Extract
Note: Please read the complete
full text with Figures and Tables at
Introduction
HIV-1 integrase (IN) is
an important enzyme in
the virus replication
cycle and an
attractive target for
antiviral drug design
because it catalyzes
the integration of the
virus genome into the
human genome. IN
catalyzes a 2-step
reaction. The first
step is the cleavage
of 3' GT dinucleotide
from each 3' end
of the virus long
terminal repeats (LTR)
DNA (donor DNA). This
reaction is termed 3'
processing (3P) and
depends on the
recognition of the
specific sequence at
the U5 and U3
ends of LTR. In
the subsequent step,
strand transfer (ST),
the 3'-processed DNA
ends acting as
nucleophilic agents, attack
phosphodiester bonds in
the host cellular DNA
(target DNA) and
splice these 3' ends
into the target DNA.
This reaction does not
absolutely require any
specific sequence within
the host DNA[1_3].
During the past decade, in vitro assays for IN activities
have been developed and applied for the screening of
inhibitors. These assays include 2 categories in general: (i)
low-throughput gel-based assays involving
radioactively-labeled
oligonucleotides[4,5]; and (ii) high-throughput
microtiter assays using biotin (BIO) or digoxin (DIG)-labeled
oligonucleotides with enzyme-coupled detection strategies
for the quantification of the
product[6_8]. Recently, a gel-based assay using microarrayed compound libraries and
1 microtiter assay based on time-resolved fluorescent
resonance energy transfer have been
developed[9,10]. These assays are established at the aim of high-throughput
screening (HTS), high sensitivity, specificity, and reliability. Among
these assays, solid-phase microtiter assays in which DNA
were immobilized on the microplate surface were most widely
used in the screening of antiviral compounds because they
are fast, convenient, and are optimized in HTS
format[6_8]. However, the need of plate coating and blocking in these
assays is time and labor consuming, and the invalidation of
plate blocking causes non-specific binding of DNA or
antibodies on the microplate and subsequent higher background
readings. Moreover, the immobilization of the DNA
substrate on the microplate limits the application of these
assays in reaction characters studies.
It is reported that small molecules that inhibit the 3P
activity of IN in vitro show little antiviral activity
in vivo, and the inhibition of ST activity is the primary key to antiviral
efficiency in vivo[11,12]. IN is the only 1 of the 3 enzymes for
which clinically useful drugs are not available to date.
Therefore, it is important to develop in
vitro assays for the detection of IN activities, especially ST activity, and apply
these assays in the screening of antiviral compounds. In the
present study, we describe a rapid and highly robust
approach to HIV-1 IN ST activity using HTS technology. This
assay was performed in a 96-well microplate. Based on the
commercially-available magnetic beads, this assay is rapid,
flexible, and specific, and there is no need for plate coating
or blocking. Furthermore, 2 kinds of detection strategies,
absorbance and fluorescence, have been conducted and 2
antiviral compounds were employed to test the
effectiveness of this assay.
Materials and methods
Materials Oligonucleotide sequences corresponding to
the U5 terminus of the HIV-1 LTR and the target substrate
were synthesized and modified by Shanghai Sangon Bio
(Shanghai, China): oligo I BIO-5' ACCCTTTTAGTCA-GTGTGGAAAATCTCTAGCA3', oligo II 5'
ACTGCTAGA-GATTTTCCACACTGACTAAAAG 3', oligo III 5'
ATGTGG-AAAATCTCTAGCGAT3'-DIG, and oligo IV 5'
ATCGCTA-GAGATTTTCCACAT3'. Taq DNA polymerase was
purchased from Dalian Takara Bio (Dalian, China); restriction
endonucleases and T4 DNA ligase were from New England
Biolabs (Beverly, MA, USA). Dynabeads M-280 streptavidin
(SA) magnetic particle and the Dynal MPC-96B
concentrator were purchased from Dynal Biotech (ASA, Oslo,
Norway). The monoclonal anti-DIG alkaline phosphatase
(AP) conjugate antibody and monoclonal anti-DIG
fluorescein isothiocyanate (FITC) conjugate antibody were
purchased from Sigma (St Louis, MO, USA). The
nickel-chelating column (Chelating Sepharose Fast Flow) was purchased
from Amersham Pharmacia Biotech (Piscataway, NJ, USA).
