Nucleic Acids Research, 1994, Vol. 22, No. 6
1121-1122
A novel assay for the DNA strand-transfer reaction of
HIV-1 integrase
Daria J.Hazuda*, Jeffrey C.Hastings, Abigail L.Wolfe and Emilio A.Emini
Department of Antiviral Research, Merck Research Laboratories, West Point, PA 19486, USA
Received January 12, 1994; Revised and Accepted February 11, 1994
An essential step in the replication of retroviruses is the integration
of a DNA copy of the viral genome into the genome of the host
cell. Integration involves a series of well defined DNA cleavage
and strand-transfer steps that are catalyzed by the virally encoded
enzyme integrase (1 —4). The sequence-specific endonucleolytic
activity of integrase removes two nucleotides from the 3' end
of each LTR sequence at either end of the double-stranded DNA
provirus. In the subsequent strand-transfer reaction, integrase
non-specifically nicks the host cell DNA, creating a staggered
break whose 5' ends are coordinately ligated to the processed
3' ends of each viral LTR. Although repair of the resultant
integrated product may require host-encoded cellular enzymes,
integrase is the sole protein responsible for both the specific and
non-specific endonucleolytic steps as well as DNA strand transfer.
The enzyme's absolute requirement for propagation of HTV-1
in cell culture defines integrase as an attractive target for antiviral
chemotherapeutic intervention (5). We designed the microtiter
plate assay shown in Figure 1 with the specific aim of developing
a high volume, non-radioisotopic assay for HTV-1 integrase which
uses unique oligonucleotides to represent the LTR donor and
target DNA substrates.
For these studies, we used recombinant HTV-1 HXB2 integrase
purified from E.coli (4). However, to increase the scale and
overall yield, the procedure for purification of integrase was
modified as follows: A 20 gm frozen pellet resuspended in 25
ml of buffer A (50 mM Tris-HCl pH 7.6, 10 mM MgCl2) was
lysed using sonication and lysozyme (final concentration of 0.5
mg/ml). Following centrifugation for 30 min at 4°C (15,000 rpm
Sorval SS34 rotor), the pellet was resuspended in 25 ml of buffer
A, and treated with DNase I (4,000 U, Boehringer Mannheim)
for 20 min at 37°C. DNAse I was used to aid in solubilization,
facilitating purification by improving the chromatographic profile
of the enzyme, and reducing the number of steps required to
achieve homogeneity.
Integrase was solubilized by the addition of 5 M NaCl to a
final concentration of 1.25 M and solid CHAPS to a final
concentration of 10 mM. The slurry was stirred gently on ice
for 30 min and then centrifuged as above. The supernatant was
diluted to reduce the NaCl concentration to 250 mM prior to
chromatography on Heparin agarose (4). Purity of the enzyme
was assessed by SDS —PAGE and enzymatic activity was
demonstrated using the assay for 3' end-processing of LTR
substrates (4) as well as the microtiter strand-transfer assay.
* To whom correspondence should be addressed
For the microtiter strand transfer assay, donor substrate
oligonucleotides representing the HTV-1 (strain HXB2) U5 LTR
(Figure 1A) were immobilized onto CovaLink polystyrene
microtiter plates (NUNC, Naperville, IL) (6). The
oligonucleotides were synthesized (Midland Certified Reagent
Co., Midland, Texas) to include a 5' phosphorylated three base
overhang on the 5' end of the strand which is specifically
processed on the 3' end by integrase. As illustrated in Figure
IB, carbodiimide mediated condensation of this phosphate with
the secondary amino group on the surface of the microwell orients
the double-stranded LTR oligonucleotide such that oligonucleotide
strand which is cleaved and eventually ligated by integrase is
immobilized on the plate with the 3' end distal to the surface.
The 5' overhang is required for maximum efficiency of the
condensation reaction (6).
The condensation reaction using the LTR donor oligonucleotide
(75 n\ of 0.067 fiM in 10 mM 1-methlyimidazole, pH 7) and
25 yX freshly prepared EDC (0.2 M l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide in 10 mM 1-methylimidazole) was
performed as described (6). Plates were stored for several months
at 4°C in blocking buffer, PBS, 1 % BSA (CalBiochem) and 0.2%
sodium azide. Coupling efficiency as estimated using a radiolabelled version of the phosphorylated LTR strand, generated with
T4 polynucleotide kinase (Boehringer Mannheim, Indianapolis,
IN) (7), was comparable to that published (6). Using 0.067 fiM
oligonucleotide for condensation, the amount of substrate
immobilized per well was determined to be 1.25 pMol.
The standard microtiter DNA strand-transfer assay outlined
in Figure IB was performed directly in these oligonucleotide
coated microtiter wells using the immobilized 30 bp U5 LTR
donor substrate and the biotinylated 20 bp target sequence shown
in Figure 1A. Each reaction of 100 /tl included 20 mM Tris-HCl
pH 7.8, 25 mM NaCl, 3 mM MnCl2, 5 mM Beta-mercaptoethanol, and 50 /xg/ml BSA, conditions comparable to published
endonuclease and integration gel assays (7). The microtiter strand
transfer assay reaction exhibited requirements for salt and
MnCl2 which were indistinguishable from those previously
determined (data not shown).
