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Nucleic Acids Research, 1991, Vol. 19, No. 10 2729-2734
© 1991

MOLECULAR BIOLOGY

A rapid in vitro assay for HIV DNA integration

Robert Craigie, kiyoshi Mizuuchi, Frederic D. Bushman and Alan Engelman

Laboratory of Molecular Biology, National Institute of Diabetes, and Digestive and Kidney Diseases, National Institutes of Health Bethesda, MD 20892, USA

Received January 11, 1991. Revised March 29, 1991. Accepted March 29, 1991.

Retroviruses synthesize a double stranded DNA copy of their RNA genome after Infection of a permissive cell and subsequent integration of this DNA copy into thehost genome is necessary for normal viral replication. Integration occurs by a specialized DNA recombination reaction, mediated by the viral IN protein. Because this reaction has no known cellular counterpart, it is a particularly attractive target in the search for specific inhibitors with low toxicity that may serve as therapeutic antiviral agents. We present a simple assay system that is suitable for screening potential inhibitors of HIV DNA integration. Only short oligonucleotides matching one end of HIV DNA and purified HIV IN protein are required as substrates. Furthermore, since each step of the assay can be carried out in the wells of microtiter plates, large numbers of reactions can be processed simultaneously.


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