Mutational analysis of the integrase protein of human immunodeficiency virus type 2.
Purified integrase protein (IN) can nick linear viral DNA at a specific site near the ends and integrate nicked viral DNA into target DNA. We have made a series of 43 site-directed point mutants of human immunodeficiency virus type 2 IN and assayed purified mutant proteins for the following activities: site-specific cleavage of viral DNA (donor cut), integration (strand transfer), and disintegration. In general, the different activities were similarly affected by the mutations. We found three mutations that (almost) totally abolished IN function: Asp-64-->Val, Asp-116-->Ile, and Glu-152-->Leu, whereas 25 mutations did not affect IN function. A few mutations affected the different activities differentially. Near the amino terminus a zinc finger-like sequence motif His-Xaa3-His-Xaa20-30-Cys-Xaa2-Cys is present in all retroviral IN proteins. Two mutations in this region (His-12-->Leu and Cys-40-->Ser) strongly inhibited donor cut but had less effect on strand transfer. The central region of IN is most highly conserved between retroviral INs. Three mutants in this region (Asn-117-->Ile, Asn-120-->Leu, and Lys-159-->Val) were inhibited in strand transfer but were inhibited less strongly in donor cut. Mutation of Asn-120 (to glycine, leucine, or glutamate) resulted in changes in integration-site preference, suggesting that Asn-120 is involved in interactions with target DNA. We did not find a mutant in which one activity was lost and the others were unaffected, supporting the notion that IN has only one active site for the catalysis of donor cut and strand transfer.
van Gent
D C
DC
Netherlands Cancer Institute, Division of Molecular Biology, Amsterdam.
Groeneger
A A
AA
Plasterk
R H
RH
D001483:Base Sequence; D001665:Binding Sites; D004254:DNA Nucleotidyltransferases / Q000737:chemistry / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D015498:HIV-2 / Q000201:enzymology*; D019426:Integrases; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D004254:DNA Nucleotidyltransferases; D019426:Integrases; D004706:Endodeoxyribonucleases