Gerton J L, Ohgi S, Olsen M, DeRisi J, Brown P O
Department of Microbiology and Immunology, Stanford University Medical Center, Stanford, California 94305-5428, USA.
J Virol. 1998 Jun;72(6):5046-55. doi: 10.1128/JVI.72.6.5046-5055.1998.
The phylogenetically conserved catalytic core domain of human immunodeficiency virus type 1 (HIV-1) integrase contains elements necessary for specific recognition of viral and target DNA features. In order to identify specific amino acids that determine substrate specificity, we mutagenized phylogenetically conserved residues that were located in close proximity to the active-site residues in the crystal structure of the isolated catalytic core domain of HIV-1 integrase. Residues composing the phylogenetically conserved DD(35)E active-site motif were also mutagenized. Purified mutant proteins were evaluated for their ability to recognize the phylogenetically conserved CA/TG base pairs near the viral DNA ends and the unpaired dinucleotide at the 5' end of the viral DNA, using disintegration substrates. Our findings suggest that specificity for the conserved A/T base pair depends on the active-site residue E152. The phenotype of IN(Q148L) suggested that Q148 may be involved in interactions with the 5' dinucleotide of the viral DNA end. The activities of some of the proteins with mutations in residues in close proximity to the active-site aspartic and glutamic acids were salt sensitive, suggesting that these mutations disrupted interactions with DNA.
人类免疫缺陷病毒1型（HIV-1）整合酶的系统发育保守催化核心结构域包含特异性识别病毒和靶DNA特征所需的元件。为了鉴定决定底物特异性的特定氨基酸，我们对HIV-1整合酶分离的催化核心结构域晶体结构中与活性位点残基紧邻的系统发育保守残基进行了诱变。构成系统发育保守的DD(35)E活性位点基序的残基也进行了诱变。使用解离底物评估纯化的突变蛋白识别病毒DNA末端附近系统发育保守的CA/TG碱基对以及病毒DNA 5'端未配对二核苷酸的能力。我们的研究结果表明，对保守A/T碱基对的特异性取决于活性位点残基E152。IN(Q148L)的表型表明Q148可能参与与病毒DNA末端5'二核苷酸的相互作用。一些活性位点天冬氨酸和谷氨酸附近残基发生突变的蛋白质的活性对盐敏感，表明这些突变破坏了与DNA的相互作用。
