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Journal of Virology, November 2007, p. 12189-12199, Vol. 81, No. 22
0022-538X/07/$08.00+0     doi:10.1128/JVI.02863-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Inhibition of Human Immunodeficiency Virus Type 1 Concerted Integration by Strand Transfer Inhibitors Which Recognize a Transient Structural Intermediate

Krishan K. Pandey,^1, Sibes Bera,^1, Jacob Zahm,^1, Ajaykumar Vora,^1, Kara Stillmock,² Daria Hazuda,² and Duane P. Grandgenett^1*

Institute for Molecular Virology, Saint Louis University Health Sciences Center, St. Louis, Missouri 63110,¹ Department of Antiviral Research, Merck Research Laboratories, West Point, Pennsylvania 19466²

Received 27 December 2006/ Accepted 24 August 2007

Human immunodeficiency virus type 1 (HIV-1) integrase (IN) inserts the viral DNA genome into host chromosomes. Here, by native agarose gel electrophoresis, using recombinant IN with a blunt-ended viral DNA substrate, we identified the synaptic complex (SC), a transient early intermediate in the integration pathway. The SC consists of two donor ends juxtaposed by IN noncovalently. The DNA ends within the SC were minimally processed (15%). In a time-dependent manner, the SC associated with target DNA and progressed to the strand transfer complex (STC), the nucleoprotein product of concerted integration. In the STC, the two viral DNA ends are covalently attached to target and remain associated with IN. The diketo acid inhibitors and their analogs effectively inhibit HIV-1 replication by preventing integration in vivo. Strand transfer inhibitors L-870,810, L-870,812, and L-841,411, at low nM concentrations, effectively inhibited the concerted integration of viral DNA donor in vitro. The inhibitors, in a concentration-dependent manner, bound to IN within the SC and thereby blocked the docking onto target DNA, which thus prevented the formation of the STC. Although 3'-OH recessed donor efficiently formed the STC, reactions proceeding with this substrate exhibited marked resistance to the presence of inhibitor, requiring significantly higher concentrations for effective inhibition of all strand transfer products. These results suggest that binding of inhibitor to the SC occurs prior to, during, or immediately after 3'-OH processing. It follows that the IN-viral DNA complex is "trapped" by the strand transfer inhibitors via a transient intermediate within the cytoplasmic preintegration complex.

Published ahead of print on 5 September 2007.

K.K.P., S.B., J.Z. and A.V. made equal contributions to this study.

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• Zahm, J. A., Bera, S., Pandey, K. K., Vora, A., Stillmock, K., Hazuda, D., Grandgenett, D. P. (2008). Mechanisms of Human Immunodeficiency Virus Type 1 Concerted Integration Related to Strand Transfer Inhibition and Drug Resistance. Antimicrob. Agents Chemother. 52: 3358-3368 [Abstract] [Full Text]