HEPES, imidazole, β-mercaptoethanol,
isopropyl-beta-D-thiogalactopyranoside, bovine serum albumin (BSA) and
piperazine-N, N'-bis-2-ethanesulfonic acid (PIPES) were
obtained from Merck (Hohenbrunn, Germany). Baicalein was
purchased from Fluka (Buchs SG, Switzerland). All other
chemicals were from Amresco (Solon, OH, USA).
Protein expression and
purification The plasmid containing the HIV NL4-3 IN gene was used as the PCR template
DNA. Site-directed mutagenesis was done by overlapping
PCR to bring the F185K/C280S double mutations for the
purpose of enhancing the protein solubility. 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. The PCR
product was digested with Nde I and
BamH I and ligated with Nde
I_BamH I-digested pET-28a to construct a pNL-IN
recombinant plasmid. After confirmation by sequencing
(Shanghai Sangon Bio, China), correctly constructed
pNL-IN was expressed in Escherichia coli strain BL21 (DE3) as a
soluble N-terminal 6-histidine tag fusion protein and
purified by a nickel affinity column as
described[13]. After elution from the nickel column, the purified protein was dialyzed
against 1 mol/L NaCl, 20 mmol/L HEPES, pH 7.5, 0.1 mmol/L
EDTA, 1 mmol/L dithiothreitol, and 10% (w/v) glycerol at 4
°C overnight and stored at -80 °C. SDS-PAGE was employed to
analyze the expression and purification of the IN protein,
and the IN concentration was measured by the Bradford
method (Bio-Rad, Hercules, CA, USA).
Microplate ST assay For high-throughput purpose, ST
assays were performed in a 96-well microplate (Corning, New
York, NY, USA) in a final volume of 50 µL. The wells were
washed once with 1×reaction buffer (25 mmol/L PIPES, pH
7.0, 10 mmol/L β-mercaptoethanol, 5% (w/v) glycerol, 0.1 g/L BSA,
and 10 mmol/L MnCl2). In total, 800 ng IN was added and
pre-incubated in reaction buffer. Subsequently, 1.5 pmol
donor DNA and 15 pmol target DNA were added and the
reaction was initiated. After incubation for 1 h at 37 °C, 1.5
mL magnetic particles (6.7×108 beads/mL) and 51.5 µL
binding buffer (10 mmol/L Tris-HCl, pH 7.6, 2 mol/L NaCl, 20
mmol/L EDTA, and 0.1% [w/v] Tween 20) were added and incubated
at 20 °C for 15 min. Then the wells holding the mixture were
placed in a magnetic concentrator, the supernatant was
discarded, and the wells were washed 3 times with
phosphate-buffered saline (PBS) containing 0.1% Tween 20
(PBST). Subsequently, 100 µL of 1:5000 diluted AP
conjugate anti-DIG antibody was added and incubated for 30 min
at 37 °C. Finally, the wells were washed 3 times with PBST
and the magnetic beads were transferred into fresh wells;
100 µL P-Nitrophenyl Phosphate (P-NPP) substrate (0.1
mol/L Na2CO3, pH 9.5, 6.7 mmol/L P-NPP, and 2 mmol/L
MgCl2) was added. The plates were read at 405 nm with a Model 680
microplate reader (Bio-Rad, USA).
Results
HIV-1 IN
purification and
characterization IN
was purified from the
soluble supernatant by
nickel-affinity chromatography.
The SDS-PAGE analysis
indicated that the
expression of recombinant
IN exceeded the
expression of any
other cellular protein
and IN was highly
soluble. The molecular
weight of the
recombinant IN was
approximately 33 kDa,
with a purity of
approximately 95% (Figure
1).
Principle of
the HTS
assay for
HIV-1 IN
ST reaction In
this HTS assay, the
31 bp duplex donor
DNA substrate was
designed to mimic the
U5 end of the
HIV-1 LTR. After
annealing, there was a
3 nucleotides overhang
at the 5' end
of oligo I for
better interaction of
BIO-SA in the
following DNA capture
step, and the 3'
GT dinucleotides were
removed from oligo I.
