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Journal of Virology, August 2001, p. 7756-7762, Vol. 75, No. 16
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.16.7756-7762.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Use of Patient-Derived Human Immunodeficiency Virus Type 1 Integrases To Identify a Protein Residue That Affects Target Site Selection

Amy L. Harper,1 Lynn M. Skinner,2 Malgorzata Sudol,2 and Michael Katzman1,2,*

Department of Microbiology and Immunology1 and Department of Medicine,2 Pennsylvania State University College of Medicine, The Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033

Received 13 December 2000/Accepted 21 May 2001

To identify parts of retroviral integrase that interact with cellular DNA, we tested patient-derived human immunodeficiency virus type 1 (HIV-1) integrases for alterations in the choice of nonviral target DNA sites. This strategy took advantage of the genetic diversity of HIV-1, which provided 75 integrase variants that differed by a small number of amino acids. Moreover, our hypothesis that biological pressures on the choice of nonviral sites would be minimal was validated when most of the proteins that catalyzed DNA joining exhibited altered target site preferences. Comparison of the sequences of proteins with the same preferences then guided mutagenesis of a laboratory integrase. The results showed that single amino acid substitutions at one particular residue yielded the same target site patterns as naturally occurring integrases that included these substitutions. Similar results were found with DNA joining reactions conducted with Mn2+ or with Mg2+ and were confirmed with a nonspecific alcoholysis assay. Other amino acid changes at this position also affected target site preferences. Thus, this novel approach has identified a residue in the central domain of HIV-1 integrase that interacts with or influences interactions with cellular DNA. The data also support a model in which integrase has distinct sites for viral and cellular DNA.

* Corresponding author. Mailing address: Department of Medicine, Division of Infectious Diseases, Pennsylvania State University College of Medicine, The Milton S. Hershey Medical Center, P.O. Box 850, Mail Services H036, Hershey, PA 17033-0850. Phone: (717) 531-8881. Fax: (717) 531-4633. E-mail: mkatzman{at}psu.edu.

Journal of Virology, August 2001, p. 7756-7762, Vol. 75, No. 16
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.16.7756-7762.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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