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Introduction
Human immunodeficiency virus (HIV) integrase (IN) is a
validated and an important target for the development of
novel drugs for several reasons. First, it is an essential
enzyme in the retroviral life
cycle[1,2]. The integration of the proviral DNA into transcriptional active sites of the host
DNA represents a point of no return[3]. Second, it has no
cellular homologue[4,5]. Drugs selectively targeting IN are
expected to show low toxicity in the host. Third, X-ray
crystal and Nuclear magnetic resonance (NMR) structures of the
catalytic domain of HIV IN are available for rational
structure-based drug design[6_8]. In addition, the combination of
IN inhibitors with those directed against protease and
reverse transcriptase was shown to be synergistic in several
tested models[9]. IN can catalyze 2 specific enzymatic
reactions known as 3'-processing and DNA strand
transfer[1,10]. In the first step, IN removes 2 nucleotides from each 3' end of
the linear viral DNA made by reverse transcription. This
step is sequence specific and dependent on the recognition
of a highly conserved 5'-CAGT-3' site in the U3 and U5
regions of the viral long terminal repeats (LTR). In the
subsequent strand transfer reaction, the 3'-hydroxyl group of
each cDNA strand attacks a strand of the cellular DNA and
splices these 3' ends into the host chromosome.
Both the 3'-processing and strand transfer reactions can
be carried out in vitro with purified recombinant IN, a
divalent metal cation such as Mg2+ or
Mn2+, a donor oligonucleotide that mimics 1 of the viral DNA ends, and a target
oligonucleotide[11_20]. Traditionally, IN activity is measured by a
radiolabeled substrate assay in which reaction products are
separated on polyacrylamide denaturing gels and subjected
to autoradiography[1,11,12]. The assay is highly sensitive and
commonly used for inhibitor
screening[11_13]. However, the disadvantages of the method are that: (i) it requires
radiolabeled substrates, special equipment, and appropriate
handling of hazardous radioactive waste; and (ii) it is
inconvenient to process a large amount of reactions due to the
low-throughput format. Compared with the radiolabeled
substrate assay, biotin (or digoxin)-labeled microtiter plate
assays are safe and high-throughput, and the products of IN
reactions are easy to measure with a
spectrophotometer[14_16]. However, the major drawbacks of these microtiter plate
assays are the low signal/noise ratio and intensive labor
because of the necessity for plate coating and repeated washing.
Fluorescence technology, with the advantages of high
specificity and sensitivity, has become an important tool for
studying nucleic acids and protein/DNA interactions. Recently,
some fluorescent-based assays were developed as
high-throughput screening protocols for IN
reactions[17_20].
A new fluorescence technique, known as molecular
beacons, was reported by Tyagi and Kramer in 1996 for the
construction of probes that are useful for real-time detection
of nucleic acids in homogenous
solutions[21]. Those probes are hairpin-shaped oligonucleotides, each with a fluorophore
covalently linked to 1 end and a quencher moiety covalently
linked to the other end. The fluorophore is quenched by
energy transfer when the stem keeps the ends in close
proximity. When the probe undergoes a spontaneous
conformational change forcing the stem apart, fluorescence
occurs. Recently, molecular beacons have been widely used
in the investigation of DNA-protein interactions including
DNA cleavage and ligation reactions because of their high
sensitivity, simplicity, and real-time
monitoring[22_25]. In this study, we describe a new high-throughput assay that
translates the IN 3'-processing reaction into a fluorescent signal
in real-time using the principle of molecular beacons. The
method uses commercially-available reagents and allows
quantitative monitoring of the 3'-proccessing in a multiwell
plate-reader format.
Materials and methods
Materials The 3'-processing substrate (5'-[FAM]-ACT
GCT AGA GAT TTT CCA CGT GGA AAA TCT CTA GCA GT-[DABCYL]-3') and a control substrate (5'-[FAM]-T GCT
AGA GAT TTT CCA CGT GGA AAA TCT CTA GCA_[DABCYL]-3') were labeled at the 5' end with a fluorophore
(carboxyfluorescein, FAM) and a quencher
(4-[4'-dimethyl-aminophenylazo] benzoic acid, DABCYL) at the 3' end. These
substrates were synthesized by Shanghai Sangon Bio Inc
(Shanghai, China). Taq DNA polymerase and all restriction
endonucleases used in these experiments were purchased
from Dalian Takara Bio Inc (Dalian, China).