The donor DNA was
labeled at the 5'
end of oligo I
with BIO, and the
target DNA was labeled
at the 3' end
of oligo III with
DIG. The ST reaction
resulted in a 5'
BIO and 3' DIG-labeled
covalent DNA product.
The DNA product was
captured by SA-coated
magnetic beads through
the specific BIO-SA
interaction, followed by
the addition of the
AP conjugate anti-DIG
antibody. Therefore, the
product was measured
by the AP-coupled
enzyme reaction (Figure 2).
Optimization of
the ST
activity assay
To optimize this HTS
assay, we varied the
concentrations of donor
DNA, target DNA, metal
ion, and the IN
protein to obtain the
optimal reaction conditions.
The optimal concentration
of donor DNA was
30 nmol/L. The IN
concentration varied between
17 and 1000 nmol/L,
and 300 nmol/L was
determined to be
optimal. As for the
target DNA, we varied
the concentration from
10 to 750 nmol/L,
and determined that
the 300 nmol/L target
DNA to be added
was optimal. Furthermore,
we investigated the
performance of our
assay in the presence
of either cationic
cofactor, Mg2+ and
Mn2+. Using this
assay, ST activity
peaked at 10 mmol/L
Mn2+ and 5
mmol/L Mg2+,
respectively. Accordingly,
the presence of
Mn2+ instead of
Mg2+ increased IN
activity by almost
double (Figure 3).
Here, 300 nmol/L IN,
30 nmol/L donor DNA,
300 nmol/L target DNA,
and 10 mmol/L
Mn2+ were
determined to be the
optimal reaction conditions
and used throughout
the subsequent study.
To test the sensitivity
and specificity of
this assay, we
measured the signal of
all reactions under
optimal reaction conditions.
Furthermore, 2 widely-used
essential parameters to
indicate the quality
of an assay: the
signal-to-noise ratio (S/N)
and signal-to-background
ratio (S/B) were
defined and calculated as:
and
As a result, the
negative controls in
the absence of either
IN, donor DNA, or
target DNA showed
background readings measured
as absorbance at 405
nm (A405),
lower than 0.05,
whereas the ST
reaction gave a signal
of more than 1.4
(Figure 4A). Accordingly,
the S/N and S/B
ratios of the assay
were 86 and 30, respectively.
To further demonstrate
the robustness of this
assay, we changed the
AP conjugate anti-DIG
antibody to the FITC
conjugate anti-DIG antibody.
After capturing the
reaction product by
magnetic beads, 100
mL of 1:500 PBS-diluted
FITC conjugate antibody
was added and
incubated for 30 min
at 37 °C.
Finally, the microplate
was washed as
described and the
plates were read with
a 1420 Multilabel
counter VICTOR reader
(Perkin Elmer, Boston,
MA, USA) at 485
nm excitation and 535
nm emission. The
results were similar
to the assay in
which the AP conjugate
antibody was used.
The ST reaction showed
a fluorescence signal
as high as almost
1400, whereas the
signals of negative
controls were lower
than 40 (Figure 4B).
The S/N and S/B
ratios were 61 and
32, respectively.
HIV-1 IN
ST reaction
character study
The HIV-1 IN 3P
reaction character was
studied using the
gel-based assay involving
the radioactively-labeled
oligonucleotide. Since IN
pre-incubated in the
presence of the DNA
substrate was more
active in the 3P
reaction than IN
pre-incubated alone or
with Mg2+ [14], we
were interested in the
ST reaction character
of IN under different
pre-incubation conditions
using our assay. We
set 3 reaction groups:
(i) IN pre-incubated
alone in the reaction
buffer without both
DNA and Mn2+;
(ii) IN pre-incubated
with Mn2+ in
the reaction buffer
without DNA; and (iii)
IN pre-incubated with
DNA in the reaction
buffer, but without
Mn2+. The
pre-incubation lasted for
30 min at 37
°C; subsequently, the
ST reactions were
induced by the
addition of both the
Mn2+ and DNA
substrate in (i), only
the DNA substrate in
(ii), and only
Mn2+ in (iii).
