The integration reaction mediated by retroviral integrase (IN) proteins proceeds in two temporally distinct steps (). In the first step called processing, two nucleotides adjacent to a conserved CA are removed from the 3′-ends of viral DNA. This reaction can occur in the cytoplasm of infected cells as soon as reverse transcriptase has completed synthesis of the viral DNA ends. The second step, joining, is a concerted cleavage and ligation reaction in which the newly processed 3′-ends of the viral DNA are joined to staggered phosphates in the backbone of host nuclear DNA. The product of this reaction is a gapped intermediate, and the length of the gap (4 to 6 nucleotides) is characteristic of the IN protein. Continuity of the host DNA is restored, and the provirus stabilized, through the action of host cell DNA damage sensing and repair functions [1
Fig. 1 Catalysis by retroviral integrase (IN). (A), Shows the steps known to occur in vivo. (B), Shows the steps that can be measured separately in vitro using short oligodeoxyribonucleotide duplexes (oligos) that represent a viral DNA end (heavy lines) and (more ...)
It has been more than two decades since the first report of a simple deoxyoligonucleotide-based assay to measure the catalytic activities of retroviral integrases in vitro
] (). In this assay, short (18-22 bp) radioactively labeled DNA duplexes comprising the sequence of either viral DNA terminus are incubated with the cognate IN protein in the presence of a required metal cofactor (Mg2+
). The processing and/or subsequent joining of the labeled processed strand to other such duplexes (self-integration), or other target DNAs added to the reaction, is then followed by electrophoresis of the mixture in polyacrylamide gels [3
]. The development of this assay was a watershed in the field, greatly facilitating the subsequent biochemical and structural analyses that have contributed to our current understanding of integrase function. Such gel assays and derivatives thereof, are still used by many laboratory investigators, and have become somewhat of a “gold standard” in that respect. Detailed protocols for these assays, reviewed some years ago [5
], are still relevant today. These gel assays have the advantage that all of the substrates, products, and byproducts of the reaction can be visualized directly and quantified. Nevertheless, they do suffer from some distinct disadvantages. They are time-consuming, labor-intensive, and for some applications, particularly quantification of joining activity, have low sensitivity.
To circumvent some of the disadvantages mentioned above, and to facilitate high throughput analyses, various modifications of the oligonucleotide assay have been developed, which eliminate the need for electrophoretic separation. In some cases, reporters other than radioactivity have been exploited and/or nucleotide modifications have been included to immobilize substrates or allow isolation of the products. For example, Surface Plasmon Resonance has been used to measure the kinetics of IN DNA binding to immobilized oligonucleotide duplexes representing viral DNA ends or target DNAs [7
] and anisotropy has been used to measure IN binding to similar substrates labeled with a fluorophore [8
]. The processing reaction has been assayed by quantitating the released radioactively-labeled dinucleotide, which is separated from uncleaved substrate [10
], or by measuring the release of a fluorescently-labeled dinucleotide product directly by anisotropy [11
]. Release from quenching of a fluorescent moiety on the 5′-end, opposite to the processing site has also been used to monitor this activity [14
]. Introduction of biotin at the 3′-end of the viral DNA strand that is not processed, was the basis of the first high throughput assay for joining [15
]. In this case, the 5′-end of the processed strand was radioactively labeled and the covalently joined products of self-integration were captured, after denaturation, by adherence to avidin-coated wells of a microtiter plate [15
], or to streptavidin agarose beads [10
]. There have been many variations on this last theme, including assays in which the biotin modified viral DNA is first immobilized on a streptavidin coated surface, and the target DNA is labeled either radioactively [16
] or with a chemical moiety (e.g. digoxygenin) that can be detected with an antibody [17
]. This type of assay has been employed extensively in high throughput screens for inhibitors of the joining reaction catalyzed by HIV-1 IN [18; see also Chapter 7 of this volume], but it is not optimal for analyzing biochemical/kinetic properties, because the substrates and enzymes cannot interact freely in solution. A microtiter plate [19
] and a magnetic bead assay [20
], in which products are selected after they are formed in solution, are more suitable for such studies. Finally, fluorescence resonance energy transfer (FRET) from donor to acceptor fluorophores incorporated into oligodeoxynucleotide substrates has been used to measure processing and joining [21
], IN-mediated fraying of viral DNA ends (R.A. Katz, unpublished observations), and their positioning an IN-DNA complex [22
]. Certain fairly expensive FRET reporters with a long half life [21
] have the advantage that they can be detected in the presence of inhibitor compounds that have an intrinsic fluorescence of their own, which can interfere with conventional fluorescent reporter detection in high throughput screening assays. While not within the scope of this chapter, assays based on long oligo or plasmid-derived substrates that measure the concerted integration of two viral DNA ends into a target DNA have also been described [23
; see Chapter 5, this volume].
As a practical alternative that circumvents both the need for electrophoresis and the use of radioactivity, we have come to rely on a suite of fluorescence assays that are especially convenient for routine laboratory analysis of DNA binding, processing, and joining by IN proteins. An obvious advantage of the fluorescence-base assays is that the substrates have long shelf lives. These solution assays are very sensitive, and suitable for kinetic studies. Furthermore, numerous samples can be analyzed in a fraction of the time required for the gel assays. To measure the DNA binding and processing activities, we have adopted the fluorescence anisotropy methods described and validated by Deprez and coworkers [11
]. For measuring joining activity we provide our standard protocol for a new assay that builds upon some of the modifications described above and a previously described selection strategy [27
]. This joining assay combines the use of biotin to isolate products and fluorescence for detection; a more extensive description of the assay and its validation will be reported separately (Andrake et al.
, in preparation). Our standard protocols for measuring HIV IN activities using these assay, as well as some typical results, follow.