Piperazine-N,N'-bis-2-ethanesulfonic acid (PIPES) was purchased from Roche
Diagnostics (Shanghai, China). Nickel-chelating column
(Chelating Sepharose Fast Flow) was purchased from
Amersham Pharmacia Biotech (Piscataway, NJ, USA).
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic (HEPES),
imidazole, and isopropyl-beta-D-thiogalactopyra-noside
(IPTG) were obtained from Merck (Hohenbrunn, Germany).
Of the 2 IN inhibitors, baicalein was purchased from Fluka
(Buchs SG, Switzerland) and myricetin was purchased from
Acros Organics (Morris Plains, NJ, USA). Tryptone and
yeast extract were purchased from Oxoid Limited
(Basing-stoke, Hampshire, UK). All other chemicals were purchased
from Sigma Chemical Company (St Louis, MO, USA).
Protein expression and purification The cDNA of
HIV-1 HXB2 (one of HIV-1 virus strains, GenBank accession
number K03455) IN was used as a template, F185K/C280S
site-directed mutagenesis was done by overlapping PCR to
enhance the protein solubility as described by Jenkins
et al[26]. The IN gene was modified to contain an
Nde I site at the 5' end and a BamH I site and a termination codon (TAG) at the
3' end, then cloned into a pET28(a)+ vector to construct a
pET-IN recombinant plasmid. The pET-IN was expressed in
the Escheria coli BL21 strain (DE3) plysS cells as a
N-terminal 6-histidine tag fusion protein. Cells were grown at 37 °C
in super broth medium (16 g/L tryptone, 10 g/L yeast extract,
and 5 g/L NaCl, pH 7.0) containing 50 mg/L kanamycin to
0.8 of the optical density (OD) for cells solution at 600 nm, and
induced with 0.4 mmol/L IPTG for 4 h. The cells were
harvested by centrifugation at 4200×g for 20 min at 4 °C and
resuspended in buffer A (20 mmol/L HEPES, pH 7.4, 1 mol/L
NaCl, 2 mmol/L β-mercaptoethanol, and 5 mmol/L imidazole)
with 0.3 g/L lysozyme and sonicated in an ice bath until lysis
was complete. The supernatant was collected by
centrifuga-tion, filtered, and applied to a Ni-affinity column equilibrated
with buffer A. The column was washed first with 20 and 60
mmol/L imidazole in buffer A, and then with a linear gradient
of 100_300 mmol/L imidazole in the same buffer. Fractions
containing HIV IN were pooled together and dialyzed against
buffer B [20 mmol/L HEPES, pH 7.4, 1 mol/L NaCl, 0.1
mmol/L EDTA, 1 mmol/L dithiothreitol, and 10%
(w/v) glycerol] for 24 h at 4 °C with intermittent changes of buffer to remove
imidazole and stored at -80 °C. The protein concentration
was determined by the Bradford assay with bovine serum
albumin (BSA) as a standard[27]. To determine the purity of
the proteins, SDS-PAGE was performed with a 5%_12%
(w/v) gradient of acrylamide and 0.2%
(w/v) N, N-methylene-bisacrylamide. Molecular weight standards used were
carbonic anhydrase from bovine erythrocytes (28 500), albumin
from chicken eggs (45 000), albumin from bovine serum
(66 200), and phosphorylase β from rabbit muscles (94 000).
3'-processing assay The 3'-processing assay was
performed at 37 °C in 96-well plates (Corning Incorporated, NY,
USA) in a final volume of 100 μL per well. The reaction
mixture contained 25 mmol/L PIPES, pH 7.0, 10 mmol/L
β-mercaptoethanol, 5% glycerol, 0.1 g/L BSA, 10 mmol/L
MnCl2, and 50 mg/L purified IN. The reaction was initiated by the
addition of 400 nmol/L of the 3'-processing substrate. The
fluorescence signal was continuously monitored in a 1420
Victor Multilabel counter (Perkin Elmer, Boston, MA, USA)
under the settings of 485 nm excitation and 535 nm emission.
Enzyme-free control wells were set under the same reaction
conditions without IN in the reaction mixture to monitor the
background signal. Substrate control wells were set with all
the reagents except for the substitution of the 3'-processing
substrate by a control substrate. These 2 kinds of control
wells were continuously observed as the 3'-processing
reaction. All the reagents used in the assay were freshly
made each time.
