Characterization of human immunodeficiency virus type 1 integrase mutants expressed in Escherichia coli.
The eight mutant integrase (IN) proteins of human immunodeficiency virus type (HIV-1), which have a single point mutation at a highly conserved central region, were prepared, and characterized in terms of their endonucleolytic activities and disintegration activities in vitro. Mutation of two highly conserved amino acids, Asp116 or Glu152, leads to complete loss of both the activities, suggesting that these two amino acids are directly associated with enzymatic functions. In addition, the mutant of the position Ser147 was found to have highly depressed endonucleolytic activity showing that the reaction was very delayed in comparison with that of the wild type. However, significant disintegration was detected in the mutant Ser147, indicating that the enzymatic mechanisms of the endonucleolytic and disintegration activities are not exactly reverse. The integrase protein with a mutation at the conserved amino acid Asn117 or Gly118 had a slight loss of the endonucleolytic activity, while a mutation at the three positions, Tyr143, Ser153, and Lys159, had no detectable effect on their enzymatic activities. These results indicate that only a few of the conserved amino acids are critical for enzymatic activities.
Oh
J W
JW
Department of Biotechnology, Chung-Ang University, Ansung, Korea.
Oh
Y T
YT
Kim
D J
DJ
Shin
C G
CG
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000821:virology; D005822:Genetic Vectors; D019427:HIV Integrase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D016662:Virus Integration / Q000235:genetics
D019427:HIV Integrase; D004706:Endodeoxyribonucleases; C058608:endonuclease-integrase