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INTRODUCTION |
Integration of a DNA copy of the retroviral genome into cellular
DNA is necessary for retrovirus replication and for the development of
retrovirus-related diseases. This recombination event is mediated by
the retroviral integrase
(IN)1 protein. Purified
integrase catalyzes one-step transesterifications in which the
nucleophilic oxygen of an OH group attacks a DNA phosphodiester bond
and covalently joins to the target DNA at the site of nicking (1). To
mediate integration in vivo, integrase must catalyze two
reactions that use distinctly different nucleophiles and targets. The
first reaction, termed processing (see Fig. 1A), prepares
the viral DNA ends for subsequent attachment to cellular DNA (2). The
target for this site-specific endonuclease reaction is the
phosphodiester linkage immediately 3' to the A of highly conserved CA
bases near the 3'-ends of unintegrated retroviral DNA. Nicking at this
site removes the terminal nucleotides (usually two) that follow the CA
and creates a recessed 3'-hydroxyl group at each DNA end. In
vitro studies indicate that the nucleophile for this reaction can
be provided by the OH group of a water molecule (3), by certain
alcohols (3, 4), or by the OH group at the 3'-end of the unintegrated
DNA itself (1) (see Fig. 2). Thus, processing is a site-specific
alcoholysis reaction that uses a variety of nucleophiles (3). The
second reaction, referred to as joining or strand transfer (see Fig.
1B), inserts the processed viral DNA ends into the two
strands of cellular DNA at sites that are separated by a few base pairs
(5, 6). Sequencing of multiple insertion sites has not revealed any
target consensus sequence, even though certain sites within any
sequence may be preferred during in vivo and in
vitro integration (7). Thus, joining can be considered nonspecific
with respect to target DNA. However, only one nucleophilic donor,
i.e. the processed viral DNA end, can be used for successful integration.
In addition to the biologically relevant processing and joining
activities, integrase exhibits two other endonuclease activities in vitro. During disintegration (see Fig. 1C),
which is a reversal of the joining reaction, the 3'-OH of an
oligonucleotide representing nicked cellular DNA attacks the bond
linking the CA at the viral DNA end to nonviral DNA. This action seals
the nick in the cellular DNA mimic and releases a processed viral DNA
end (8). Because the nucleophile and target for this reaction are
determined by the oligonucleotide complex, which juxtaposes the
reactive groups, disintegration is specific for both substrates. The
final and most recently described endonuclease activity of integrase is nonspecific alcoholysis (see Fig. 1D). During this activity,
integrase uses a variety of small alcohols as nucleophiles that nick
and join to any internal 5'-phosphate group of DNA (9). As with DNA
joining, any position in nonviral DNA can be attacked even though
certain sites within a given target are preferred. Both disintegration
and nonspecific alcoholysis can be catalyzed by the isolated central
domain of the integrase protein, unlike processing and joining, which
generally require the complete enzyme (9, 10). The biological
significance of disintegration and nonspecific alcoholysis is unknown.
However, because integrase has a single catalytic site (11), these
reactions have proved useful for analyzing the mechanism and substrate
interactions of this important enzyme.
Despite their mechanistic similarities, the four reactions catalyzed by
integrase represent all possible combinations of specificity for the
attacking nucleophile and the target DNA (see Fig. 1). In particular,
processing uses various nucleophiles and a specific target site,
joining uses a specific nucleophile and various target sites,
disintegration uses a specific nucleophile and a specific target site,
and nonspecific alcoholysis uses various nucleophiles and various
target sites. How a single enzyme accomplishes this feat is unclear,
but study of the interactions between integrase and its various
substrates should shed light on its catalytic mechanism and may
identify potential antiviral targets. In the current report, we
describe quantitative analyses, in three retroviral systems, of the two
integrase reactions that use a variety of nucleophiles (Fig. 1,
A and D). The
experiments tested two hypotheses: (i) that the target DNA influences
the choice of attacking nucleophile and (ii) that the nature of the
nucleophile influences the choice of target DNA sites. The data show
that the integrase proteins of human immunodeficiency virus type 1 (HIV-1), visna virus, and Rous sarcoma virus (RSV) exhibit distinct
preferences for water or other nucleophiles during processing and
nonspecific alcoholysis assays. Moreover, although the target DNA
influenced the choice of nucleophile, the nucleophile did not affect
the choice of target sites. These results have implications for models
of integration.

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Fig. 1.
Endonuclease activities of integrase.
Curved arrows denote the coupled nucleophilic cleavage and
joining that is common to all actions catalyzed by integrase.
Heavy lines represent viral DNA and light lines
represent any DNA. 5'-Phosphate groups are depicted by closed
circles and 3'-hydroxyl groups by open circles. The
invariant CA bases near the viral DNA end are in boldface.
During processing (A), the terminal nucleotides
(NN) can be removed by a variety of nucleophiles
(represented as ROH). During joining (B), the
recessed 3'-end of the processed viral DNA acts as the nucleophile to
nick and join to any site in target DNA (similar reactions occur on
each cellular DNA strand). During disintegration (C), which
is a reversal of the joining reaction, a juxtaposed 3'-DNA-end attacks
after the CA to seal the nick and regenerate the processed viral DNA
end. During nonspecific alcoholysis (D), a variety of
nucleophiles can nick and join to any internal site in DNA. The first
two reactions occur in vivo, whereas all four reactions
occur in vitro. Characteristics of the attacking nucleophile
and target DNA are indicated at the right.