Inhibition of the integrase 3'-processing activity
To determine the inhibition of the integrase 3'-processing, the
reactions were carried out in the 100 μL total volume with 25
mmol/L PIPES, pH 7.0, 10 mmol/L β-mercaptoethanol, 5%
glycerol, 0.1 g/L BSA, 10 mmol/L MnCl2, 50 mg/L purified IN,
and various concentrations of inhibitors diluted in DMSO.
The reactions were initiated by the addition of 400 nmol/L of
the 3'-processing substrate. The reagents mixed with 10%
DMSO, but no inhibitor was set as the drug-free control; the
background control was set as mentioned in the
3'-processing assay. Rate data were collected at 37 °C in the 1420
Victor Multilabel counter. IC50 values were determined by a
nonlinear regression curve fit of the rate data to the
following equation:
Results
HIV IN expression and purification HIV-1 IN was
expressed in the E coli BL21 (DE3) plysS strain and purified
from the soluble supernatant using nickel affinity
chromatography as described in the Material and methods section.
The 12% SDS-PAGE used to separate the total protein of the
cell lysate (lanes 1_3) indicated that after 4 h inducement,
the expression of IN significantly exceeded the expression
level of other cellular proteins (Figure 1). The molecular
weight of purified IN was about 33 kDa, with a purity of
about 95%.
Principles of high-throughput real-time assay based on
molecular beacons In the present study, a real-time assay
for the integrase 3'-processing reaction in solution was
proposed (Figure 2). It is an in vitro cell-free and virus-free
system in which the recombinant HIV-1 IN is incubated with
a 38 mer oligonucleotide substrate with a sequence identical
to the U5 end of HIV-1 LTR. As part of the 3'-processing
reaction, the terminal 3'-dinucleotide, containing the
quencher, is cleaved from the oligonucleotide, and the
quenching effect is abrogated to result in a marked increase
in the fluorescence intensity of the reaction mixture. The
rate of the increase in fluorescence intensity is proportional
to the velocity of the 3'-processing reaction because the
change in fluorescence intensity is dependent on the
release of the quencher-containing dinucleotide. This assay
is specific to the IN 3'-processing reaction.
Optimization of 3'-processing assay
To optimize the robustness of this assay, we performed tests to investigate
the effects of substrates, enzyme, and the metal ion
concentrations on IN activity. In the assay, substrate
concentrations ranged from 10 to 1000 nmol/L, the recombinant IN
concentrations ranged from 10 to 100 mg/L, and the metal
ion concentrations were chosen from 1 to 10 mmol/L. At the
same time, the reference controls of the substrate and metal
ion concentrations were set with all the reagents without IN.
At substrate concentrations below 400 nmol/L, the
fluorescence intensity grew in proportion to the increase in the
substrate concentration. The fluorescence intensity of the
reaction mixture grew slower than that of the control without
IN at substrate concentrations exceeding 400 nmol/L. The
assay showed that the reaction rate was also dependent on
the protein concentration, and the optimal IN concentration
was 50 mg/L. The fluorescence intensity was enhanced with
increasing Mn2+ concentrations, but no obvious change was
observed at Mn2+ concentrations greater than 10 mmol/L.
Here, 10 mmol/L Mn2+ was chosen as an optimal
concentra-tion.
Under the optimal reaction conditions, a continuous and
sharp fluorescence signal growth representing the
accumulation of the 3'-processing product was observed in the first
2 h, but the growth rate slowed down and the real-time curve
reached a plateau after that point in time (Figure 3A). The
3'-processing reaction under optimal conditions showed
560 000 cps of the fluorescence intensity during 3.5 h, while
the enzyme-free control and the substrate control remained
under 30 000 and 60 000 cps, respectively, during the entire
reaction.
