Analysis of integration activity of human immunodeficiency virus type-1 integrase.
The integration activity of human immunodeficiency virus type-1 (HIV-1) integrase was characterized in vitro by using pre-processed oligonucleotide substrates. The highest level of integration activity was found at pH 6.5 to 7.0, while the endonucleolytic activity was highest at pH 7.4 to 8.0. Although the endonucleolytic and integration reactions are consecutive in retroviral integration, our result indicates that the optimal conditions of the two reactions are quite different. In addition, it is suggested that the endonucleolytic and integration steps can be separated by control of the cellular physiological state in retroviral therapy. Strong integration was detected in the presence of 0.5-10 mM Mn2+ ion, but weak integration at around 10 mM Mg2+ ion. This observation explains that the Mn2+ ion is preferred to the Mg2+ ion as a cofactor in the integration reaction. Although there was no sequence-specificity in the integration site of the target DNA, integration was found to frequently occur at particular regions of the target DNA. Furthermore, the mutant integrases such as Asp116, Ser147, and Glu152, which had been reported previously, were shown to lose integration activity completely, indicating that these residues are critically involved in catalytic action.
Kim
D J
DJ
Department of Biotechnology, Chung-Ang University, Ansung, Korea.
Oh
Y T
YT
Lee
S K
SK
Shin
C G
CG
D001483:Base Sequence; D002021:Buffers; D004305:Dose-Response Relationship, Drug; D019427:HIV Integrase / Q000378:metabolism*; D016325:HIV Long Terminal Repeat / Q000502:physiology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008345:Manganese / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism; D017354:Point Mutation; D016662:Virus Integration / Q000235:genetics*
D002021:Buffers; D009841:Oligonucleotides; D008345:Manganese; D019427:HIV Integrase