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EXPERIMENTAL PROCEDURES |
Purified Integrases--
The integrase coding regions of
HIV-1BH10 and visna virus were cloned into plasmid pQE-30
and expressed in Escherichia coli M15[pREP4] (Qiagen,
Inc., Chatsworth, CA), as described previously (12, 13). The RSV
integrase coding region from a derivative of plasmid pBH-RCAN-HiSV
(14), which was obtained from Rebecca Craven of Pennsylvania State
University, was cloned and expressed similarly. Proteins were purified
as described previously (12, 13) except that glycerol was omitted from
all buffers when the integrases were to be tested in the absence of
alcohols or with other alcohols. Such glycerol-free proteins were not
frozen. The concentrations of purified proteins were measured by
comparison to a series of Coomassie Blue-stained standards following
SDS-polyacrylamide gel electrophoresis and densitometry of dried gels.
The concentrations of the purified HIV-1 and visna virus integrases
were ~200-700 ng/µl (6-20 pmol/µl), yielding a final
concentration in the typical 10-µl reaction of ~1-2 pmol/µl
(1-2 µM). Because RSV integrase always purified at
higher concentrations, it was diluted 5- to 25-fold to yield comparable
concentrations in all reactions.
Oligodeoxynucleotides--
The sequences of the substrates for
the processing assays were as follows (only the labeled strand
is shown, and the CA is underlined): HIV-1 U5 plus
strand 18-mer, TGGAAAATCTCTAGCAGT; visna virus U3 minus
strand 18-mer, GTTCTCTGTCCTGACAGT; RSV U3 minus strand
18-mer, ATTGCATAAGACTACATT. The sequence for the labeled strand of the nonspecific DNA substrate is shown below (see
Fig. 9D). All oligodeoxynucleotides were gel-purified
following synthesis and again after being radiolabeled.
Oligonucleotides were 5'-end-labeled with [
-32P]ATP
(3000 Ci/mmol, PerkinElmer Life Sciences) by T4 polynucleotide kinase.
For labeling near the 3'-end, DNA was annealed to a complementary oligonucleotide that created a 5'-overhang, and a 3'-fill-in reaction was performed by the Klenow fragment of DNA polymerase I using either
[
-32P]dTTP (800 Ci/mmol) for the viral DNA substrates
(to reconstitute the 18-mers) or [
-32P]dCTP (800 Ci/mmol) for the nonviral 24-mer. Sequence-specific markers for gel
analysis were produced by titrating the 3'- to 5'-exonuclease activity
of snake venom phosphodiesterase (Sigma Chemical Co., St. Louis, MO) on
5'-radiolabeled oligonucleotides (2) or the endonuclease activities of
DNase I (Roche Molecular Biochemicals, Indianapolis, IN) and mung bean
nuclease (New England BioLabs, Beverly, MA) on 3'-radiolabeled
oligonucleotides. Assignment of marker positions to the 3'-labeled
nonspecific DNA was confirmed by synthesizing and 5' radiolabeling
oligonucleotides that correspond to the 6-, 9-, and 10-mer positions.
In Vitro Integrase Assays--
Double-stranded DNA substrates
were prepared by annealing the labeled strand with 4-fold excess
unlabeled complementary oligonucleotide by sequentially incubating the
DNA at 95 °C for 5 min, 37 °C for 30 min, and 4 °C for at
least 10 min. Standard 10-µl reaction mixtures contained 0.5 pmol of
double-stranded DNA, 25 mM Tris-HCl (pH 8.0), 10 mM dithiothreitol, and 1.0 µl of integrase or protein storage buffer. Unless otherwise indicated, reactions contained 10 mM MnCl2. Some reactions were supplemented with
nucleophilic alcohols that were added before integrase. Reaction
mixtures were incubated for 60-120 min at 37 °C, then stopped by
addition of 10-20 µl of loading buffer (95% formamide, 20 mM EDTA, 0.05% bromphenol blue, 0.05% xylene cyanol) and
heating at 95 °C for 5 min; all reactions in any set received the
same amount of loading buffer. Equal-sized aliquots were loaded onto
20% polyacrylamide (acrylamide to methylene-bisacrylamide ratio,
19:1)/7 M urea-denaturing gels, followed by electrophoresis
at 75 watts until the bromphenol blue dye had migrated 21-28 cm. Wet
gels were autoradiographed at
70 °C.
Quantitation of Results--
Results were quantified by
measuring the radioactivity of bands in wet gels with a Betascope
(Betagen, Waltham, MA) or PhosphorImager (Molecular Dynamics,
Sunnyvale, CA) or by measuring the intensities of bands on
autoradiograms with a laser densitometer (Molecular Dynamics). To
calculate the yield of various products, counts per minute,
phosphorimaging, or densitometry units for the entire gel lane were
used as the denominator, and corrections were made for appropriate
control reactions at zero time or that did not contain integrase or the
exogenous alcohols.
