A shaded box) was located at the corner of a loop attached to a long stem structure. The replacement of bases was anticipated to disrupt the stem of the presumed structure. In addition, the fact that HIV preferably integrates into transcriptionally active genes [1] suggests that the target segment used in the present study, which is part of a gene involved in T cell development, is probably transcriptionally active. As such, the segment may therefore be accessible to DNA-binding proteins such as transcription factors or components of the transcriptional apparatus. It is also possible that the double strand in the target segment may be rewound into a single strand following formation of the loop-like structure by hybridization within the strand. Therefore, it appears that both the focal nucleotides at theAffinity of Viral Integrase for Target SequencesQuartz crystal microbalance (QCM) technology was applied to measure the affinity of viral integrase for host CD27 DNA. Integrase binding activity was evaluated by determining the weight (ng) of integrase bound to the oscillator-detection sensor (Fig. 3A). In addition, the 59-T:GCA-39 sequences in the repeat unit segments were removed and AG-221 biological activity replaced (“replaced i and ii) and the resulting products were examined using the assay. The weight of integrase that bound to the replaced i modified DNA was lower than the weight of integrase that bound to the native DNA sequence, but the difference was not significant, strongly suggesting that the binding affinity of integrase is dependent upon the 59-T:GCA-39 sequence in the target DNA (Fig. 3B).Suppression of Retroviral Erastin integration by Modified Target Sequence DNAsModified substrate DNA (replaced i or replaced ii) was mixed with an equal concentration of native target CD27 DNA and an in vitro integration assay was performed. Unexpectedly, integration into the native target DNA was significantly repressed in the presence of modified replaced i DNA (Fig. 4A, *P , 0.01). TheTarget Sequence of HIV-1 IntegrationFigure 2. In vitro integration site in the target CD27 sequence DNA. (A) Percentage of integration at individual sites in the target CD27 sequence DNA. A shaded box indicates the frequent integration site in the in vitro integration assay in (B). (B) Anticipated secondary structure formation in the target CD27 sequence DNA as determined by m-fold analysis. An arrowhead indicates the integration site in the previously reported target nucleotide (Genbank Accession No. AF038363) [6]. Blue, green, and red arrows represent mutations of replaced i, replaced ii, and replaced iii, respectively. Gibbs’ free energy is given by DG = DH -TDS, where DG = 27.34 kcal/mol at 37uC, DH = 2114.80 kcal/mol, DS = 2346.4 cal/(K?mol), Tm = 58.1uC under ionic conditions in which [Mg2+] = 1.0 mol/L. The same concentration of Mg2+ was used in the in vitro integration assay. A shaded box indicates the frequent integration site in the in vitro integration assay. (X) Percentage of integration at various sites in the target CD27 sequence DNA, control random DNA, and modified DNAs (replaced i and replaced ii). Length ratio percentage indicates the ratio of percentage integration into the target sequence DNA to that of integration into the whole substrate DNA. The percentage integration into the CD27 sequence DNA was significantly higher than that into random sequence and replaced DNAs (i) and (ii) (*, **, ***, P , 1662274 0.01). (D) Percentage of integration into the target sequence DNA r.A shaded box) was located at the corner of a loop attached to a long stem structure. The replacement of bases was anticipated to disrupt the stem of the presumed structure. In addition, the fact that HIV preferably integrates into transcriptionally active genes [1] suggests that the target segment used in the present study, which is part of a gene involved in T cell development, is probably transcriptionally active. As such, the segment may therefore be accessible to DNA-binding proteins such as transcription factors or components of the transcriptional apparatus. It is also possible that the double strand in the target segment may be rewound into a single strand following formation of the loop-like structure by hybridization within the strand. Therefore, it appears that both the focal nucleotides at theAffinity of Viral Integrase for Target SequencesQuartz crystal microbalance (QCM) technology was applied to measure the affinity of viral integrase for host CD27 DNA. Integrase binding activity was evaluated by determining the weight (ng) of integrase bound to the oscillator-detection sensor (Fig. 3A). In addition, the 59-T:GCA-39 sequences in the repeat unit segments were removed and replaced (“replaced i and ii) and the resulting products were examined using the assay. The weight of integrase that bound to the replaced i modified DNA was lower than the weight of integrase that bound to the native DNA sequence, but the difference was not significant, strongly suggesting that the binding affinity of integrase is dependent upon the 59-T:GCA-39 sequence in the target DNA (Fig. 3B).Suppression of Retroviral Integration by Modified Target Sequence DNAsModified substrate DNA (replaced i or replaced ii) was mixed with an equal concentration of native target CD27 DNA and an in vitro integration assay was performed. Unexpectedly, integration into the native target DNA was significantly repressed in the presence of modified replaced i DNA (Fig. 4A, *P , 0.01). TheTarget Sequence of HIV-1 IntegrationFigure 2. In vitro integration site in the target CD27 sequence DNA. (A) Percentage of integration at individual sites in the target CD27 sequence DNA. A shaded box indicates the frequent integration site in the in vitro integration assay in (B). (B) Anticipated secondary structure formation in the target CD27 sequence DNA as determined by m-fold analysis. An arrowhead indicates the integration site in the previously reported target nucleotide (Genbank Accession No. AF038363) [6]. Blue, green, and red arrows represent mutations of replaced i, replaced ii, and replaced iii, respectively. Gibbs’ free energy is given by DG = DH -TDS, where DG = 27.34 kcal/mol at 37uC, DH = 2114.80 kcal/mol, DS = 2346.4 cal/(K?mol), Tm = 58.1uC under ionic conditions in which [Mg2+] = 1.0 mol/L. The same concentration of Mg2+ was used in the in vitro integration assay. A shaded box indicates the frequent integration site in the in vitro integration assay. (X) Percentage of integration at various sites in the target CD27 sequence DNA, control random DNA, and modified DNAs (replaced i and replaced ii). Length ratio percentage indicates the ratio of percentage integration into the target sequence DNA to that of integration into the whole substrate DNA. The percentage integration into the CD27 sequence DNA was significantly higher than that into random sequence and replaced DNAs (i) and (ii) (*, **, ***, P , 1662274 0.01). (D) Percentage of integration into the target sequence DNA r.