Aliquots were taken
after 5, 10, 15,
20, 30, 40, and
50 min, and the
product of the
formation of the
reactions versus the
time plot were
analyzed (Figure 5A).
The result showed that
Mn2+ strongly
enhanced the ST
activity of IN, with
a signal 1.5 times
that of IN
pre-incubated alone. IN
pre-incubated in the
presence of DNA was
almost the same active
as IN pre-incubated
alone. We subsequently
performed the experiments
in the presence of
Mg2+ instead of
Mn2+. The time
plot curves of the
Mg2+-dependent ST
reactions were partially
different from previous
Mn2+-dependent reactions.
Compared with IN
pre-incubated alone, the
pre-incubation of IN
with Mg2+ also
enhanced the ST
activity notably, but
not as significant as
that of the
Mn2+-dependent reaction.
Unlike the
Mn2+-dependent reaction
in which the
pre-incubation of IN
with DNA had no
effect on ST activity,
the pre-incubation of
IN with DNA enhanced
the Mg2+-dependent
ST activity remarkably
(Figure 5B).
Antiviral compounds
screening efficiency
Baicalein and L-708906
were diluted in DMSO
to a final
concentration of 10%
DMSO into the reaction
volume and pre-incubated
with IN at 37°
C in the reaction
buffer in the absence
of Mn2+ for
10 min, followed by
the addition of
Mn2+ and the
DNA substrates. The
reactions were carried
out at 37 °C
for 1 h and the
AP-coupled anti-DIG antibody
and subsequent detection
procedure was applied
to detect the assay
signals. The inhibition
percentage and
IC50 values were
calculated based on
the assay results
using a non-linear
regression curve fit.
The IC50 values
of baicalein and
L-708906 for the IN
ST reaction in this
assay were 1.06 and
0.77 µmol/L,
respectively (Figure 6).
Statistical evaluation
of assay
performance The
screening window coefficient,
Z (or Z') factor,
has been defined to
evaluate effective HTS
assays[15]. It is
capable of reflecting
the assay signal
dynamic range as well
as the data variation
associated with the
signal measurements. In
the present assay, all
Z' factors were
determined as
described[15]:
Z'=1-[3×(SDPOS+SDNEG
)/(MPOS-MNEG)]
Calculations were based
on the standard
deviations (SD) and
intensity means (M) of
the controls. The
highest values were
used as the positive
control and the no
IN reactions (or no
metal ion reactions)
were used as the
negative control. An
analysis of the data
resulting from the
experiments present in
these Figures showed
that the assay we
developed for IN ST
activity had a Z'
score ranging from 0.6
to 0.9 (Table 1).
Together with the S/N
and S/B ratios, these
statistical parameters
reflect the high
sensitivity, specificity, and
robustness of the assay.
Discussion
In the present study, we describe a novel assay that can
be employed to measure the ST activity of IN as well as both
the 3P and ST activities together. It is reported that ST
reaction is the primary key to the effective suppression of
viral replication, and compounds inhibiting the IN activity
in vitro by interfering the 3P reaction alone lack antiviral
activity[11,12]. Therefore, we applied this assay only in the ST
reaction measurement in this work.
Several improvements have been acquired in this assay.
First, the BIO-DIG combination was used and the assay was
conducted in a 96-well microplate. This could be conducted
in all kinds of multiwell microplate formats if desired,
ensuring no radioactive waste and a high-throughput format.
Second, the magnetic beads were used to capture the
reaction product, therefore, all the reagents were freely suspended
in solution instead of solid-phase assays in which donor or
target DNA was attached to the microplate. Without the
immobilization of DNA or the protein, each reagent can be
added at any given time; it is more flexible to investigate the
interaction of all reagents and it is easy to study the
pharmacology of inhibitors targeting IN. Furthermore, with the
application of magnetic beads, neither the precoating nor the
blocking of microplate was required; it was less laborious
and time consuming. After the last wash before the detection,
the magnetic beads were easily transferred into fresh wells,
and the background caused by the non-specific binding of
DNA or the antibody was almost totally eliminated. Third,
we applied this assay in 2 different detection systems. Both
the AP conjugate and FITC conjugate anti-DIG antibodies
and their following detection strategies were used. The
result from both absorbance and fluorescence proves that the
assay is specific and sensitive. Furthermore, the application
of the fluorescent antibody needs no extra enzyme-coupled
detection procedure, and the product formation is directly
quantified by a fluorescent reader or flow cytometer. The
entire assay can be accomplished in approximately 2 h; it is
fast and convenient.
