Identification of amino acid residues critical for endonuclease and integration activities of HIV-1 IN protein in vitro.
HIV-IN protein, tagged with a hexahistidine tail was expressed in Escherichia coli and purified by a one-step nickel chelate affinity chromatography procedure. The purified IN protein was characterized in terms of its endonuclease and integrase properties in vitro. Specific cleavage and integration of HIV U5 LTR ends were observed in the presence of 2-5 mM Mg2+ or Ca2+. In the presence of 2 mM Mn2+, cleavage and integration occurred at additional sites indicating a decreased specificity. The properties of mutant IN proteins were examined in vitro. Deletion of 39 amino acids from the N-terminus and a minimum of 25 residues from the C-terminus impaired IN-mediated cleavage and integration activities. The results of site-directed mutagenesis experiments showed that residues critical for IN function are highly conserved. Mutations of conserved residues Asp64, Pro109, Asp116, and Glu152 adversely affected IN function in vitro. Mutations of nonconserved residues Gly189 and Thr112 had no effect. Mutation of a conserved Thr115 to Ala caused a near complete loss of Mg(2+)-dependent integration activity, but only partially effected endonucleolytic cleavage activity of IN. These results suggest that not all conserved residues are involved in both endonucleolytic cleavage and integration activities of HIV-IN.
Drelich
M
M
Department of Biology, F. Hoffmann-LaRoche, Ltd., Basel, Switzerland.
Wilhelm
R
R
Mous
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D004252:DNA Mutational Analysis; D004254:DNA Nucleotidyltransferases / Q000737:chemistry*; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology*; D019426:Integrases; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D016662:Virus Integration
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D004254:DNA Nucleotidyltransferases; D019426:Integrases; D004706:Endodeoxyribonucleases