Role of metal ions in catalysis by HIV integrase analyzed using a quantitative PCR disintegration assay

Abstract

Paired metal ions have been proposed to be central to the catalytic mechanisms of RNase H nucleases, bacterial transposases, Holliday junction resolvases, retroviral integrases and many other enzymes. Here we present a sensitive assay for DNA transesterification in which catalysis by human immunodeficiency virus-type 1 (HIV-1) integrase (IN) connects two DNA strands (disintegration reaction), allowing detection using quantitative PCR (qPCR). We present evidence suggesting that the three acidic residues of the IN active site function through metal binding using metal rescue. In this method, the catalytic acidic residues were each substituted with cysteines. Mn2+ binds tightly to the sulfur atoms of the cysteine residues, but Mg2+ does not. We found that Mn2+, but not Mg2+, could rescue catalysis of each cysteine-substituted enzyme, providing evidence for functionally important metal binding by all three residues. We also used the PCR-boosted assay to show that HIV-1 IN could carry out transesterification reactions involving DNA 5' hydroxyl groups as well as 3' hydroxyls as nucleophiles. Lastly, we show that Mn2+ by itself (i.e. without enzyme) can catalyze formation of a low level of PCR-amplifiable product under extreme conditions, allowing us to estimate the rate enhancement due to the IN-protein scaffold as at least 60 million-fold.

Figure 1

Disintegration and the PCR-based disintegration assay. (A) Diagram of integration and disintegration. During normal integration, IN first cleaves 2 nt off the 3′ end of each LTR DNA. IN then catalyzes a coupled DNA breaking and joining reaction, which covalently joins the viral DNA 3′ end to host cell DNA. Disintegration is the reversal of the integration reaction. In disintegration, the 3′ end in the target DNA at the junction with the viral DNA attacks the nearby phosphodiester, rejoining the target DNA segment and releasing the viral DNA. For standard disintegration assays in vitro, a ³²P-radiolabel is attached at the indicated 5′ end (*), allowing detection of the larger product formed by disintegration (34). (B) The qPCR-based disintegration assay. The assay substrate contains four annealed oligonucleotides resembling the integration product (upper). DNA transesterification can occur on this substrate, yielding the 97mer disintegration product (middle). The product can be quantitated by TaqMan qPCR (lower). Nucleic acid 5′ ends are represented by black dots. LTR ends are represented by thick lines, while target is represented by thin lines.

Figure 2

Sensitivity of the PCR-based disintegration assay. (A) A standard curve was generated using 4-fold dilutions of the synthetic 97mer product oligonucleotide (Dis Full-97). The standard curve was linear and reproducible in the range tested (from ∼50 to 200 000 copies). (B) Disintegration reactions were carried out with dilutions of HIV-1 IN and analyzed in triplicate by qPCR. The IN reaction products were diluted by the factor shown. The threshold cycles for the IN reactions were compared to the standard curve for quantitation of product copies. (C) Disintegration reactions were carried out on a substrate with the Ttop-60 oligonucleotide ³²P-labeled at the 5′ end. Reactions were analyzed by 8% denaturing-polyacrylamide gel and PhosphorImager. (D) The total number product copies for reactions with radiolabeled substrate were calculated after measurement of percent product conversion at each IN concentration using ImageQuant software. Assay background was determined from the reaction with no IN added.

Figure 3

Functional metal binding by all three residues of the conserved D,DX₃₅E motif of HIV-1 IN. (A) Two divalent metal ions proposed to bind to the active site are shown interacting with the conserved acidic residues of HIV-1 IN. This model was adapted from the structure of avian sarcoma leukosis virus (1VSJ.pdb) in complex with cadmium ions (8). (B) Assays of WT IN and substitutions at D64 and D116. (C) Assays of substitutions at E152. PCR-based disintegration assays were carried out with the indicated IN derivatives in the presence of 10 mM Mn²⁺ or 20 mM Mg²⁺. Disintegration reactions were incubated for 4 h at 37°C. The amount of product copies was determined using 250 000-fold dilutions of WT IN, 50 000-fold dilutions of D64 and D116 substituted INs and 10 000-fold dilutions or E152 substituted INs in the qPCR. The total product copies generated in the disintegration reaction is graphed. A one-tailed Mann–Whitney U-test was used to compare three independent disintegration reactions with qPCR in at least triplicate for each of the E152A and E152C Mn²⁺ reactions or the E152C Mn²⁺ and Mg²⁺ reactions to show that the difference between the sets was statistically significant with P < 0.0001. (D) A time course for disintegration activity with E152C IN. All error bars indicate one standard deviation around the mean of qPCR performed in triplicate. (E) Verification of the sizes of disintegration reaction products by PCR amplification and end labeling.

Figure 4

DNA transesterification activity of HIV-1 on a reverse polarity substrate. (A) Schematic of the reverse polarity substrate, which contains a free 5′ hydroxyl at the DNA three-way junction in place of the usual 3′ hydroxyl. The same primers and probe are used to measure product copies for the two substrates. The 5′ ends are noted by black dots. (B) A time course for DNA transesterification activity of WT IN on the reversed polarity substrate in the presence of 10 mM MnCl₂ or 20 mM MgCl₂. Error bars indicate one standard deviation around the mean of qPCR performed in triplicate. To show that the difference seen between reactions with Mn²⁺ and Mg²⁺ were significantly different, three independent reactions from the 2 h time point (the time point for which we had the most replicates) with triplicate qPCR for each were compared using a one-tailed Mann–Whitney U-test (P < 0.0001).

Figure 5

DNA transesterification reactions in the presence of Mn2 alone. DNA transesterification reactions were carried out on the standard substrate in the absence of IN. (A) Reactions with 50 mM MnCl₂ or 100 μM EDTA were not treated (‘no dehyd’) or dehydrated in a SpeedVac (‘dehyd’) followed by heating for the indicated amount of time at 95°C and 1/100 dilutions were analyzed by qPCR. The total product copies generated during the transesterification reaction are graphed. Error bars indicate one standard deviation around the mean of two independent transesterification reactions each analyzed in triplicate by qPCR. The difference between the reactions with Mn²⁺ versus EDTA was compared for each time point and found to be significant with a P-value of 0.0011, based on a one-tailed Mann–Whitney U-test, for all time points after 1 h. (B) Verification of the sizes of reaction products by PCR amplification with end-labeled primer and 8% denaturing gel electrophoresis. Products for PCRs containing 10-fold dilutions of reactions from the 22 h time point were compared to amplifications of the 97mer oligonucleotide representing reaction product and used in the standard curve. The number of copies of 97mer standard added is indicated above the autoradiogram.