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RESULTS |
Activity of Enzymes Purified without Glycerol--
The integrase
proteins from HIV-1, visna virus, and RSV were purified from a standard
bacterial expression system. To permit testing of the enzymes in the
presence of various nucleophilic alcohols, most purifications were
performed without glycerol in the extraction or dialysis buffers, which
necessitated storage of the proteins at 4 °C. Such preparations had
comparable protein concentrations and enzymatic activity to our
standard preparations purified in the presence of 10% glycerol and
stored in 40% glycerol at
70 °C (data not shown). Although
glycerol-free RSV integrase remained active for weeks when stored at
4 °C, the HIV-1 and visna virus proteins lost activity during
similar storage and thus were usually tested immediately following
purification. Each integrase was tested on oligonucleotides derived
from one end of its cognate viral DNA in the presence of different
concentrations of various nucleophilic alcohols. To facilitate
comparisons, the viral DNA end that was previously established as most
susceptible to each enzyme was used as the appropriate substrate for
processing, i.e. the HIV-1 U5 end (the downstream or right
end) and the visna virus and RSV U3 ends (the upstream or left ends)
(12, 13, 15). Alcohols that were used for these experiments included
three diols previously shown to support integrase-mediated alcoholysis
(3, 9): 1,2-ethanediol (ethylene glycol), 1,2-propanediol (propylene glycol), and 1,2,3-propanetriol (glycerol). Reactions conducted with
5'-labeled substrates demonstrated that the presence of these alcohols
at concentrations up to 30% generally had minimal or no effect on the
extent of processing by any of the three integrases during standard
reactions. Similarly, the yield and patterns of the longer
strand-transfer products were not influenced by the exogenous alcohols
(data not shown).
Nucleophiles Used for Processing Viral DNA Ends--
The departing
nucleotides at the viral DNA ends are released by integrase as a simple
dinucleotide when water serves as the nucleophile, as an alcohol adduct
when an exogenous alcohol is the nucleophile, or as a cyclic
dinucleotide when the OH at the 3'-DNA-end loops around to create the
nick after the CA (Fig. 2). Although
assays performed with 5'-labeled substrates cannot reveal which
nucleophiles are used for this reaction, the various products can be
distinguished if the DNA is radiolabeled near the 3'-end (Fig. 2) (1,
3). Under such conditions, the linear dinucleotide comigrates on
denaturing polyacrylamide gels with a specific oligonucleotide marker,
the alcohol adducts migrate more slowly to novel positions that are a
function of the particular alcohol (i.e. the identity of the
R group in Fig. 2), and the cyclic product migrates at an intermediate
position just ahead of the alcohol adducts. We first confirmed that
each of the integrases created all three types of products. Initial
reactions were conducted in the presence of Mn2+, because
all retroviral integrases exhibit maximal activity in standard
oligonucleotide assays when this divalent cation is present. For the
HIV-1 and visna virus integrases, three major products were evident
(Fig. 3, A and B).
These bands were previously identified as the alcohol adducts
(A), the cyclic dinucleotide (C), and the linear
dinucleotide (L). None of these bands were observed when reactions were conducted without enzyme, whether or not an exogenous alcohol was present (lanes 2 and 3 in Fig. 3,
A and B). Under these conditions, the HIV-1 and
visna virus integrases predominantly used water as the nucleophile for
processing but also used the exogenous alcohols efficiently, whereas
the cyclic products were produced to a lesser extent (lanes
5-7 in Fig. 3, A and B). When reactions were conducted in the absence of the alcohols, each of these
enzymes preferentially used water rather than the DNA end as the
attacking nucleophile (lane 4 in Fig. 3, A and
B).

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Fig. 2.
Alternative nucleophiles for processing viral
DNA ends. Integrase uses the electron pair (depicted as two
dots) of certain nucleophilic molecules to nick after invariant CA
bases (shown in boldface) near each of the viral DNA
3'-ends. Depending on whether water (HOH), an alcohol
(ROH), or the viral DNA 3'-OH end acts as the nucleophilic
donor for the nicking reaction (denoted by arrows above the
substrate DNA), the terminal nucleotides (NN) leave either
as a linear dinucleotide, bound to an alcohol, or circularized,
respectively. The products can be distinguished by gel electrophoresis
if the CA-containing strand is 32P-labeled near the 3'-end
(as indicated by the circle); the products cannot be
distinguished if the substrate is labeled at the 5'-end (as indicated
by the asterisk).
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Fig. 3.
Choice of nucleophile during
Mn2+-dependent processing by three
integrases. An autoradiogram of a denaturing polyacrylamide gel is
shown. Double-stranded 18-mers derived from the indicated viral DNA
ends were labeled near the 3'-end of the strand with the invariant CA
and used as the substrate for the corresponding integrase
(IN) in the presence of 10 mM Mn2+
and only one nucleophilic alcohol (E, ethylene glycol,
lanes 5; P, propylene glycol, lanes 6;
or G, glycerol, lanes 7). Control reactions were
without alcohol or IN (lanes 2), with one alcohol (ethylene
glycol) but without IN (lanes 3), or with IN but without
alcohol (lanes 4). Sequence-specific markers are in
lanes 1. Only one intervening sample lane was removed
between lanes 6 and 7; thus, each panel
accurately represents the mobility of the various bands. Specific
cleavage products are indicated: L, linear dinucleotide;
C, cyclic product; A, alcohol adducts. For RSV IN
(panel C), the trinucleotide product (T) that is
prominent in Mn2+-dependent reactions also is
indicated, the cyclic products migrate as a broad band, and
visualization of the alcohol adducts required a longer exposure. The
nature of the integrase-dependent but alcohol-independent
band migrating just behind the 9-position in panel B is
unknown (12). Other minor bands are present in the negative control
lanes or reflect secondary cleavage sites.
