**1. Introduction**

#### **1.1 Summary**

304 Biochemistry

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The biochemistry of retroviral integration selectivity is not fully understood. We modified the previously reported *in vitro* integration reaction protocol and developed a novel reaction system with higher efficiency. We used a DNA target composed of a repeat sequence DNA, 5'-(GTCCCTTCCCAGT)*6*(ACTGGGAAGGGAC) *6*-3', that was ligated into a circular plasmid. Target DNA was reacted with a pre-integration (PI) complex that was formed by incubation of the end cDNA of the HIV-1 genome and recombinant integrase. It was confirmed that integration selectively occurred in the middle segment of the repeat sequence. On the other hand, both frequency and selectivity of integration markedly decreased when target sequences were used in which CAGT bases in the middle position of the original target sequence were deleted. Moreover, upon incubation with a combination of these deleted DNAs and the original sequence, the integration efficiency and selectivity towards the original target sequence were significantly reduced, which indicated interference effects by the deleted sequence DNAs. Efficiency and selectivity were also found to vary with changes in the manganese dichloride concentration of the reaction buffer, probably due to induction of fluctuation in the secondary structure of the substrate DNA. Such fluctuation may generate structural isomers that are favorable for selective integration into the target sequence DNA. In conclusion, there is considerable selectivity in HIV-integration into the specified target sequence. The present *in vitro* integration system will therefore be useful for monitoring viral integration activity or for testing of integrase inhibitors.

#### **1.2 Background**

Retroviral integration into host DNA is a critical step in the viral life cycle. Once integrated, the proviral genome will be stably duplicated along with the host cellular DNA duplication and will be transmitted to the daughter cells. Retroviruses can thus serve as powerful tools for the integration of foreign genes into a host genome (Fig. 1), and an MLV vector can be used to examine the function of introduced genes and the development of induced pluripotent stem (iPS) cells [1]. However, integrated retroviral genomes also have the potential to cause unexpected transformation through up-regulation of target genes by retroviral promoter elements located at the long terminal repeat (LTR) following integration [2]. Although

Fig. 1. Scheme of viral integration into target DNA: 3'-processing, join reaction, and gap repair. After the HIV-1 RNA genome has been transcribed into double-stranded DNA, the viral protein integrase binds to the termini of the viral DNA ends in a tetrameric fashion and the integrase creates overlapping 5'-ends by removing two nucleotides from the 3'-ends (3'endprocessing). The HIV-1 DNA and the host cell DNA are ligated by synthesis of phosphodiester bonds between the terminal nucleotides of the viral 3'-ends and overlapping 5'-ends of the host chromosome. The non-homologous 5'-ends from the viral DNA are removed by integrase. Finally, the gaps are filled up by host cellular repair proteins, which recognize single strand breaks. A five base-repeat is observed in the flanking sequence after the gap repair reaction.

integration events have long been considered to be random, several recent findings have shown that integration of the murine leukemia retrovirus (MLV) and of HIV-1 is detected more frequently in actively transcribed genes [3, 4] or in promoter regions [5]. Previous statistical studies have also demonstrated that weak palindromic sequences are a common feature of the sites targeted for retroviral integration [6, 7]. Similar target preferences have also been reported for human T cell leukemia retrovirus type I (HTLV-I) integration sites [8]. Because of these findings, we investigated the biophysical mechanisms underlying *in vitro* integration. In the present study, we aimed to establish an *in vitro* integration assay using

Fig. 1. Scheme of viral integration into target DNA: 3'-processing, join reaction, and gap repair. After the HIV-1 RNA genome has been transcribed into double-stranded DNA, the viral protein integrase binds to the termini of the viral DNA ends in a tetrameric fashion and the integrase creates overlapping 5'-ends by removing two nucleotides from the 3'-ends (3'end-

phosphodiester bonds between the terminal nucleotides of the viral 3'-ends and overlapping 5'-ends of the host chromosome. The non-homologous 5'-ends from the viral DNA are removed by integrase. Finally, the gaps are filled up by host cellular repair proteins, which recognize single strand breaks. A five base-repeat is observed in the flanking sequence after

integration events have long been considered to be random, several recent findings have shown that integration of the murine leukemia retrovirus (MLV) and of HIV-1 is detected more frequently in actively transcribed genes [3, 4] or in promoter regions [5]. Previous statistical studies have also demonstrated that weak palindromic sequences are a common feature of the sites targeted for retroviral integration [6, 7]. Similar target preferences have also been reported for human T cell leukemia retrovirus type I (HTLV-I) integration sites [8]. Because of these findings, we investigated the biophysical mechanisms underlying *in vitro* integration. In the present study, we aimed to establish an *in vitro* integration assay using

processing). The HIV-1 DNA and the host cell DNA are ligated by synthesis of

the gap repair reaction.

retroviral cDNA and integrase. Yoshinaga et al. previously reported the development of an *in vitro* integration assay using recombinant HIV-1 integrase, and short viral and target DNA sequences [9]. Using this method, they successfully detected retroviral cDNA-target DNA complexes *in vitro* and reported that the dinucleotide motif 5'-CA that is located at the proviral genome termini was essential for HIV-1 integration. Yoshinaga et al. called these dinucleotides the integration signal sequence. We modified Yoshinaga's method of *in vitro* integration in order to identify the precise HIV-1 integration sites using a target DNA that corresponds to an actual gene sequence.

In our previous study, we identified a common MLV integration site within the *signal transducer and activators of transcription 5*a (*Stat5a*) gene in MLV-induced spontaneous murine lymphoma in an inbred strain of mice, SL/Kh [10]. This is the first report of MLV integration target sequence. It has also been previously demonstrated that the *Stat5a* gene represents one of the common integration sites of MLV (the Mouse Retrovirus Tagged Cancer Gene Database (MRTCGD) (http://rtcgd.ncifcrf.gov/cgi-bin/mm7/easy\_search.cgi) [11]. The encoded STAT5A protein is a transcription factor that is known to play an essential role in the development of myelo- and lympho-proliferative disease [12, 13]. In the current study, we modified this *Stat5a* gene sequence for use as a target for HIV-1 integration *in vitro*. This target gene consists of a 5'-CA-rich sequence, which may provide a useful clue for preparation of target DNA sequences, because terminal CA dinucleotide motifs are shared by MLV and HIV-1, as well as by HTLV-I proviruses.
