Bushman F, Mizuuchi K, Engelman A, Craigie R; International Conference on AIDS.

Int Conf AIDS. 1991 Jun 16-21; 7: 24 (abstract no. W.A.11).

National Institutes of Health, Bethesda, MD, USA

OBJECTIVE: Our goal is to facilitate the development of therapeutic agents that block the integration of reverse transcribed HIV DNA into host DNA, a step in the viral life cycle that is known to be required for replication. Here we present an in vitro integration system that is suitable for rapid screening of potential inhibitors. METHODS: The in vitro integration reaction is quite simple, containing only the HIV-encoded integration protein (IN) and a labeled DNA oligonucleotide matching the ends of the unintegrated HIV DNA, in a simple reaction mix. We have prepared IN protein by overexpressing the IN coding region in E. coli or baculoviral expression systems, and purified active IN from these sources. Oligonucleotide DNA molecules also serve as the integration target. To facilitate rapid screening of candidate inhibitors, we have incorporated biotin into the target oligonucleotide; integration of a labeled substrate oligonucleotide into the biotinylated target DNA yields a biotinylated and labeled product DNA strand that can be recovered on avidin-coated microtiter plates and readily quantitated. RESULTS: Quantitation of integration reactions using the biotin-avidin method consistently yields results that match the results of integration assays using previously established physical methods. Recovery of biotinylated products on avidin-coated plates is highly selective and efficient. CONCLUSIONS: The integration step is a particularly attractive point in the HIV life cycle for therapeutic intervention: the HIV integration reaction has no known cellular analogs, and so some inhibitors may exhibit relatively low toxicity. Because the assay described here can be carried out entirely in microtiter plates, this assay offers the possibility of partial automation of the inhibitor screening process.

Publication Types:

• Meeting Abstracts

Keywords:

• AIDS Vaccines
• Anti-HIV Agents
• Biological Assay
• Biotin
• Case-Control Studies
• DNA
• DNA Nucleotidyltransferases
• HIV
• HIV Integrase
• HIV Integrase Inhibitors
• HIV Long Terminal Repeat
• In Vitro
• Oligonucleotides
• analysis
• genetics
• methods
• reverse transcriptase, Human immunodeficiency virus 1

Other ID:

• 3001191

UI: 102195971

From Meeting Abstracts