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In contrast to the above findings, RSV integrase had a strong
preference for using the 3'-OH at the DNA end to make cyclic products,
whether or not exogenous alcohols were present (Fig. 3C,
lanes 4-7). Moreover, the alcohol adducts were
produced inefficiently and their detection required a darker
autoradiographic exposure than that shown. As expected, the bands
identified as the cyclic products and alcohol adducts were resistant to
alkaline phosphatase and polynucleotide kinase (data not shown). RSV
integrase also created a moderate amount of linear trinucleotide
products (T), and the cyclic products likely are a mixture
of circular di- and trinucleotides. This result was expected, because
the avian retroviral integrases are known to nick between the conserved
C and A nucleotides in addition to the biologically relevant site after
the A when reactions contain Mn2+ (2, 6, 16). In fact, RSV
integrase created comparable amounts of 15- and 16-mer products under
these reaction conditions when the 18-mer substrate was labeled at the
5'-end (not shown). Combining this observation with the finding that
the amount of linear dinucleotides (L) evident in Fig.
3C was less than the amount of linear trinucleotides
(T) indicates that most of the cyclic products resulted from
circularization of a departing dinucleotide rather than a
trinucleotide. This conclusion is consistent with the observation
that the cyclic products sometimes resolved into a predominant faster
migrating component and a minor slower component.
We also examined the choice of nucleophile for processing by all three
enzymes in reactions that contained Mg2+. Although RSV
integrase is very active when Mg2+ serves as the cofactor,
most integrases are inactive with Mg2+ under standard
reaction conditions. However, we recently reported that our
preparations of HIV-1 and visna virus integrase exhibit high levels of
Mg2+-dependent activity when processing assays
are supplemented with 30-40% dimethyl sulfoxide
(Me2SO) (17). By combining such conditions with the
use of 3'-labeled substrates, we have now found that both of these
integrases create all three types of dinucleotide products in the
presence of Mg2+ (Fig. 4, A and
B). However, the absolute and
relative yields of the cyclic products made by the HIV-1 and visna
virus enzymes were very low in reactions with Mg2+
(lanes 3-6 of Fig. 4, A and
B). These faint bands, which are better appreciated on the
original autoradiograms, align with the cyclic dinucleotide markers and
are not evident from the reactions that did not contain integrase. The
yield of alcohol products also was low, perhaps because the large
amount of Me2SO in these reactions permitted the addition
of relatively low alcohol concentrations (data not shown). That
retroviral integrases can create the alternative products in reactions
performed with Mg2+ is made clear by reactions using RSV
integrase. This enzyme was active with Mg2+ even without
the addition of Me2SO and readily made all three types of
processing products (Fig. 4C). As expected, greater
specificity for nicking after the CA was exhibited with
Mg2+ (compare the amounts of dinucleotides and
trinucleotides in lanes 6 and 7 of Fig.
4C), which was confirmed in reactions that used 5'-labeled
substrates (not shown). Taken together, Figs. 3 and 4 demonstrate that
each of these integrases can use all three types of nucleophilic donors
for the processing reaction, albeit with different efficiencies,
whether reactions are conducted with Mn2+ or
Mg2+.

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Fig. 4.
Choice of nucleophile during
Mg2+-dependent processing. Double-stranded
18-mers derived from the indicated viral DNA ends were labeled near the
3'-end of the strand with the invariant CA and used as the substrate
for the corresponding IN in the presence of one nucleophilic alcohol
(E, ethylene glycol, lanes 4; P,
propylene glycol, lanes 5; or G, glycerol,
lanes 6). Control reactions were without alcohol or IN
(lanes 1), with one alcohol (ethylene glycol) but without IN
(lanes 2), or with IN but without alcohol (lanes
3). Reactions in lanes 1-6 of panel A were
conducted with 7.5 mM MgCl2 and 30%
Me2SO, those for lanes 1-6 in panel
B contained 10 mM MgCl2 and 35%
Me2SO, and those for lanes 1-6 in panel
C had 5 mM MgCl2 and no Me2SO.
As markers for the various products, reactions conducted with 10 mM MnCl2 and one alcohol are included as
lanes 7 in each panel. The 18-mer substrate and specific
cleavage products (as in Fig. 3) are indicated. Additional bands are
due to the extended autoradiographic exposures necessary to demonstrate
the various products.
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Quantitative Analysis of Nucleophile Selection during
Processing--
The results in Figs. 3 and 4 suggest distinct
preferences by the three integrases for using different nucleophiles
during processing. These impressions were confirmed and extended when the data from many experiments were quantified and normalized to the
total amount of processing (Table I). The
results are shown for all three enzymes with Mn2+
and for RSV integrase with Mg2+ (meaningful comparisons
during Mg2+-dependent processing by HIV-1 or
visna virus integrase were precluded by the low yield of cyclic and
alcohol products in those experiments). The relative utilization of
water or the 3'-OH end of DNA in the absence of exogenous alcohols is
indicated by the columns labeled "-" in Table I. The data reveal
that the HIV-1 and visna virus integrases used water as the nucleophile
8-fold more often than they used the DNA end (89% or 90% compared
with 11%). Very different results were obtained for RSV integrase.
This enzyme used water as the nucleophile only about one-third as often
as it used the viral DNA end during
Mn2+-dependent reactions, even when
trinucleotides were included in the calculations (27% for the
total of linear di- and trinucleotides versus 74% as cyclic
products). When Mg2+ served as the divalent cofactor, RSV
integrase used water only about 50% more often than the viral DNA end
(61% versus 39%).
