In Vitro HIV-1 Selective Integration into the Target Sequence and Decoy-Effect of the Modified Sequence

Tatsuaki Tsuruyama,Kenichi Yoshikawa,Guang Jin,Tonau Nakai,Takuya Hiratsuka,Takuro Nakamura

doi:10.1371/journal.pone.0013841

Tatsuaki Tsuruyama, Kenichi Yoshikawa + Show 4 more

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https://doi.org/10.1371/journal.pone.0013841

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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(ACTG GGAAGGGAC)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.

Highlights

Integration into the host cell genome is an important process in the life cycle of HIV-1
We found that the ratios of the post-integration amplification product (PIAP) copy numbers into the random sequences of 144 bp were not significantly different from the base length percentage
The copy numbers of PIAPs arising from the target sequence were significantly greater than those when applying random sequences (Fig. 2A, B)

Summary

Introduction

Integration into the host cell genome is an important process in the life cycle of HIV-1. Schroeder et al performed a genome-wide screening of integration sites using a cell culture system with HIV-1 infection and identified integration sites throughout whole chromosome [4]. Following statistical analysis, they reported that integration preferentially occurred at transcriptionally active genes, and similar data on murine leukemia retroviral integration were reported [5,6,7]. Recent data provide evidence that selective integration can occur via a tethering mechanism through the recruitment of the lentiviral integrase by the cellular LEDGF/ p75 protein, which have been recognized as the target of antiintegration therapy [2]

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Results

Conclusion