Rapid solution assays for retroviral integration reactions and their use in kinetic analyses of wild-type and mutant Rous sarcoma virus integrases.

Muller, B.; Jones, Kathryn; Merkel, George; Skalka, Anna Marie

doi:10.1073/pnas.90.24.11633

Cited by 22 publications

(20 citation statements)

References 23 publications

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“…We previously reported that the optimal pH for the full-length wild type ASV IN was ϳ9 (16). Using the IN catalytic core domain as well as the full-length wild type IN protein, we carried out a standard assay for IN processing and endonuclease activity.…”

Section: Conformation Of Asp/asn-64 -mentioning

confidence: 99%

Structural Basis for Inactivating Mutations and pH-dependent Activity of Avian Sarcoma Virus Integrase

Łubkowski¹,

Yang²,

Alexandratos³

et al. 1998

Journal of Biological Chemistry

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Crystallographic studies of the catalytic core domain of avian sarcoma virus integrase (ASV IN) have provided the most detailed picture so far of the active site of this enzyme, which belongs to an important class of targets for designing drugs against AIDS. Recently, crystals of an inactive D64N mutant were obtained under conditions identical to those used for the native enzyme. Data were collected at different pH values and in the presence of divalent cations. Data were also collected at low pH for the crystals of the native ASV IN core domain. In the structures of native ASV IN at pH 6.0 and below, as well as in all structures of the D64N mutants, the side chain of the active site residue Asx-64 (Asx denotes Asn or Asp) is rotated by ϳ150°around the C␣OC␤ bond, compared with the structures at higher pH. In the new structures, this residue makes hydrogen bonds with the amide group of Asn-160, and thus, the usual metal-binding site, consisting of Asp-64, Asp-121, and Glu-157, is disrupted. Surprisingly, however, a single Zn 2؉ can still bind to Asp-121 in the mutant, without restoration of the activity of the enzyme. These structures have elucidated an unexpected mechanism of inactivation of the enzyme by lowering the pH or by mutation, in which a protonated side chain of Asx-64 changes its orientation and interaction partner. Integrase (IN)1 (1) is one of only four enzymes encoded by retroviruses, such as human immunodeficiency virus type 1 and avian sarcoma virus (ASV), and it is absolutely essential for the support of the viral life cycle. For these reasons, IN is currently a target for the design of antiretroviral drugs. Retroviral INs contain approximately 300 amino acids, organized into three domains: zinc-binding N terminus, catalytic core domain, and DNA-binding C terminus. IN catalyzes the incorporation of the reverse-transcribed viral DNA into the host genome in two steps, processing and joining (1-3), both of which involve a nucleophilic attack by a hydroxyl group on a DNA phosphate. In the processing step, a water molecule attacks near the end of the viral DNA, two nucleotides from the 3Ј-ends of both viral DNA strands. In the joining step, the exposed viral DNA deoxyribose 3Ј-OH is activated to attack the host DNA at a relatively nonspecific location, thereby inserting viral DNA into the host genome. In vitro, these reactions require only virus-like DNA, IN, and metal cations.Although the isolated ASV IN catalytic core domain is defective for the processing and joining activities, it retains two activities: disintegration, the reverse of the joining step that uses a preformed DNA substrate, and an endonuclease activity that cleaves between the highly conserved C and A (Ϫ3 activity) at the termini of the viral DNA (CATT-3Ј) (4). The endonuclease activity is distinct from the normal processing activity of IN, which cleaves between the A and T (Ϫ2 activity). Both the endonuclease and disintegration activities of the catalytic domain are dependent on the D, D(35)E catalytic triad, corresponding to Asp-...

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Section: Conformation Of Asp/asn-64 -mentioning

confidence: 99%

Structural Basis for Inactivating Mutations and pH-dependent Activity of Avian Sarcoma Virus Integrase

Łubkowski¹,

Yang²,

Alexandratos³

et al. 1998

Journal of Biological Chemistry

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“…) INs (30,31). However, a much lower value for the processing rate (0.24 hr Ϫ1 ) was measured for HIV-1 IN using fluorescence resonance energy transfer (32).…”

mentioning

confidence: 99%

Mode of Interaction of G-Quartets with the Integrase of Human Immunodeficiency Virus Type 1

