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Journal of Virology, July 2004, p. 6715-6722, Vol. 78, No. 13
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.13.6715-6722.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Metal Binding by the D,DX35E Motif of Human Immunodeficiency Virus Type 1 Integrase: Selective Rescue of Cys Substitutions by Mn2+ In Vitro

Kui Gao, Steven Wong, and Frederic Bushman*

Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6076

Received 7 November 2003/ Accepted 5 March 2004

The D,DX35E motif characteristic of retroviral integrase enzymes (INs) is expected to bind the required metal cofactors (Mg2+ or Mn2+), but direct evidence for a catalytic role has been lacking. Here we used a metal rescue strategy to investigate metal binding. We established conditions for analysis of an activity of IN, disintegration, in both Mg2+ and Mn2+, and tested IN mutants with cysteine substitutions in each acidic residue of the D,DX35E motif. Mn2+ but not Mg2+ can bind tightly to Cys, so if metal binding at the acidic residues is mechanistically important, it is expected that the Cys-substituted enzymes would be active in the presence of Mn2+ only. Of the three acidic residues, a strong metal rescue effect was obtained for D116C, a weaker rescue was seen for D64C, and no rescue was seen with E152C. Modest rescue could also be detected for D116C in normal integration in vitro. Comparison to Ser and Ala substitutions at D116 established that the rescue was selective for Cys. Further studies of the response to pH suggest that the metal cofactor may stabilize the deprotonated nucleophile active in catalysis, and studies of the response to NaCl titrations disclose an additional role for the metal cofactor in stabilizing the IN-DNA complex.

* Corresponding author. Mailing address: University of Pennsylvania School of Medicine, Department of Microbiology, 3610 Hamilton Walk, Philadelphia, PA 19104-6076. Phone: (215) 573-8732. Fax: (215) 573-4856. E-mail: bushman{at}mail.med.upenn.edu.

Journal of Virology, July 2004, p. 6715-6722, Vol. 78, No. 13
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.13.6715-6722.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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