Upon the addition of integrase (20 yX, final concentration 50
nM), specific cleavage of the 3' end of the immobilized strand
processed the LTR donor for subsequent strand transfer. After
30 min at 37 °C, 5 y.\ of the heterologous, biotinylated oligonucleotide added to the reaction provided the target substrate for
1122 Nucleic Acids Research, 1994, Vol. 22, No. 6
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Figure 1. Microtiter assay for strand transfer. (A) Sequences of the LTR donor
and 'random' sequence target oligonucleoudes used in the microciter assay. (B)
Schematic representation of the microtiter assay, detailed in the text.
Figure 2. Donor and target substrate dependence. (A) Microtiter plates were coated
with either the precleaved ( • ) . 5'p ACC CTT TTA GTC AGT GTG GAA AAT
CTC TAG CA 373' GAA AAT CAG TCA CAC CTT TTA GAG ATC GTC
A 5' or uncleaved (H, see Figure 1) U5 LTR substrate, resulting in the final
concentrations for each donor substrate of 0 to 50 nM as indicated. The mkrouter
assay was performed in duplicate for each concentration. (B) Plates were coated
with either the wild-type precleaved U5 LTR donor substrate ( • ) . or a mutant
precleaved donor substrate ( • ) , 5'p ACC CTT TTA GTC AGT GTG GAA
AAT CTC TAG CA37'3'GAA AAT CAG TCA CAC CTT TTA GAG ATC
GTT A5'. See text. The concentration of the target substrate was varied from
0 to 300 nM as shown. ( O The wild-type uncleaved U5 LTR oligonucleocide
was used as the donor substrate. Four 20-mer duplex target oligonucleotides were
tested: 3' biotinylated-poly d(GT)10/poly d(CA)10 (D), 3' bioonylated-poly d(GA)10/poly d(CT)10 ( • ) . 3' biotinylated-poly(dA)20/poly(dT)20 (O), and
poly(dA)20/3' biotinylated-poly(dT)20 (A). The concentration of each target was
varied as shown.
strand transfer. Integrase nicks the target DNA 'randomly' and
mediates the formation of a new phosphodiester linkage between
the 5' end of this nick and the processed 3' end of the immobilized
LTR donor oligonucleotide, thereby transferring the 3' biotin
group of the target oligonucleotide to the plate.
The strand transfer reaction was performed for 30 min at 37°C.
The wells were washed three times with 200 y\ each of PBS
containing 0.05% Tween 20. After two washes with PBS to
eliminate residual detergent, plates were blocked with 1 % BSA
in PBS, and the strand transfer products were detected and
quantified using a colorimetic avidin-linked alkaline phosphatase
reporter (alkaline phosphatase-conjugated avidin, Boehringer
Mannheim, Indianapolis, IN, and PNP substrate, Sigma Chemical
Co., St Louis, MO, as per the manufacturer's directions).
The microtiter integration assay displayed hyperbolic saturation
with respect to both the concentration of immobilized donor LTR
oligonucleotide and added target DNA substrate (Figure 2). Either
the blunt-ended U5 LTR 30-mer oligonucleotide or the analogous
preprocessed US LTR oligonucleotide (which does not contain
the terminal dinucleotide normally removed by integrase, ref.
8) could be employed as donor substrates in this assay. As shown
in Figure 2A, both DNAs were equally efficient donor substrates
in the reaction and can, therefore, be used to discriminate between
the specific 3' processing and non-specific nicking and strandtransfer activities of the enzyme.
In addition, since the microtiter assay uncouples the
requirements for donor and target DNAs, the assay can also be
used to investigate the sequence requirements for each substrate.
As shown in Figure 2B, mutation of the donor oligonucleotide
at the - 2 position of the 5' overhang reduced the strand-transfer
activity by approximately three-fold. This suggests that the
enzyme may interact with this strand even though this portion
of the substrate is not directly involved in the reaction. The result
is consistent with previous studies which demonstrated that strand
transfer requires at least a one base 5' overhang on the
unprocessed strand (9). In Figure 2C, four oligonucleotides of
equivalent length, but different composition, were tested as target
substrates. Comparison of these target substrates demonstrates
that strand transfer occurs preferentially on target DNAs
containing GC base pairs. Poly(dA)/poly(dT) 20-mers were poor
integration target substrates when compared to either the 'random'
sequence target (Figure 2A and B) or alternating copolymers
containing GC base pairs (Figure 2C).
In conclusion, we have developed a novel rapid assay for the
DNA strand-transfer activity of HTV-1 integrase. Since the assay
is designed to uncouple donor and target DNA selection, it can
be used for the detailed characterization of both donor and target
requirements. The assay requires the 3' end processing, nonspecific nicking and strand-transfer activities of the enzyme. The
ease with which this assay can be performed using readily
available reagents should facilitate the identification of selective
inhibitors for any of these processes. Moreover, the general
format of the assay can easily be adapted to the study of similar
enzymes such as other retroviral integrases as well as
recombinases, transposases and ligases.
ACKNOWLEDEGMENTS
The authors would like to thank Dr Robert LaFemina for helpful
discussions, Dr Wayne Herber for supplying bacterial cell paste,
and Dolores Wilson for preparation of the manuscript.
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