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J Virol, June 1998, p. 5046-5055, Vol. 72, No. 6
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Effects of Mutations in Residues near the Active Site of Human Immunodeficiency Virus Type 1 Integrase on Specific Enzyme-Substrate Interactions

Jennifer L. Gerton,1,dagger Sharron Ohgi,2 Mari Olsen,2 Joseph DeRisi,3 and Patrick O. Brown2,3,*

Howard Hughes Medical Institute,2 Department of Biochemistry,3 and Department of Microbiology and Immunology,1 Stanford University Medical Center, Stanford, California 94305-5428

Received 27 June 1997/Accepted 16 February 1998

The phylogenetically conserved catalytic core domain of human immunodeficiency virus type 1 (HIV-1) integrase contains elements necessary for specific recognition of viral and target DNA features. In order to identify specific amino acids that determine substrate specificity, we mutagenized phylogenetically conserved residues that were located in close proximity to the active-site residues in the crystal structure of the isolated catalytic core domain of HIV-1 integrase. Residues composing the phylogenetically conserved DD(35)E active-site motif were also mutagenized. Purified mutant proteins were evaluated for their ability to recognize the phylogenetically conserved CA/TG base pairs near the viral DNA ends and the unpaired dinucleotide at the 5' end of the viral DNA, using disintegration substrates. Our findings suggest that specificity for the conserved A/T base pair depends on the active-site residue E152. The phenotype of IN(Q148L) suggested that Q148 may be involved in interactions with the 5' dinucleotide of the viral DNA end. The activities of some of the proteins with mutations in residues in close proximity to the active-site aspartic and glutamic acids were salt sensitive, suggesting that these mutations disrupted interactions with DNA.

* Corresponding author. Mailing address: B253 Beckman Center, Stanford University Medical Center, Stanford, CA 94305-5428. Phone: (650) 723-0039. Fax: (650) 723-1399. E-mail: pbrown{at}cmgm.stanford.edu.

dagger Present address: Department of Biology, University of North Carolina, Chapel Hill, NC 27599-3280.

J Virol, June 1998, p. 5046-5055, Vol. 72, No. 6
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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