MODE OF INTERACTION OF G-QUARTETS WITH THE INTEGRASE OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1

Oligonucleotides that can form a highly stable intramolecular four-str anded DNA structure containing two stacked guanosine-quartets (G-quart ets) have been reported to inhibit the replication of the human immuno deficiency virus type 1 (HIV-1) in cell culture. Two possible mechanis ms for the observed antiviral activity have been proposed: interferenc e with virus adsorption to the cell and/or inhibition of HIV-1 integra se. We investigated the molecular interaction of G-quartet-containing oligonucleotides with HIV-1 integrase in comparison with random oligon ucleotides and dextran sulfate. The prototypical G-quartet-containing oligonucleotide, T30177 (Zintevir), inhibited the overall integration reaction with an IC50 value of 80 nM. A random oligonucleotide was 10- fold less potent, but dextran sulfate was more potent, with an IC50 va lue of 7 nM. We developed novel kinetic assays to dissect the overall integration reaction in three steps: the formation of the initial stab le complex (ISC), the 3'-processing reaction, and the DNA strand-trans fer step. We then analyzed the kinetics of the ISC formation and 3'-pr ocessing. The rate constant determined for the conversion of ISC into the cleaved product was 0.08 +/- 0.01 min(-1). T30177 did not inhibit 3'-processing or DNA strand transfer, whereas dextran sulfate inhibite d DNA strand transfer to some extent. Binding studies using surface pl asmon resonance technology revealed that both T30177 and dextran sulfa te 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 betwe en integrase and substrate DNA. Finally, we selected for an HIV-1 stra in 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 ant iviral activity in cell culture of G-quartets, the study of their spec ific inhibition of HIV-1 integrase may lead to the development of effe ctive integrase inhibitors.