Divalent ion is necessary for both in
vivo and in vitro activities of IN.
In vitro assays use either Mg2+ or
Mn2+ as the cofactor in reactions.
Mg2+ is widely considered to be the biological relevant divalent cation cofactor for IN
activity in vivo[16_19], but IN shows very low activity with
Mg2+ as a cofactor, and Mn2+ is frequently used in most in vitro
assays[8,10,20,21]. We optimized this assay in the presence of
either Mg2+ or Mn2+ and found that the presence of
Mn2+ instead of Mg2+ doubled the ST activity, but not as
significantly as other reported works in which the
Mn2+-dependent activity was approximately 4_6 times that of the
Mg2+-dependent
activity[13,20,21]. Using
Mg2+ as the cofactor still showed a signal as high as 0.7 of
A405. Lee et al reported that the 3P activity of IN alters depending on the structure and
length of the oligonucleotide substrates, and increases in
the length of the substrate cause alterations in the efficiency
of Mg2+-dependent ST
activity[22]. Hwang et al optimized
their ST activity assay in the presence of
Mg2+ using 35 bp donor DNA and suggested that the lengthening of donor
DNA allowed Mg2+ as the
cofactor[7]. It is clear that an increase in the length of the DNA substrate could enhance the
Mg2+-dependent IN activities as
suggested[7,22_24]. With the lengthening of donor DNA to 31 bp, the assay can be
conducted in the presence of either cationic cofactor and
extends the assay utility.
Previous in vitro studies for IN 3P reactions using
Mg2+ as a cationic cofactor have reported that the pre-incubation
of IN with DNA increases enzymatic activity, and IN-DNA
interaction does not require a metal ion
cofactor[14]. In contrast, Vink et
al used Mn2+ in their study as the cationic
cofactor and reported that the stable binding of IN to DNA
requires Mn2+ and IN shows increased activity upon
pre-incubation with Mn2+[25]. We observed complicated
interactions between IN, DNA, and divalent ions in the ST reaction.
When Mg2+ was used as the reaction cofactor, the
pre-incubation of DNA with IN enhanced the ST activity remarkably,
as compared with IN pre-incubated alone (Figure 5B). This
indicates an IN-DNA binding step before ST reaction. The
same observation has been reported and the DNA-binding
step has been determined by steady-state fluorescence
anisotropy[14,26]. However, when
Mn2+ was used as the reaction cofactor, no difference was observed after
pre-incubation of DNA with IN, as compared with IN pre-incubated
alone (Figure 5B). The result indicates that the IN-DNA
interaction requires Mn2+ when
Mn2+ is involved as the cationic cofactor. As previously suggested by Vink
et al, Mn2+ is necessary for the effective binding and correct
positioning of DNA in the active site of
IN[25]. Furthermore, when either
Mn2+ or Mg2+ was used as the cofactor,
pre-incubation of metal ion with IN increased the ST activity, but
pre-incubation of IN with Mn2+ instead of
Mg2+ changed the ST activity more significantly (Figure 5). This information
indicates the existence of the metal ion-IN interaction and metal
ion coordination step before the ST reaction, and the
interaction is more important for the
Mn2+-dependent ST reaction. Yi et
al reported that the affinity of IN for DNA is increased
in the presence of metal ions. The relative order of this effect
is Mn2+>Mg2+, and it is suggested that the different metal
ion effect may explain why Mn2+ is generally more effective
than Mg2+ as a cofactor for catalysis in the
in vitro assays[27]. Taken together, the information presented from our
experiments indicates that the Mg2+-dependent and
Mn2+-dependent ST activity is not equivalent. The difference of IN
activities in the presence of either 2 cations has been
reported and widely studied[23,25_30]. Engelman et al reported
that IN displays more non-specific nuclease activity and less
ionic strength in the presence of Mn2+ than in the presence
of Mg2+ [23]. Moreover, it has been demonstrated that
mutations located in the IN binding site significantly affect the
Mg2+-dependent IN activity, but not the
Mn2+-dependent activity[28],
suggesting that the IN-DNA contact is quite different with these
2 cations and IN coordinates these 2 cofactors differently. The
coordination property differences of
Mg2+ and Mn2+ may result in different conformational changes of IN, therefore causing
differences in the specificity of IN-DNA interactions and
accounting for differences in the Mg2+-dependent and
Mn2+-dependent IN activities as
suggested[26,28,30]. It is confirmed that
our assay can be adapted to study the ST reaction
mechanism in a high-throughput format.