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Table I
Relative yields of processing products
Numbers represent the average amount of each product formed (normalized
to 100%) in reactions with the indicated integrase (IN) and divalent
cation. The percentage of substrate that was shortened by two
nucleotides is shown in parentheses at the bottom as Total processing,
except that nicking to release trinucleotides also was included in the
calculations for RSV IN with Mn2+. Reactions with alcohol
typically contained 25-40% of ethylene glycol, propylene glycol, or
glycerol. The calculations for reactions with alcohol used the maximum
amount of each alcohol adduct measured in 8-10 experiments. Reactions
without alcohol were done four to five times.
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We also quantified the choice of nucleophile in the presence of
alcohols. To assess the extent to which the different integrases were
able to utilize exogenous alcohols as the nucleophilic donor under
optimal conditions, we included in the calculations only data from the
reaction that yielded the maximum amount of each alcohol adduct in any
set of experiments. Of note, the amount of processing did not change
substantially or in any consistent manner when reactions were
supplemented with alcohols at these concentrations (Table I, bottom
row). A striking finding from these experiments was that the relative
utilization of the 3'-OH group at the DNA end was not influenced by the
presence of the alcohols, remaining at ~10% for HIV-1 and visna
virus integrase with Mn2+, ~75% for RSV integrase with
Mn2+, and ~40% for RSV integrase with Mg2+.
In contrast, use of water as the nucleophile was influenced by the
presence of the alcohols. For example, HIV-1 integrase substituted
exogenous alcohols for water about half the time (Table I, compare the
89% figure in the first column for HIV-1 with the 40% figure in the
second column). These conclusions are supported by an experiment that
compared yields of the three dinucleotide products as a function of the
alcohol concentration (Fig. 5). When the
amount of 1,2-ethanediol was increased from 0% to 40%, the amount of
cyclic products remained stable, whereas the alcohol adducts
progressively replaced the linear dinucleotide products until
approximately equal amounts were produced (Fig. 5, A and B). In a similar manner, visna virus integrase substituted
the alcohols for water as the attacking nucleophile about one-third of
the time (Table I, compare the 90% in the first column for visna virus
with the 32% in the second column). In contrast, RSV integrase
substituted the alcohols for water less than 20% of the time,
such that 5-7% of the products were formed by the use of these
nucleophiles in the presence of either cation (Table I).

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Fig. 5.
Quantitative analysis of nucleophile choice
during processing by HIV-1 integrase. A,
double-stranded 18-mers derived from the HIV-1 U5 DNA end were labeled
near the 3'-end of the strand with the invariant CA and incubated with
glycerol-free HIV-1 IN in the presence of 10 mM
Mn2+ and increasing amounts of ethylene glycol, as
indicated by % ROH. A control reaction with 40% ethylene
glycol but without integrase is shown in lane 1. The 18-mer
substrate, alcohol adduct (A), cyclic product
(C), and linear dinucleotide product (L) are
indicated at the right. B, reactions shown in panel
A were quantified by densitometry, and the relative amounts of the
various dinucleotide products were normalized to 100%. The efficiency
of processing averaged 69% (range, 58-81%), and no consistent effect
on total processing from the exogenous alcohol was evident.
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In addition to varied usage of different types of nucleophiles, varied
usage of different exogenous alcohols was observed (Fig.
6). The HIV-1 and visna virus integrases
readily used glycerol and 1,2-ethanediol (ethylene glycol), whereas
1,2-propanediol (propylene glycol) was used somewhat less efficiently.
Firm conclusions about the relative usage of these three alcohols by
RSV integrase could not be drawn from the data. Each of these diols
presents OH groups on adjacent carbon atoms. Previously, Vink et
al. (3) reported that 1,3-propanediol, which does not have
adjacent OH groups, was not used by HIV-2 integrase for processing. We
therefore tested this chemical with all three enzymes and found that it was used much less efficiently than the 1,2-diols. However, each of the
enzymes was able to utilize it as the nucleophile for processing (Fig.
6). Thus, retroviral integrase does not have an absolute requirement
for adjacent OH groups on acceptable nucleophiles, although there is a
marked preference for such substrates.

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Fig. 6.
Usage of alternative nucleophiles during
viral DNA processing. The yields of the various alcohol adducts
were quantified from all experiments in which at least 10% of the
indicated nucleophilic alcohol was present and >5% of the appropriate
viral DNA substrate was processed by the corresponding IN. Data are
normalized to the amount of processing; similar results were obtained
when the absolute amounts of alcohol adducts were compared. The data
are presented as the mean ± S.D. of multiple experiments with the
indicated combinations of integrase and divalent cation; each reaction
was conducted an average of 5 times (range, 2-8).
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Nucleophile Selection for Nicking Nonviral DNA--
Integrase can
use water or various exogenous alcohols as the nucleophile for
nonspecific nicking of nonviral DNA (Figs. 1D and
7). Any site that is not close to the end
of the DNA substrate can be nicked by this action of integrase.
Nonetheless, different integrases exhibit distinct preferences for
nicking certain sites in the target DNA (9). The pattern of target site
selection is readily apparent when substrates are labeled at the
5'-end, whereas the relative choice of nucleophile is revealed by the use of substrates labeled near the 3'-end. Thus, the two types of
experiments yield complementary information. Nonspecific alcoholysis assays were performed with Mn2+ as the divalent cation,
because RSV integrase catalyzes this reaction inefficiently in the
presence of Mg2+ (9) and we have not detected
Mg2+-dependent nonspecific alcoholysis by HIV-1
or visna virus integrase.

