Specific interactions between retroviral integrase (IN) and long terminal repeats are required for insertion of viral DNA into the host genome. To characterize quantitatively the determinants of substrate specificity, we used a method based on a stepwise increase in ligand complexity. This allowed an estimation of the relative contributions of each nucleotide from oligonucleotides to the total affinity for IN. The interaction of HIV-1 integrase with specific (containing sequences from the LTR) or nonspecific oligonucleotides was analyzed using a thermodynamic model. Integrase interacted with oligonucleotides through a superposition of weak contacts with their bases, and more importantly, with the internucleotide phosphate groups. All these structural components contributed in a combined way to the free energy of binding with the major contribution made by the conserved 3'-terminal GT, and after its removal, by the CA dinucleotide. In contrast to nonspecific oligonucleotides that inhibited the reaction catalyzed by IN, specific oligonucleotides enhanced the activity, probably owing to the effect of sequence-specific ligands on the dynamic equilibrium between the oligomeric forms of IN. However, after preactivation of IN by incubation with Mn(2+), the specific oligonucleotides were also able to inhibit the processing reaction. We found that nonspecific interactions of IN with DNA provide approximately 8 orders of magnitude in the affinity (Delta G degrees approximately equal to -10.3 kcal/mol), while the relative contribution of specific nucleotides of the substrate corresponds to approximately 1.5 orders of magnitude (Delta G degrees approximately equal to - 2.0 kcal/mol). Formation of the Michaelis complex between IN and specific DNA cannot by itself account for the major contribution of enzyme specificity, which lies in the k(cat) term; the rate is increased by more than 5 orders of magnitude upon transition from nonspecific to specific oligonucleotides.
The DNA polymerase of the human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) is a target widely used to inhibit HIV-1 replication. In contrast, very few inhibitors of the RNase H activity associated with RT have been described, despite the crucial role played by this activity in viral proliferation. DNA ligands with a high affinity for the RNase H domain of HIV-1 RT were isolated by systematic evolution of ligands by an exponential enrichment strategy (SELEX), using recombinant RTs with or without the RNase H domain. The selected oligonucleotides (ODNs) were able to inhibit in vitro the HIV-1 RNase H activity, while no effect was observed on cellular RNase H. We focused our interest on two G-rich inhibitory oligonucleotides. Model studies of the secondary structure of these ODNs strongly suggested that they were able to form G-quartets. In addition to the inhibition of HIV-1 RNase H observed in a cell free system, these ODNs were able to strongly diminish the infectivity of HIV-1 in human infected cells. Oligonucleotides described here may serve as leading compounds for the development of specific inhibitors of this key retroviral enzyme activity.
Retroviral integrase (IN) catalyzes the integration of double-stranded viral DNA into the host cell genome. The reaction can be divided in two steps: 3P P-end processing and DNA strand transfer. Here we studied the effect of short oligonucleotides (ODNs) on human immunodeficiency virus type 1 (HIV-1) IN. ODNs were either specific, with sequences representing the extreme termini of the viral long terminal repeats, or nonspecific. All ODNs were found to competitively inhibit the processing reaction with K i values in the nM range for the best inhibitors. Our studies on the interaction of IN with ODNs also showed that: (i) besides the 3P P-terminal GT, the interaction of IN with the remaining nucleotides of the 21-mer specific sequence was also important for an effective interaction of the enzyme with the substrate; (ii) in the presence of specific ODNs the activity of the enzyme was enhanced, a result which suggests an ODNinduced conformational change of HIV-1 IN.z 1999 Federation of European Biochemical Societies.
4-(Arylthio)-pyridin-2(1H)-ones variously substituted in their 3-, 5-, and 6-positions have been synthesized as a new series of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT)-pyridinone hybrid molecules. Biological studies revealed that some of them show potent HIV-1 specific reverse transcriptase inhibitory properties. Compounds 16 and 7c, the most active ones, inhibit the replication of HIV-1 at 3 and 6 nM, respectively.