The assay we developed
was designed to screen
antiviral compounds targeting
IN. Therefore, we
tested the inhibitor
screening efficiency of
the microplate assay
using 2 known
integrase inhibitors:
baicalein and 1
compound of the diketo
acid family L-708906.
The IC50 values
are comparable to
previous experiment data
using other
assays[8,20], indicating
that this microplate
assay is efficient and
reliable in antiviral
compound identification.
This assay is
optimized in multiwell
microplate format, therefore
has the potential of
screening antiviral drug
candidates in a
high-throughput format.
In summary, the assay
we presented here can
be used for the
rapid and specific
detection of HIV-1 IN
ST activity as well
as for the efficient
identification of drug
candidates targeting IN.
Furthermore, this assay
can be also adapted
to study the reaction
character in a
high-throughput manner.
Future efforts will
focus on using this
assay to screen
compound libraries to
test HTS efficiency of
the assay and identify
new IN inhibitor candidates.
Acknowledgements
We thank Dr Li-ming
HU, Department of Drug
Synthesis, College of
Life Science and
Bioengineering, Beijing
University of Technology,
for providing the
L-708906 compound.
References
1 Craigie R. HIV integrase, a brief overview from chemistry to
therapeutics. J Biol Chem 2001; 276: 213_16.
2 Chiu TK, Davies DR. Structure and function of HIV-1 integrase.
Curr Top Med Chem 2004; 4: 965_77.
3 Pluymers W, De Clercq E, Debyser Z. HIV-1 integration as a
target for antiretroviral therapy: a review. Curr Drug Targets
Infect Disord 2001; 1: 133_49.
4 Marchand C, Neamati N, Pommier Y. In
vitro human immunodeficiency virus type 1 integrase assays. Methods Enzymol 2001;
340: 624_33.
5 Chow SA. In vitro assays for activities of retroviral integrase.
Methods 1997; 12: 306_17.
6 Hazuda DJ, Hastings JC, Wolfe AL, Emini EA. A novel assay for
the DNA strand-transfer reaction of HIV-1 integrase. Nucleic
Acids Res 1994; 22: 1121_22.
7 Hwang Y, Rhodes D, Bushman F. Rapid microtiter assays for
provirus topoisomerase, mammalian type IB topoisomerase and
HIV-1 integrase: application to inhibitor isolation. Nucleic Acids
Res 2000; 28: 4884_92.
8 John S, Fletcher TM III, Jonsson CB. Development and
application of a high-throughput screening assay for HIV-1 integrase
enzyme activities. J Biomol Screen 2005; 10: 606_14.
9 David CA, Middleton T, Montgomery D, Lim HB, Kati W, Molla
A, et al. Microarray compounds screening (microARCS) to
identify inhibitors of HIV integrase. J Biomol Screen 2002; 7:
259_66.
10 Wang Y, Klock H, Yin H, Wolff K, Bieza K, Niswonger K,
et al. Homogeneous high-throughput screening assays for HIV-1
integrase 3'-processing and strand transfer activities. J Biomol
Screen 2005; 10: 456_62.
11 Hazuda DJ, Felock PJ, Witmer M, Wolfe A, Stillmock K, Grobler
JA, et al. Inhibitors of strand transfer that prevent integration
and inhibit HIV-1 replication in cells. Science 2000; 287:
646_50.