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Fig. 7.
Hydrolysis versus
alcoholysis of nonviral DNA. A single nick on one DNA strand
is shown. The nucleophilic oxygen (with its donor electrons indicated
by two dots) is provided by an OH group of water
(HOH) or an alcohol (ROH). In both schemes, the
attacking oxygen nicks on one side of a phosphodiester bond and joins
to the newly exposed 5'-phosphate group at the site of DNA cleavage. If
the radiolabel is on the 5'-end of the substrate (as indicated by
asterisks), all labeled products from either pathway
comigrate on gels with similarly labeled oligonucleotide markers. If
the label is near the 3'-end of the DNA (as indicated by
circles), only the labeled hydrolysis products comigrate
with these markers and the labeled alcoholysis products migrate as a
function of the attached alcohol.
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Several important observations resulted from experiments that used a
5'-labeled nonviral DNA substrate. Perhaps most striking, the total
amount of DNA nicking was dramatically augmented by increasing
concentrations of exogenous alcohols. In the presence of no or minimal
amounts of the alcohols, HIV-1 integrase (Fig. 8, A and
D) and visna virus integrase
(Fig. 8, B and E) nicked about 15% of the DNA
substrate by hydrolysis, whereas more than 50% of the substrate was
nicked with high concentrations of the alcohols. Even RSV integrase,
which nicked about 50% of the DNA in the absence of alcohols, was
stimulated to nick an additional 20% of the substrate with optimal
amounts of the alcohols (Fig. 8, C and F). When
reactions were performed with lower concentrations of RSV integrase to
reduce the baseline amount of hydrolysis, stimulation similar to that
seen for the HIV-1 and visna virus enzymes was observed (data not
shown). Importantly, no nicking occurred when the DNA was incubated
with high concentrations of alcohol in the absence of integrase
(lane 1 in Fig. 8, A-C). Similar results were
obtained with glycerol, 1,2-ethanediol, 1,2-propanediol, and
1,3-propanediol (although total nicking was not as high with the last
chemical). Thus, these agents do not merely compete with water but also
stimulate the nonspecific nuclease activity of integrase.

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Fig. 8.
Alcohol stimulates nonviral DNA nicking by
integrase. A-C, double-stranded nonviral 23-mers (the
sequence is shown in Fig. 9D) were 5'-labeled on one strand
and incubated with glycerol-free preparations of the indicated
integrase (IN) and increasing amounts of 1,2-propanediol, as
indicated by % ROH. Sequence-specific markers
(M) in panel A are relevant for all three sets of
reactions, which are from adjacent parts of a single gel. Numbers
beneath the lanes are aligned with the lower parts of the lanes. The
sizes in nucleotides of the preferential cleavage sites for each IN are
indicated to the right of the panels. D-F, the total amount
of DNA nicking by the indicated enzymes in the presence of various
nucleophilic alcohols was quantified. Each curve represents the average
of all experiments in which >5% of the substrate was nicked. The
average number of reaction sets for each curve was 3 (range,
2-7).
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The distinct target site preferences of the various integrases are
evident in Fig. 8. HIV-1 integrase preferentially nicked this substrate
at positions that are 8, 11, 15, and 17 nucleotides from the 5'-end,
whereas visna virus integrase preferred positions 5, 15, and 19 and RSV
integrase preferred positions 8, 12, 15, and 19. The patterns of
prominent products were generally independent of the presence or amount
of the alcohols (for example, compare lanes 2-4 with
lanes 7-9 in panels A, B, and
C of Fig. 8). The only apparent exceptions are the
diminished amounts of 19-mers and 15-mers produced by RSV integrase in
reactions with the highest alcohol concentrations (Fig. 8C,
lanes 8 and 9). However, time-course studies
proved that these findings actually were due to further nicking of the
19- and 15-mers to yield the very prominent 8-mer product under
conditions in which the 23-mer substrate was being depleted (not
shown). Thus, the nicking pattern was independent of the alcohol
concentration. It should also be noted that, although 1,2-propanediol
was used for Fig. 8, the same patterns of preferential sites were
obtained when the other three alcohols were used (not shown). These
data suggest that the selection of target sites is not influenced by
which nucleophile integrase uses to nick nonviral DNA. However, the
experiments do not reveal which nucleophile actually was used, because
it is possible that all of the nicked products resulted from hydrolysis.
To ascertain which nucleophile was used, experiments were performed
with substrates labeled near the 3'-end (as depicted in Fig. 7). Under
these conditions, products of hydrolysis comigrate with an
oligonucleotide ladder (Fig. 9A, lane
M), whereas alcohol adducts migrate
to novel positions that are a function of the particular alcohol (Fig.
9, results are shown only for 1,2-propanediol). As expected, each
integrase exhibited the same site preferences with the 3'-labeled
substrate as with the 5'-labeled DNA. The positions of the prominent
alcoholysis products in Fig. 9 were interpolated from the
oligonucleotide positions. Each of these bands correlates with a
prominent band produced from the 5'-labeled DNA. For example, the band
migrating as a "9.7-mer," created from the 3'-labeled 24-mer
substrate, reflects the same nicking event that created a 15-mer
product from the 5'-labeled 23-mer substrate (as demonstrated
diagrammatically in Fig. 9D). However, additional information was provided by the experiments with 3'-labeled substrates. For example, the nick at the position 15 nucleotides from the 5'-end,
which created the labeled 15-mer in Fig. 8A, produced bands
at the 9 and 9.7 positions in Fig. 9A, reflecting hydrolysis and alcoholysis, respectively. Similarly, the nick at the position 11 nucleotides from the 5'-end, which created the labeled 11-mer in Fig.