(1987) BiolTechnology 5,486-4891 has been substantially modified, leading to an increased yield and a higher degree of purity. Several biochemical properties of the enzyme are described (template specificity, effect of DNA synthesis inhibitors); interestingly, HIV reverse transcriptase is highly resistant to N-ethylmaleimide. A complex between the human retroviral enzyme and the bovine tRNALyS was shown, using a direct approach, by glycerol gradient centrifugation, as well as by the protective and specific effect of the tRNALyS against enzyme inactivation by thermal denaturation and trypsin digestion. A competitive type of inhibition of HIV reverse transcriptase by tRNALys, but not by tRNAVa', is observed when viral RNA or activated DNA are used as templates.Human immunodeficiency virus (HIV) is widely recognized as the etiological agent of the acquired immunodeficiency syndrome [l, 21. This retrovirus encodes an RNAdependent DNA polymerase or reverse transcriptase which copies its genomic RNA into complementary DNA using a lysine-specific tRNA as primer (tRNAiY5) [3]. Retroviral-directed DNA synthesis is initiated from the 3'hydroxyl of the terminal adenosine of the primer tRNA.Experimental evidence has accumulated over the last ten years, mainly in the avian retroviral system, indicating that avian reverse transcriptase forms a stable and specific complex with primer tRNATrp [4-81. In our laboratory we have studied the topography of the complex between avian myeloblastosis virus (AMV) reverse transcriptase and beef tRNATrp, as well as the role of the AMV reverse transcriptase in the annealing of primer tRNA to the AMV genome after partial unwinding of primer tRNA [9, lo]. Moreover, we showed that AMV reverse transcriptase was able to deacylate tryptophanyl-tRNA, thus increasing the number of free 3'-OH-tRNA termini able to initiate cDNA synthesis [ l l ] (for a Correspondence zo L.
Removal of 3-azido-3deoxythymidine (AZT) 3-azido-3-deoxythymidine 5-monophosphate (AZTMP) from the terminated primer mediated by the human HIV-1 reverse transcriptase (RT) has been proposed as a relevant mechanism for the resistance of HIV to AZT. Here we compared wild type and AZT-resistant (D67N/K70R/ T215Y/K219Q) RTs for their ability to unblock the AZTMP-terminated primer by phosphorolysis in the presence of physiological concentrations of pyrophosphate or ATP. The AZT-resistant enzyme, as it has been previously described, showed an increased ability to unblock the AZTMP-terminated primer by an ATP-dependent mechanism. We found that only mutations in the p66 subunit were responsible for this ability. We also found that three structurally divergent non-nucleoside reverse transcriptase inhibitor (NNRTI), nevirapine, TIBO, and a 4-arylmethylpyridinone derivative, were able to inhibit the phosphorolytic activity of the enzyme, rendering the AZT-resistant RT sensitive to AZTTP. The 4-arylmethylpyridinone derivative proved to be about 1000-fold more potent in inhibiting phosphorolysis than nevirapine or TIBO. Moreover, combinations of AZTTP with NNRTIs exhibited an exceptionally high degree of synergy in the inhibition of AZT-resistant enzyme only when ATP or PP i were present, indicating that inhibition of phosphorolysis was responsible for the synergy found in the combination. Our results not only demonstrate the importance of phosphorolysis concerning HIV-1 RT resistance to AZT but also point to the implication of this activity in the strong synergy found in some combinations of NNRTIs with AZT.Human immunodeficiency virus, type 1 (HIV-1) 1 reverse transcriptase (RT) is responsible for the conversion of singlestranded viral RNA into double-stranded DNA prior to integration into the genome of the human host. Numerous compounds that inhibit the DNA polymerase activity of RT have been described. They can be divided into two broad classes. The first group, that of nucleoside analogs, includes dideoxynucleoside compounds, such as 2Ј,3Ј-dideoxycytidine and AZT, that inhibit viral replication by acting as chain terminators of DNA synthesis (1). The second group, the non-nucleoside reverse transcriptase inhibitors (NNRTI), includes a large number of structurally dissimilar hydrophobic compounds that bind to a site on the RT palm subdomain adjacent to but distinct from the polymerase active site (2).The FDA-approved HIV-1 therapies involve drugs that inhibit two viral enzymes, reverse transcriptase and protease. AZT was the first drug approved against HIV-1 and is still widely used in combination with other antiretroviral drugs. The prolonged clinical use of this nucleoside analog in monotherapy gives rise to highly resistant viruses containing mutations in the RT enzyme, D67N, K70R, T215F/Y, K219E/Q (3), and, in some cases, M41L and L210W. Viruses carrying at least four mutations are more than 100-fold less sensitive to AZT than wild type virus in cell culture. Although the genotype for AZT resistance is well character...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
334 Leonard St
Brooklyn, NY 11211
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.