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J Virol, May 1998, p. 3916-3924, Vol. 72, No. 5
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Mutational Scan of the Human Immunodeficiency Virus Type 2 Integrase Protein

Fusinita M. I. van den Ent, Arnold Vos, and Ronald H. A. Plasterk*

Division of Molecular Biology, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands

Received 7 November 1997/Accepted 1 February 1998

Retroviral integrase (IN) cleaves linear viral DNA specifically near the ends of the DNA (cleavage reaction) and subsequently couples the processed ends to phosphates in the target DNA (integration reaction). In vitro, IN catalyzes the disintegration reaction, which is the reverse of the integration reaction. Ideally, we would like to test the role of each amino acid in the IN protein. We mutagenized human immunodeficiency virus type 2 IN in a random way using PCR mutagenesis and generated a set of mutants in which 35% of all residues were substituted. Mutant proteins were tested for in vitro activity, e.g., site-specific cleavage of viral DNA, integration, and disintegration. Changes in 61 of the 90 proteins investigated showed no phenotypic effect. Substitutions that changed the choice of nucleophile in the cleavage reaction were found. These clustered around the active-site residues Asp-116 and Glu-152. We also found alterations of amino acids that affected cleavage and integration differentially. In addition, we analyzed the disintegration activity of the proteins and found substitutions of amino acids close to the dimer interface that enhanced intermolecular disintegration activity, whereas other catalytic activities were present at wild-type levels. This study shows the feasibility of investigating the role of virtually any amino acid in a protein the size of IN.

* Corresponding author. Mailing address: Division of Molecular Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. Phone: 31-20-5122081. Fax: 31-20-5122086. E-mail: rplas{at}nki.nl.

J Virol, May 1998, p. 3916-3924, Vol. 72, No. 5
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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