 integrase. Binding of tyrphostins at or near the integrase catalytic site was also suggested by experiments showing a global inhibition of the choice of attacking nucleophile in the 3'-processing reaction. None of the tryphostins tested inhibited eukaryotic topoisomerase I, even at 100 microM, suggesting selectivity for integrase inhibition. Molecular-modeling studies have revealed that, after energy minimization, several tyrphostins may adopt folded conformations. The similarity of the tyrphostin family to other families of inhibitors is discussed. Tyrphostins may provide lead compounds for development of novel antiviral agents for the treatment of acquired immunodeficiency syndrome based upon inhibition of HIV-1 integrase.

Characterization of Endonucleolytic Activity of HIV-1 Integrase Using a Fluorogenic Substrate

Retroviruses require viral DNA to be synthesized by reverse transcription in the cytoplasm followed by integration of the resulting viral DNA into the host chromosome in the nucleus. Reverse transcription and integration, essential steps in the life cycle of retroviruses, are possible targets in the development of antiviral reagents. One attractive target is the integrase protein, a product of the retroviral pol gene which is solely responsible for the retroviral integration process through cutting and joining reactions. When screening for massive numbers of antiviral agents, a rapid and precise assay is ideal. We report the application of fluorescence resonance energy transfer (FRET) with fluorescein and eosin as the energy transfer pair to characterize HIV-IN-mediated DNA cleavage reactions. Past concerns with applications of FRET to DNA were due to interactions of the fluorophore with the DNA, resulting in quenched fluorescence. However, in this study these concerns have been resolved with the use of a nucleotide analog with a 12-carbon linker arm, 5-amino (12)-2′-deoxyuridine β-cyanoethyl phosphoramidite. Steady-state fluorescence studies show that cleavage of the fluorogenic substrate by integrase results in enhancement of quenched donor fluorescence intensity. The fluorescence assay was confirmed by autoradiographic analysis of the cleavage reaction with radiolabeled fluorogenic substrate. This fluorescence assay will facilitate both detailed kinetic studies and the rapid screening of novel integrase inhibitors.

Processing of deoxyuridine mismatches and abasic sites by human immunodeficiency virus type-1 integrase.

We have examined the activities of HIV-1 integrase on substrates containing mismatches, composed of deoxyuridine at different positions in either the processed or nonprocessed strand of viral DNA, within and near the conserved CA dinucleotide of the U5 end of the HIV-1 LTR. Substitution in the processed strand of either the C or A of the CA dinucleotide or of the G 5' to the CA reduced strand transfer six-, three- and seven-fold respectively. 3'-processing was also reduced by substitution at the GC but not at the A. Substitution in the nonprocessed strand of the G nucleotide at the processing site abolished strand transfer while substitution of the T had no effect. DNA binding of HIV-1 integrase was not affected by deoxyuridine substitutions. Deoxyuridine substitution outside the trinucleotide remained compatible with enzyme activity. Enzymatically generated abasic sites were created at each mismatch to determine the effect of a missing base on integrase activity. Consistent with the deoxyuridine mismatch observations, 3'-processing and strand transfer were abolished when the abasic site was substituted for either of the nucleotides of the GCA trinucleotide. Integrase was, however, able to tolerate mismatches within this trinucleotide during the disintegration reaction. Taken together, these results suggest that base-mismatched or base-deleted substrates, which can be created by the proofreading-deficient HIV-1 RT, can be tolerated by HIV-1 integrase when located outside of the GCA trinucleotide at the U5 end of the LTR.

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

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.

Methylphosphonodiester substitution near the conserved CA dinucleotide in the HIV LTR alters both extent of 3'-processing and choice of nucleophile by HIV-1 integrase.

We present evidence suggesting that the 3'-processing activity of HIV-1 integrase is dramatically affected by electrostatic and/or steric perturbations 3' to the conserved CA dinucleotide. When the phosphodiester bond 3' to the scissile phosphodiester is replaced by a methylphosphonodiester linkage, 3'-processing decreases by two orders of magnitude. This block of cleavage can be somewhat overcome by increasing the pH of the reaction. Labeling of the substrates at the 3'-end revealed blockage of water and glycerol, but stimulation of the viral DNA 3'-hydroxyl, acting as the nucleophile with the methylphosphonodiester substrate. Interestingly, a circular trinucleotide was formed using the phosphodiester and methylphosphonodiester substrates when the terminal nucleotide was 3'-deoxyadenosine but not 2'-deoxyadenosine. Mutagenesis of the enzyme active site has previously been shown to alter the choice of nucleophile in the 3'-processing reaction. Taken together, the results in this study suggest that 'mutagenesis' of the DNA backbone can also alter the choice of nucleophile.

Erratum

AIDS Research and Human RetrovirusesVol. 8, No. 5 Erratumis erratum ofLocalization of DNA Binding Activity of HIV-1 Integrase to the C-Terminal Half of the ProteinPublished Online:16 Mar 2009https://doi.org/10.1089/aid.1992.8.669AboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail AbstractPlease note that in the article "Localization of the DNA Binding Activity of HIV-1 Integrase to the C-terminal Half of the Protein", by Amy M. Woerner, Michael Klutch, Judith G. Levin, and Carol J. Marcus-Sekura (AIDS Research and Human Retroviruses Volume 8, Number 2, pp 297-304), the illustrations for Figure 2 and Figure 5 were interchanged at a point between the page proofs and publication in the Journal. As published, the illustration for Figure 2 should be read with the legend for Figure 5, and the illustration for Figure 5 should be read with the legend for Figure 2. The corrected figures and legends are as shown below:FiguresReferencesRelatedDetailsRelated articlesLocalization of DNA Binding Activity of HIV-1 Integrase to the C-Terminal Half of the Protein16 Mar 2009AIDS Research and Human Retroviruses Volume 8Issue 5May 1992 To cite this article:Erratum.AIDS Research and Human Retroviruses.May 1992.669-669.http://doi.org/10.1089/aid.1992.8.669Published in Volume: 8 Issue 5: March 16, 2009PDF download