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...

Pregastric esterase (PGE) (EC 3.1.1.3) was purified to homogeneity from calf pharyngeal tissue. The enzyme had an apparent molecular mass of 50 kDa, as determined by SDS/PAGE. The serine-binding reagent diethyl p-nitrophenyl phosphate was a potent inhibitor of PGE. This is in accordance with the claim that a functional serine residue is necessary for the lipolytic activity of lipases. PGE was not inhibited by the thiol reagents 5,5'-dithiobis(2-nitrobenzoic acid) or 4,4'-dithiopyridine. A partial inhibition with dodecyldithio-5-(2-nitrobenzoic acid) was observed, but the same degree of inhibition was caused by the non-esterified fatty acid C(12:0). PGE shows a great sequence similarity to gastric lipases. Gastric lipases have three cysteine residues, and two of these form a disulphide bridge. Blocking the remaining free cysteine with thiol reagents inactivates the gastric lipases. The fact that PGE is not inhibited by thiol reagents indicates that PGE has no functional free thiol group. The PGE cDNA codes only for two cysteines, and their involvement in the formation of a disulphide bridge was demonstrated.