8A, produced bands at the 13 and 13.5 positions in Fig. 9A. Thus, the amounts of hydrolysis and alcoholysis at
specific sites can be compared.

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Fig. 9.
Choice of nucleophile with nonviral DNA.
A-C, double-stranded nonviral 24-mers labeled near the
3'-end of one strand were incubated with glycerol-free preparations of
the indicated integrases and increasing amounts of 1,2-propanediol,
as indicated by % ROH. Hydrolysis markers (M) in
panel A are relevant for all three sets of reactions, which
are from adjacent parts of a single gel. The apparent sizes in
nucleotides of prominent products that migrate differently from the
hydrolysis markers were interpolated from the autoradiogram and are
indicated to the right of the panels. D, the sequence of the
labeled strand of the nonviral 24-mer substrate used for the reactions
in panels A-C is shown; the radiolabel is near the 3'-end
(as indicated by the circle). The 23-mer substrate for the
reactions shown in Fig. 8 was labeled at the 5'-end (as depicted by the
asterisk) and did not contain the final 3'-base pair. Also
indicated are the relationships of the prominent alcoholysis products
seen in panels A-C (numbered from the 3'-end and shown
above the sequence) to the preferred cleavage products seen
in Fig. 8 (numbered from the 5'-end and shown below the
sequence). Note that the effect of the attached alcohol on migration
through the gel diminished as the length of the 3'-end-labeled products
increased.
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Inspection of the autoradiograms in Fig. 9 and similar experiments
permitted several conclusions. First, the presence of the alcohols
generally had little effect on the amount of hydrolysis by integrase.
Almost all of the additional DNA nicking was from integrase-mediated
alcoholysis, which was evident with as little as 4% of the alcohols.
We have even detected alcohol adducts with 0.1% of exogenous alcohol
(data not shown). Second, at high alcohol concentrations, integrase
sometimes used the exogenous nucleophile in place of water, as
indicated by diminished amounts of hydrolysis and increased amounts of
alcoholysis at the same position (e.g. compare the bands at
the 16 and 16.4 positions in Fig. 9C). Third, under optimal
conditions, the integrases used the alcohols comparably to or more
often than water. This fact is revealed by comparing the sum of the
intensities of the alcoholysis products with the corresponding
hydrolysis products in Fig. 9 (more evident in panels A and
B but also seen in other experiments with RSV integrase). Finally, the target site preferences exhibited by each integrase were
the same for hydrolysis and alcoholysis (compare lanes 2 and
9 in each of Figs. 9A, B, and
C). Moreover, these preferences were independent of the type
of alcohol used as the nucleophile, because similar results were
obtained with all four alcohols tested (data not shown). These results
prove that selection of target sites was not influenced by which
nucleophile integrase used to nick nonviral DNA.
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DISCUSSION |
Retroviral integrase is a multifunctional enzyme that is
responsible for the permanent incorporation of a DNA copy of the retroviral genome into cellular DNA. Knowledge of the biochemistry of
how integrase functions is critical for obtaining a more complete view
of the virus life cycle and for new treatments for the acquired immunodeficiency syndrome and other retrovirus-related diseases. In
particular, how integrase interacts with its various substrates and how
these substrates interact with each other are poorly understood. To
examine how the target DNA and nucleophile affect each other during
reactions catalyzed by integrase, the experiments in this report
compared nucleophile selection by different integrases during the
activities that accommodate a variety of nucleophiles.
Processing of viral DNA ends in preparation for insertion into cellular
DNA is the first enzymatic action required of integrase. In
vitro studies have identified three types of nucleophiles that can
provide the OH group for nicking near the viral DNA ends, i.e. water (3), the 3'-end of the unintegrated DNA (1), and
certain alcohols. Exogenous alcohols shown previously to participate in
this reaction either have OH groups on adjacent carbon atoms, including
glycerol, 1,2-ethanediol, and 1,2-propanediol (3), or have OH and amino
groups on adjacent carbons, such as the amino acids serine and
threonine (3, 4). In contrast to a report that 1,3-diols do not
participate, we found that the retroviral integrases from two
lentiviruses (HIV-1 and visna virus) and an oncovirus (RSV) can use
1,3-propanediol for processing, although less efficiently than
1,2-diols (Fig. 6). It is likely that the structure of acceptable
nucleophiles reflects the configuration of the site on the protein that
interacts with the nucleophilic donor before or during catalysis.
