Comparison of metal-dependent catalysis by HIV-1 and ASV integrase proteins using a new and rapid, moderate throughput assay for joining activity in solution

Abstract

Background: HIV-1 integrase (IN) is an attractive target for the development of drugs to treat AIDS, and inhibitors of this viral enzyme are already in the clinic. Nevertheless, there is a continuing need to devise new approaches to block the activity of this viral protein because of the emergence of resistant strains. To facilitate the biochemical analysis of wild-type IN and its derivatives, and to measure the potency of prospective inhibitory compounds, a rapid, moderate throughput solution assay was developed for IN-catalyzed joining of viral and target DNAs, based on the detection of a fluorescent tag.

Results: A detailed, step-by-step description of the new joining assay is provided. The reactions are run in solution, the products captured on streptavidin beads, and activity is measured by release of a fluorescent tag. The procedure can be scaled up for the analysis of numerous samples, and is substantially more rapid and sensitive than the standard radioactive gel methods. The new assay is validated and its utility demonstrated via a detailed comparison of the Mg++- and Mn++-dependent activities of the IN proteins from human immunodeficiency virus type 1 (HIV-1) and the avian sarcoma virus (ASV). The results confirm that ASV IN is considerably more active than HIV-1 IN, but with both enzymes the initial rates of joining, and the product yields, are higher in the presence of Mn++ than Mg++. Although the pH optima for these two enzymes are similar with Mn++, they differ significantly in the presence of Mg++, which is likely due to differences in the molecular environment of the binding region of this physiologically relevant divalent cation. This interpretation is strengthened by the observation that a compound that can inhibit HIV-1 IN in the presence of either metal cofactors is only effective against ASV in the presence of Mn++.

Conclusion: A simplified, assay for measuring the joining activity of retroviral IN in solution is described, which offers several advantages over previous methods and the standard radioactive gel analyses. Based on comparisons of signal to background ratios, the assay is 10-30 times more sensitive than gel analysis, allows more rapid and accurate biochemical analyses of IN catalytic activity, and moderate throughput screening of inhibitory compounds. The assay is validated, and its utility demonstrated in a comparison of the metal-dependent activities of HIV-1 and ASV IN proteins.

Figure 1

The retroviral DNA integration reaction. Panel A. The processing and joining steps catalyzed by retroviral integrases produce a gapped recombination intermediate. The shaded region represents an IN multimer, heavy lines the viral DNA, and thin lines host DNA. The position of the conserved CA dinucleotides at the ends of the viral DNA is shown and the position of the processing cleavage sites are marked with straight arrows. The curved arrows indicate the staggered phosphodiester bonds cleaved during the joining reaction. Panel B. Simple in vitro assays for IN activity represent reactions at a single viral DNA end. Viral(donor) or host(target) DNAs are distinguished as in A. Filled circles mark the 5' phosphate ends and open circles the 3' hydroxyl ends.

Figure 2

Moderate-throughput solution assay for integrase joining activity. Panel A. Principles of a solution assay to measure integrase joining activity by fluorescence. Labeling and symbols are as in Figure 1. FAM stands for carboxyfluorescein labeled DNA, a circle with B denotes a biotin modified 3' end in the target oligodeoxynucleotide. Panel B. Comparison of HIV-1 and ASV IN joining activities in Mg⁺⁺ and Mn⁺⁺. The dashed lines with squares show the activity of ASV IN and the solid lines with triangles show the activity of HIV-1 IN expressed as RFUs versus time. Filled and open symbols represent activity in Mn⁺⁺ and Mg⁺⁺, respectively. The inset shows results from the same experiment, after 40 min. and up to 180 min. incubation. Panel C. Comparison of the joining activity of ASV IN with the recessed versus the blunt-ended donor oligodeoxynucleotides in the presence of Mg⁺⁺ (recessed donor oligodeoxynucleotide, dashed line with filled squares; blunt-ended donor oligodeoxynucleotide, solid line with filled circles).

Figure 3

Joining activity confirmed with gel electrophoresis. Left, sequences of the donor oligodeoxynucleotides used in the joining assay. The location of carboxyfluorescein (FAM), 5' radioactive ³²P, and 3' biotin are shown. The -A substrate removes only the A of the conserved CA dinucleotide while the -CA substrate removes both residues. Right, lanes 1 through 3 show HIV-1 IN joining activity on its substrate after 0, 60, 120 min of incubation, respectively. Lanes 4 through 6, 7 through 9, and 10 through 12, show ASV IN joining activity after 0, 15, 30 min of incubation.

Figure 4

Tests of the metal cofactor effects of HIV-1 IN inhibitors on HIV-1 and ASV IN joining activities. A. Dose response curves showing the joining activities of HIV-1 and ASV IN (at 1 μM concentration) as a function of increasing concentration of compound 1. Triplicate data are plotted for each inhibitor concentration and the curves show non-linear regression fitting of the data using Visual Enzymics software. The solid and open triangles represent HIV-1 IN activity in the presence of Mn⁺⁺ or Mg⁺⁺ cofactors, respectively. The solid and open squares represent ASV IN activity in the presence of Mn⁺⁺ or Mg⁺⁺ cofactors, respectively. B. Comparison of the IC₅₀ values obtained by gel and solution based methods. The structure of the inhibitors is shown to the left of the table. Previously published values for IC₅₀s with HIV-1 IN are shown on the left, while values on the right for both HIV-1 and ASV IN were obtained with the solution assay described here. The latter values were determined from non-linear fitting of the triplicate data to a four parameter sigmoidal dose response equation, with the standard error of the fit shown for compounds 1 and 3. Data for compound 2 are from a single experiment.