Elative to the percentage of integration into the whole substrate DNA

Elative to the percentage of integration into the whole substrate DNA (*P , 0.01 vs. target CD27 DNA segment). The site of the nucleotide replacement in the Replaced (iii) segment is shown by the red arrow in Fig. 2A. Letters next to the arrows in 2A denote the replacement nucleotides. Results are representative of 5 independent assays. The means 6 SD are shown. The notations (iii)+(i) and (iii)+ (ii) signify segments with both types of modification. doi:10.1371/journal.pone.0049960.gintegration site and the secondary structure generated by the sequences flanking the integration site play a role in determining the accessibility for integrase. The QCM assay was used to directly determine which segment of the target DNA sequence is preferentially bound by Dimethylenastron web integrase [15]. The results of QCM assays demonstrated that approximately twice as much integrase was bound to CD27 DNA, including the TGCA sequence, than was bound to the modified DNAs weexamined. Studies indicate that HIV-1 proviruses and other proviruses such as HTLV-I and MLV share the dinucleotide motif 59-CA and 59-TG at their termini [16]. It is therefore likely that interaction between the TGCA sequence in the target DNA and the viral DNAs is a common occurrence. In our previous study [7], we found that the modified target sequence favored in HIV-1 cDNA integration affected integration into the native target sequence. The cause of this in vitroTarget Sequence of HIV-1 IntegrationFigure 3. Assessment of integrase binding using a K162 quartz crystal microbalance. (A) Scheme depicting the quartz crystal microbalance assay. DNA is deposited on the electrode. (B) Representative graphs of results of assays using target CD27 DNA and replaced i DNA. Downward arrows represent the frequency (Hz) at the plateau phase after integrase binding. (C) Graph showing the weight of integrase bound to CD27 target, random, replaced i, and replaced ii DNAs which were fixed onto the QCM sensor chip. (*, **P , 0.01). Results are representative of 5 independent assays. The means 6 SD are shown. doi:10.1371/journal.pone.0049960.gFigure 4. Decoy effect of the CD27 modified sequence. (A) The percentage of integration into the native target DNA was significantly suppressed in the presence of the modified DNAs replaced i and replaced ii (*, **P , 0.01). Results are representative of 5 independent assays. The means 6 SD are shown. (B) Scheme depicting the proposed mechanism of the decoy effect of the modified DNA. doi:10.1371/journal.pone.0049960.gTarget Sequence of HIV-1 Integrationinterference, termed the decoy effect, remains unclear. One plausible explanation is that the 24786787 modified DNA segment has some affinity for the HIV-1 integrase-cDNA complex, and that this low affinity interferes with integration through competition. This possibility is supported by QCM assay results indicating that the affinity of the modified segments for the integrase complex is about half of that of the native DNA segment. As a result, the modified sequence DNA competes with the native DNA for integrase. The CD27 antigen is involved in the activation of T cells and plays a role in the infection of T cells by HIV-1. Integration of HIV-1 into CD27 disrupts the CD27 translational region. Integration into the genome of CD4+ T cells renders the host cell unable to differentiate through the CD27 signal [17]. CD27 plays a supportive role in T(H)1 differentiation in vivo, without 1662274 modulating the classical T(H)2 response. In addition, CD27 instruc.Elative to the percentage of integration into the whole substrate DNA (*P , 0.01 vs. target CD27 DNA segment). The site of the nucleotide replacement in the Replaced (iii) segment is shown by the red arrow in Fig. 2A. Letters next to the arrows in 2A denote the replacement nucleotides. Results are representative of 5 independent assays. The means 6 SD are shown. The notations (iii)+(i) and (iii)+ (ii) signify segments with both types of modification. doi:10.1371/journal.pone.0049960.gintegration site and the secondary structure generated by the sequences flanking the integration site play a role in determining the accessibility for integrase. The QCM assay was used to directly determine which segment of the target DNA sequence is preferentially bound by integrase [15]. The results of QCM assays demonstrated that approximately twice as much integrase was bound to CD27 DNA, including the TGCA sequence, than was bound to the modified DNAs weexamined. Studies indicate that HIV-1 proviruses and other proviruses such as HTLV-I and MLV share the dinucleotide motif 59-CA and 59-TG at their termini [16]. It is therefore likely that interaction between the TGCA sequence in the target DNA and the viral DNAs is a common occurrence. In our previous study [7], we found that the modified target sequence favored in HIV-1 cDNA integration affected integration into the native target sequence. The cause of this in vitroTarget Sequence of HIV-1 IntegrationFigure 3. Assessment of integrase binding using a quartz crystal microbalance. (A) Scheme depicting the quartz crystal microbalance assay. DNA is deposited on the electrode. (B) Representative graphs of results of assays using target CD27 DNA and replaced i DNA. Downward arrows represent the frequency (Hz) at the plateau phase after integrase binding. (C) Graph showing the weight of integrase bound to CD27 target, random, replaced i, and replaced ii DNAs which were fixed onto the QCM sensor chip. (*, **P , 0.01). Results are representative of 5 independent assays. The means 6 SD are shown. doi:10.1371/journal.pone.0049960.gFigure 4. Decoy effect of the CD27 modified sequence. (A) The percentage of integration into the native target DNA was significantly suppressed in the presence of the modified DNAs replaced i and replaced ii (*, **P , 0.01). Results are representative of 5 independent assays. The means 6 SD are shown. (B) Scheme depicting the proposed mechanism of the decoy effect of the modified DNA. doi:10.1371/journal.pone.0049960.gTarget Sequence of HIV-1 Integrationinterference, termed the decoy effect, remains unclear. One plausible explanation is that the 24786787 modified DNA segment has some affinity for the HIV-1 integrase-cDNA complex, and that this low affinity interferes with integration through competition. This possibility is supported by QCM assay results indicating that the affinity of the modified segments for the integrase complex is about half of that of the native DNA segment. As a result, the modified sequence DNA competes with the native DNA for integrase. The CD27 antigen is involved in the activation of T cells and plays a role in the infection of T cells by HIV-1. Integration of HIV-1 into CD27 disrupts the CD27 translational region. Integration into the genome of CD4+ T cells renders the host cell unable to differentiate through the CD27 signal [17]. CD27 plays a supportive role in T(H)1 differentiation in vivo, without 1662274 modulating the classical T(H)2 response. In addition, CD27 instruc.