title: In vitro HIV-1 selective integration into the target sequence and decoy-effect of the modified sequence.
abstract: Although there have been a few reports that the HIV-1 genome can be selectively integrated into the genomic DNA of cultured host cell, the biochemistry of integration selectivity has not been fully understood. We modified the in vitro integration reaction protocol and developed a reaction system with higher efficiency. We used a substrate repeat, 5'-(GTCCCTTCCCAGT)(n)(ACTGGGAAGGGAC)(n)-3', and a modified sequence DNA ligated into a circular plasmid. CAGT and ACTG (shown in italics in the above sequence) in the repeat units originated from the HIV-1 proviral genome ends. Following the incubation of the HIV-1 genome end cDNA and recombinant integrase for the formation of the pre-integration (PI) complex, substrate DNA was reacted with this complex. It was confirmed that the integration selectively occurred in the middle segment of the repeat sequence. In addition, integration frequency and selectivity were positively correlated with repeat number n. On the other hand, both frequency and selectivity decreased markedly when using sequences with deletion of CAGT in the middle position of the original target sequence. Moreover, on incubation with the deleted DNAs and original sequence, the integration efficiency and selectivity for the original target sequence were significantly reduced, which indicated interference effects by the deleted sequence DNAs. Efficiency and selectivity were also found to vary discontinuously with changes in manganese dichloride concentration in the reaction buffer, probably due to its influence on the secondary structure of substrate DNA. Finally, integrase was found to form oligomers on the binding site and substrate DNA formed a loop-like structure. In conclusion, there is a considerable selectivity in HIV-integration into the specified sequence; however, similar DNA sequences can interfere with the integration process, and it is therefore difficult for in vivo integration to occur selectively in the actual host genome DNA.
authors.affiliation: Department of Forensic Medicine and Molecular Pathology, Graduate School of Medicine, Kyoto University, Kyoto, Kyoto Prefecture, Japan. tsuruyam@fp.med.kyoto-u.ac.jp
authors.forename: Tatsuaki
authors.initials: T
authors.lastname: Tsuruyama
authors.forename: Tonau
authors.initials: T
authors.lastname: Nakai
authors.forename: Takuya
authors.initials: T
authors.lastname: Hiratsuka
authors.forename: Guang
authors.initials: G
authors.lastname: Jin
authors.forename: Takuro
authors.initials: T
authors.lastname: Nakamura
authors.forename: Kenichi
authors.initials: K
authors.lastname: Yoshikawa
mesh_terms: D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002712:Chlorides / Q000494:pharmacology; D004279:DNA, Viral / Q000235:genetics* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D019427:HIV Integrase / Q000235:genetics / Q000378:metabolism; D016325:HIV Long Terminal Repeat / Q000235:genetics; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D017895:Manganese Compounds / Q000494:pharmacology; D008957:Models, Genetic; D009841:Oligonucleotides / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D016662:Virus Integration / Q000187:drug effects / Q000235:genetics*
chemical_list: D002712:Chlorides; D004279:DNA, Viral; D017895:Manganese Compounds; D009841:Oligonucleotides; D019427:HIV Integrase; C025340:manganese chloride