et al. 1997

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SUMMARYOligonucleotides that can form a highly stable intramolecular four-stranded DNA structure containing two stacked guanosine-quartets (G-quartets) have been reported to inhibit the replication of the human immunodeficiency virus type 1 (HIV-1) in cell culture. Two possible mechanisms for the observed antiviral activity have been proposed: interference with virus adsorption to the cell and/or inhibition of HIV-1 integrase. We investigated the molecular interaction of G-quartet-containing oligonucleotides with HIV-1 integrase in comparison with random oligonucleotides and dextran sulfate. The prototypical G-quartet-containing oligonucleotide, T30177 (Zintevir), inhibited the overall integration reaction with an IC 50 value of 80 nM. A random oligonucleotide was 10-fold less potent, but dextran sulfate was more potent, with an IC 50 value of 7 nM. We developed novel kinetic assays to dissect the overall integration reaction in three steps: the formation of the initial stable complex (ISC), the 3Ј-processing reaction, and the DNA strandtransfer step. We then analyzed the kinetics of the ISC formation and 3Ј-processing. The rate constant determined for the conversion of ISC into the cleaved product was 0.08 Ϯ 0.01 min Ϫ1. T30177 did not inhibit 3Ј-processing or DNA strand transfer, whereas dextran sulfate inhibited DNA strand transfer to some extent. Binding studies using surface plasmon resonance technology revealed that both T30177 and dextran sulfate were capable of preventing the binding of integrase to specific DNA. We propose a model in which the interaction of HIV-1 integrase with G-quartets results in the inhibition of the formation of the ISC between integrase and substrate DNA. Finally, we selected for an HIV-1 strain that was resistant to T30177 in cell culture. DNA sequence analysis revealed mutations in the envelope glycoprotein gp120 but not in the integrase gene. Although gp120 seems to be the main target for the antiviral activity in cell culture of G-quartets, the study of their specific inhibition of HIV-1 integrase may lead to the development of effective integrase inhibitors.For the treatment of infection with HIV-1, a number of inhibitors of both the reverse transcriptase and HIV protease have been formally approved (1). Targeting the third viral enzyme IN with effective inhibitors remains an elusive goal. The IN enzyme inserts the viral cDNA copy into the host cell chromosome; this step is essential for the replication of the virus (2, 3). Because no human counterpart of the enzyme is known, there is considerable interest in developing effective and selective inhibitors of the HIV integration process. The recent establishment of high-throughput microtiter plate assays (4, 5), on the one hand, and the elucidation of the three-dimensional structure of the catalytic domain of HIV-1 IN, on the other hand (6), will likely boost the antiviral screening of chemical libraries as well as the structure-based design of enzyme inhibitors.The only enzyme required for HIV-1 integration is IN, a protein o...

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“…The initial exponential phase represents the actual reactivity of the enzyme with respect to both binding specificity and catalytic rate. In addition, the initial exponential phase arises as a direct consequence of a slow post-chemistry step in the reaction mechanism and generally reflects a slow rate-limiting product release step (35). In these instances, a faster steadystate turnover typically reflects only a faster rate of product release (21, 36 -39).…”

Section: Presteady-state Analysis Of a Novel Splicing Reaction-mentioning

confidence: 99%

Presteady-state Analysis of Avian Sarcoma Virus Integrase

Bao

Skalka

Wong

2002

Journal of Biological Chemistry

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Integrase catalyzes insertion of a retroviral genome into the host chromosome. After reverse transcription, integrase binds specifically to the ends of the duplex retroviral DNA, endonucleolytically cleaves two nucleotides from each 3-end (the processing activity), and inserts these ends into the host DNA (the joining activity) in a concerted manner. In first-turnover experiments with synapsed DNA substrates, we observed a novel splicing activity that resembles an integrase joining reaction but uses unprocessed ends. This splicing reaction showed an initial exponential phase (k splicing ‫؍‬ 0.02 s ؊1 ) of product formation and generated products macroscopically indistinguishable from those created by the processing and joining activities, thus bringing into question methods previously used to quantitate these reactions in a time regime where multiple turnovers of the enzyme have occurred. With a presteady-state assay, however, we were able to distinguish between different pathways that led to formation of identical products. Furthermore, the splicing reaction allowed characterization of substrate binding and specificity. Although integrase requires only a 3 hydroxyl with respect to nucleophiles derived from DNA, it specifically favors the cognate sequence CATT as the electrophile. These experimental results support a two-site "switching" model for binding and catalysis of all three integrase activities.After infection, retroviruses create a linear DNA copy of their RNA genome that, through the strand-transfer mechanism of reverse transcriptase, places the U3 region of the LTR sequence at one terminus and the U5 region of the LTR sequence at the other terminus (1). Retroviral replication is dependent on the viral protein integrase catalyzing the recombination of the viral DNA genome into the host genomic DNA. Integrase binds to the two blunt-ended viral LTRs, hydrolyzes the terminal two nucleotides to expose a recessed 3Ј-OH of the conserved CA dinucleotide at each of the ends (the processing activity), and inserts these "processed" ends into the host DNA (the joining activity) at sites separated by a virus-specific stagger of six base pairs for avian sarcoma virus (ASV).1 The location of the insertion is nearly random as there is little sequence specificity for the site of recombination within the host genome (2-4). The processing and joining activities are biochemically similar in that both use a hydroxyl group as the nucleophile in an endonucleolytic cleavage. In the case of the processing activity (3Ј-dinucleotide removal), the enzyme is specific in its choice of electrophile (the cognate CATT), whereas the nucleophile (the processed CA-OH) is specified in the joining reaction (strand transfer). Both the ends-processing and joining activities have been reproduced in vitro with purified recombinant integrase and oligonucleotides whose sequences are derived from the retroviral U3 and U5 LTR sequences (5-7). Detailed examination of the processing activity in vitro has revealed that integrase requires the physiol...

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Rapid solution assays for retroviral integration reactions and their use in kinetic analyses of wild-type and mutant Rous sarcoma virus integrases.

Cited by 22 publications

References 23 publications

Structural Basis for Inactivating Mutations and pH-dependent Activity of Avian Sarcoma Virus Integrase

Structural Basis for Inactivating Mutations and pH-dependent Activity of Avian Sarcoma Virus Integrase

Mode of Interaction of G-Quartets with the Integrase of Human Immunodeficiency Virus Type 1

Presteady-state Analysis of Avian Sarcoma Virus Integrase

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