HIV-1 Selectively Integrates Into Host DNA In Vitro

Abstract

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.