To demonstrate that the cleavage observed in the
presence of the enzyme is really a 3'-processing activity, but not
a non-specific hydrolysis, a control substrate was designed
as an oligonucleotide without AC dinucleotide at the 5' end
and GT dinucleotide at the 3' end. HIV-1 IN proved to be
sensitive and specific to its substrate during the
3'-processing reaction. If this cleavage of 5'-fluorophore or 3'-quencher
results from the 3'-processing reaction, no fluorescence
signal increase can be observed for the reaction of IN with the
control substrate. If not, fluorescence intensity would
increase with time. In this assay, no notable fluorescence
signal increase was observed for the substrate control wells,
and the substrate control signal was approaching that of the
enzyme-free control. Compared with the 2 controls, the
fluorescence intensity increased significantly with the reaction
time in the system of IN with the 3'-processing substrate
(Figure 3A). It is clear that the increase of fluorescence
signal in this assay is specifically due to the IN-mediated
3'-processing reaction.
This enzymatic assay for IN 3'-processing activity
described here does not strictly correspond to
Michaelis_Menten conditions as well as most of the enzymatic assays
for IN activities[11_20]. The time-dependent fluorescence
intensity for the 3'-processing reaction in this assay displayed
3 distinct phases (Figure 3A): the lag in phase I lasted about
20_30 min, followed by an apparent linear increase (phase II)
for about 1.5 h, and an equilibrium phase (phase III) after 2 h.
This phenomenon is similar to the observation made by
Smolov et al[20]. This lag may be due to the slow binding of
IN to DNA for about 20 min to form a stable DNA-IN
complex[20,29] because the DNA substrate was added at the end to initiate
the reactions and there was no pre-incubation of DNA with
IN in our assay. The DNA-binding step can be measured by
steady-state fluorescence anisotropy
[20,28_30], which is based on the principle of depolarization of light by a fluorescent
probe caused by the rotational diffusion that occurs between
excitation and emission. IN-bound DNA gave a higher
anisotropy value than free DNA, and the equilibrium reached after
a 20 min incubation of IN with the DNA
substrate[20]. Furthermore, phase I was reduced when IN was
pre-incubated with DNA for 10_20 min at 37 °C prior to the addition
of the metal ion cofactor, and pre-incubation did not
significantly influence the product formation in phases II and III.
The effects of Mn2+and
Mg2+ on the IN 3'-processing activity in this assay were compared (Figure 3B).
Mg2+ is believed to be relevant for viral replication
in vivo, but Mn2+ is frequently used in most
in vitro assays instead of Mg2+ because higher activity of HIV-1 IN is observed with
Mn2+. The optimum concentration of
Mg2+ is 20 mmol/L, while the optimum concentration of
Mn2+ is 10 mmol/L. The
Mn2+-dependent activity is about 4 times that of
Mg2+-dependent activity and is similar to the observation by Tramontano
et al[31].
Integrase inhibitor screening Baicalein and myricetin,
reported to be HIV-1 IN 3'-processing
inhibitors[31], were employed to test the performance of this assay in IN
inhibitor screening (Figure 4). The
IC50 values of baicalein and myricetin obtained in our assay are 0.89 µmol/L and 5.6
µmol/L, respectively, which are similar to the values (0.7 µmol/L for
baicalein and 7.0 µmol/L for myricetin) obtained from a
radiolabeled substrate assay by Tramontano et
al[31]. This shows that the assay can be used for the identification of HIV-1 IN
3'-processing inhibitors.
Discussion
In the present work, we propose a high-throughput
real-time assay for the 3'-processing activity of IN. Based on the
principle of molecular beacons, the assay has significant
advantages. First, since no precoating of the assay plates
with enzyme substrate or capture reagents was required, the
measurements of the homogeneous assay yielded highly
reproducible data with enzyme reactions performed in
solution and no loss of enzyme activity. Second, the
identification of rate-limiting steps in the overall catalytic process is
important, especially for pharmacological purposes. The
assay has high sensitivity and real-time monitoring, and it can
be used for the kinetic study of the IN 3'-processing reaction.
Third, compared to current assays, this assay can be used
for different purposes because it can be conducted in
microplates, microtubes, or other reaction containers, and
the results can be monitored by any fluorescence detecting
instruments. To test the performance of this assay in
antiviral compounds screening, 2 reported 3'-processing
inhibitors were used as examples. The
IC50 values of baicalein and myricetin observed in our assay were similar to the previous
experiment data[31]. In summary, the assay presented here
can be used both for monitoring the HIV-1 IN 3'-processing
activity and for inhibitor screening.
Acknowledgements
We thank Dr Xue-mei MA, Mr Chuan-chen LU, and Mr
Fei XIE of Beijing University of Technology for their helpful
discussion for the fluorescence intensity measurements.
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