Substrate-length-dependent activities of human immunodeficiency virus type 1 integrase in vitro: differential DNA binding affinities associated with different lengths of substrates.

Human immunodeficiency virus type 1 integrase (HIV-IN) is an enzyme essential for the integration of viral DNA into the host chromosome, a process that is an attractive target for drug development. In vitro assays have been developed to study both components of the integration process, the 3'-processing and strand transfer reactions. However, major discrepancies between results obtained from in vivo and in vitro events raise concerns as to the biological relevance of activities observed in vitro. These discrepancies include the size of the substrate and the nature of the divalent cation used. In this study, we characterized activities of HIV-IN with oligonucleotide substrates varying in length. Our previous studies indicate that the preferred cation in vitro for 3'-processing is altered from Mn2+ to Mg2+ by increasing the length of the oligonucleotide substrate. This study demonstrates that HIV-IN efficiently catalyzes Mg(2+)-dependent 3'-processing while repressing the strand transfer reaction. Substrate competition studies indicate that longer substrates preferentially bind to the viral DNA binding site of the integrase, whereas the shorter substrate has much less specificity. In addition, the shorter substrate requires a higher concentration of Mg2+, indicating that there is an alteration in the metal binding affinity associated with the varying substrates. Our results show that substrate-length-dependent differential activities are due to differences in the divalent metal binding and DNA binding affinities associated with the different substrates. These results suggest that the structure of the viral DNA is an important factor in differentiating the donor and target substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of Mg(2+)-dependent 3'-processing activity for human immunodeficiency virus type 1 integrase in vitro: real-time kinetic studies using fluorescence resonance energy transfer.

Human immunodeficiency virus type 1 integrase (HIV-1 IN) catalyzes the integration of HIV-1 DNA into the host chromosome. In vitro reactions with endogenous viral DNA require Mg2+ as the metal cofactor, whereas in vitro studies performed with short oligonucleotide substrates utilize Mn2+. In this study, we report that the donor processing activity of HIV-1 IN alters depending on the structure and length of the oligonucleotide substrates. Increases in the length of the substrate cause alterations in the efficiency of Mg(2+)-dependent donor processing activity, thereby reconciling this discrepancy between the in vivo and in vitro HIV-1 IN mediated reactions. We have also found that the 3'-processing activity of HIV-IN is responsible for cleaving the junction between the viral and target sequences of the recombination intermediate. Its mechanism differs from the previously described disintegration reaction in that the donor strands are regenerated without a joining reaction of the target strands. Kinetic studies of 3'-processing activity suggest that the kcat (0.24/h) is very low. This implies that HIV-1 IN remains as a complex with the processed DNA prior to the strand transfer reaction.

Characterization of endonucleolytic activity of HIV-1 integrase using a fluorogenic substrate.

Retroviruses require viral DNA to be synthesized by reverse transcription in the cytoplasm followed by integration of the resulting viral DNA into the host chromosome in the nucleus. Reverse transcription and integration, essential steps in the life cycle of retroviruses, are possible targets in the development of antiviral reagents. One attractive target is the integrase protein, a product of the retroviral pol gene which is solely responsible for the retroviral integration process through cutting and joining reactions. When screening for massive numbers of antiviral agents, a rapid and precise assay is ideal. We report the application of fluorescence resonance energy transfer (FRET) with fluorescein and eosin as the energy transfer pair to characterize HIV-IN-mediated DNA cleavage reactions. Past concerns with applications of FRET to DNA were due to interactions of the fluorophore with the DNA, resulting in quenched fluorescence. However, in this study these concerns have been resolved with the use of a nucleotide analog with a 12-carbon linker arm, 5-amino (12)-2'-deoxyuridine beta-cyanoethyl phosphoramidite. Steady-state fluorescence studies show that cleavage of the fluorogenic substrate by integrase results in enhancement of quenched donor fluorescence intensity. The fluorescence assay was confirmed by autoradiographic analysis of the cleavage reaction with radiolabeled fluorogenic substrate. This fluorescence assay will facilitate both detailed kinetic studies and the rapid screening of novel integrase inhibitors.