12 Farnet CM, Wang B, Lipford JR, Bushman FD. Differential
inhibition of HIV-1 preintegration complexes and purified integrase
by small molecules. Proc Natl Acad Sci USA 1996; 93: 9742_7.
13 He HQ, Ma XH, Liu B, Zhang XY, Chen WZ, Wang CX,
et al. High-throughput real-time assay based on molecular beacons for
HIV-1 integrase 3'-processing reaction. Acta Pharmacol Sin 2007;
28: 811_7.
14 Smolov M, Gottikh M, Tashlitskii V, Korolev S, Demidyuk L.
Kinetic study of the HIV-1 DNA 3'-end processing
single-turnover property of integrase. FEBS J 2006; 273: 1137_51.
15 Zhang JH, Chung TD, Oldenburg KR. A simple statistical
parameter for use in evaluation and validation of high throughput
screening assays. J Biomol Screen 1999; 4: 67_73.
16 Leh H, Brodin P, Bischerour J, Deprez E, Tauc P, Brochoon JC,
et al. Determinants of Mg2+-dependent activities of recombinant
human immunodeficiency virus type 1 integrase. Biochemistry
2000; 39: 9285_94.
17 Hindmarsh P, Lei J. Retroviral DNA integration. Microbiol Mol
Biol Rev 1999; 63: 836_43.
18 Sherman MP, Greene WC. Slipping through the door: HIV entry
into the nucleus. Microbes Infect 2002; 4: 67_73.
19 Andrake MD, Skalka AM. Retroviral integrase, putting the pieces
together. J Biol Chem 1996; 271: 19 633_6.
20 Tramontano E, Onidi L, Esposito F, Badas R, Colla PL. The use
of a new in vitro reaction substrate reproducing both U3 and U5
regions of the HIV-1 3'-ends increases the correlation between
the in vitro and in vivo effects of the HIV-1 integrase inhibitors.
Biochem Phar 2004; 67: 1751_61.
21 Debyser Z, Cherepanov P, Pluymers W, De Clercq E. Assays for
the evaluation of HIV-1 integrase inhibitors. Methods Mol Biol
2001; 160: 139_55.
22 Lee SP, Kim HG, Censullo ML, Han MK. Characterization of
magnesium-dependent 3'-processing activity for human
immunodeficiency virus type 1 integrase in
vitro: real-time kinetic studies using fluorescence resonance energy transfer.
Biochemistry 1995; 34: 10 205_14.
23 Engelman A, Craigie R. Efficient magnesium dependent human
immunodeficiency virus type 1 integrase activity. J Virol 1995;
69: 5908_11.
24 Miller M, Bor YC, Bushman FD. Target DNA capture by HIV-1
integration complexes. Curr Biol 1995; 5: 1047_56.
25 Vink C, Lutzke RAP, Plasterk RHA. Formation of a stable
complex between the human immunodeficiency virus integrase
protein and viral DNA. Nucleic Acids Res 1994; 22: 4103_10.
26 Agapkina J, Smolov M, Barbe S, Zubin E, Zatsepin T, Deprez E,
et al. Probing of HIV-1 integrase/DNA interactions using novel
analogs of viral DNA. J Biol Chem 2006; 281: 11 530_40.
27 Yi J, Asante-Appiah E, Skalka AM. Divalent cations stimulate
preferential recognition of a viral DNA end by HIV-1 integrase.
Biochemistry 1999; 38: 8458_68.
28 Esposito D, Craigie R. Sequence specificity of viral end DNA
binding by HIV-1 integrase reveals critical regions for
protein/DNA interaction. EMBO J 1998; 17: 5832_43.
29 Diamond TL, Bushman FD. Role of metal ions in catalysis by
HIV integrase analyzed using a quantitative PCR disintegration
assay. Nucleic Acids Res 2006; 34: 6116_25.
30 Grobler JA, Stillmock K, Hu B, Witmer M, Felock P, Espeseth
AS, et al. Diketo acid inhibitor mechanism and HIV-1 integrase:
implications for metal binding in the active site of
phosphotrans-ferase enzymes. Proc Natl Acad Sci USA 2002; 99: 6661_6.
|
