Characterization of mutant HIV-1 integrase carrying amino acid changes in the catalytic domain.
To gain insight into the importance of conserved residues in the core domain of HIV-1 IN, we performed site-directed mutagenesis of the full-length enzyme, overexpressed the mutant proteins in E. coli, purified and analyzed their 3'-processing, integration and disintegration activities in vitro. Change of E152V in the DD(35)E motif abolished all detectable activities of IN. Alteration of two highly conserved residues, P145 and K156, by isoleucine, resulted in a substantial loss or completely abolished the three activities of the enzyme. Mutant P90D weakly reduced the 3'-processing but severely affected the two other IN activities. Results obtained from double and triple mutations, P90D/P1451 and P145I/F185K/C280S, clearly suggest a crucial role of P145 in the catalytic function of IN, whereas the mutants V150E, L158F and L172M had no detectable effect on any of the IN activities. Taken together, these results allowed us to conclude that all the conserved amino acids in the core domain of IN are not equally important for catalytic functions: like D64, D116 and E152, our data suggest that P90, P145 and K156 are also essential for all three enzymatic activities of HIV-1 IN in vitro, whereas V150, L158 and L172 appear to be less critical.
Sayasith
K
K
Armand-frappier Institute, Virology Research Center, Laval, Quebéc, Canada. sayasik@yahoo.ca
Sauvé
G
G
Yelle
J
J
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004926:Escherichia coli; D019427:HIV Integrase / Q000737:chemistry* / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D019427:HIV Integrase