We found that the three integrases studied in this report exhibited
different nucleophile selectivities during processing. Consistent with
previously published data for Mn2+-dependent
reactions (1, 3, 12, 18), HIV-1 integrase and visna virus integrase
primarily used water rather than the viral DNA ends as the nucleophile,
whereas RSV integrase had a strong preference for using the 3'-OH at
the viral DNA end (Table I). These results have now been documented in
reactions performed in the absence of exogenous nucleophiles by using
glycerol-free integrase preparations, which had previously been done
only for the HIV-1 enzyme (3). Moreover, when reactions were
supplemented with optimal concentrations of alcohols, HIV-1 integrase
used the alcohols almost as often as water, whereas visna virus
integrase used the alcohols somewhat less often than water. In marked
contrast, RSV integrase used the alcohols inefficiently. When
processing reactions were conducted with Mg2+ as the
divalent cofactor, the preference of the HIV-1 and visna virus
integrases for water became even stronger and minimal amounts of the
other products were detected, consistent with previous reports for
HIV-1 or HIV-2 integrase (3, 19, 20). Although the use of water by RSV
integrase also was facilitated in reactions with Mg2+, as
described by others (18), we found that the avian enzyme made cyclic
products and alcohol adducts even with this cation. The distinct
preferences of the three enzymes are unlikely to be due to membership
in different retrovirus subfamilies, because preferential usage of the
3' viral DNA end as the nucleophile for processing has also been
described for murine leukemia virus (21) and feline immunodeficiency
virus integrases (22). Rather, these preferences must reflect subtle
differences in the structure of the various proteins, as suggested by
crystallographic studies of the HIV-1 and RSV enzymes (23-25). This
conclusion is consistent with the finding that certain amino acid
substitutions in the central region of HIV-1 or HIV-2 integrase affect
the relative amounts of the various dinucleotide products created by
processing (19, 26). Similarly, the influence of the divalent cation on
nucleophile selection for processing may reflect metal-induced conformational changes of the enzyme (27). Steric effects and electrostatic interactions between integrase and substrate DNA also
have been suggested to contribute to the choice of nucleophile during
processing (20).
Nonspecific alcoholysis, the recently discovered fourth activity of
integrase, is the most potent action of several integrase proteins (13,
28). This activity uses a variety of nucleophiles (as does processing),
nicks almost any target DNA site (as does joining), and only requires
the central domain of the protein (as does disintegration). Thus,
nonspecific alcoholysis has a unique combination of characteristics
that makes it a useful tool for understanding how integrase selects
nucleophiles and target sites for catalysis. We have previously
emphasized the relationship of this activity to joining, because both
activities attack multiple DNA sites, lack a target consensus sequence,
avoid 5'-ends of substrates, and display site preferences that are a
function of the target DNA sequence and the viral source of integrase
(29). We have now found that HIV-1 integrase and visna virus integrase exhibit similar patterns of nucleophile selection during nonspecific alcoholysis reactions, whereas RSV integrase performs greater hydrolysis. The high baseline level of hydrolysis by RSV integrase was
not due to contaminating nucleases, because it demonstrated the
signature nicking pattern (9) characteristic of the avian enzyme (Figs.
8C and 9C). In fact, the patterns of preferential nicks created by each enzyme were the same whether the enzymes were
catalyzing hydrolysis or alcoholysis, consistent with the idea that
these reactions reflect one catalytic mechanism. Moreover, the nicking
patterns were independent of which exogenous alcohol was used. Thus,
the nucleophile did not affect the choice of target DNA sites.
In contrast to the above conclusion, the target DNA did affect
selection of the nucleophile. In fact, several differences in
nucleophile usage were noted between processing and nonspecific alcoholysis, reactions that differ merely by the DNA that is presented to integrase. Most striking was the finding that exogenous nucleophiles stimulated the nonspecific nuclease activity of integrase. Much of the
additional nicking of nonviral DNA was due to the use of exogenous
alcohols as the attacking nucleophile (Fig. 9), whereas the same
alcohols only competed with water during processing (e.g. Fig. 5). In addition, the various 1,2-diols were used more equivalently during nonspecific alcoholysis than during processing (Figs. 6 and 8).
Moreover, the difference in usage of a 1,3-diol compared with the
1,2-diols appeared to be less during nonspecific alcoholysis than
during processing. Finally, RSV integrase used water relatively inefficiently as the nucleophile for processing viral DNA ends (Table
I) but performed high levels of hydrolysis on nonviral DNA (Figs. 8 and
9).
The ability to accommodate a diversity of nucleophiles suggests
considerable flexibility for the active site of integrase (1). Indeed,
we observed that the use of exogenous alcohols could exceed the use of
water during processing or nonspecific alcoholysis (Figs. 5 and 9).
Although the alcohol concentrations reached in these experiments are
relatively high (40% glycerol or propylene glycol corresponds to 5.4 M and 40% ethylene glycol corresponds to 7.2 M), they are much less than the 33 M
concentration of water in a solution that is 60% water. How integrase
chooses various nucleophiles for these reactions is unclear, but
current evidence indicates that the nucleophiles interact with
integrase as specific substrates rather than merely as solvent
molecules attacking DNA that is appropriately positioned by the enzyme. Most importantly, use of the nucleophiles involves specificity because
not all compounds are used equivalently. For example, ethanol and
2-propanol were not used by integrase (3, 9). Moreover, many compounds
that inhibit integrase in vitro have adjacent OH groups (30,
31), as do the most active alcohols in these assays, suggesting that
these agents act at a similar protein site.
It is important to note that the cyclic dinucleotide and alcohol
adducts described in these experiments are stable products that do not
convert to hydrolysis products. This fact was confirmed by purifying
the different products from gels, incubating them under various
conditions, including exposure to alkali, and re-examining them by gel
electrophoresis (data not shown). Thus, our measurements reflect the
true distribution of the various products. Moreover, the relative
distribution of the various products was not affected by the extent of
the reaction. In particular, time-course studies of the processing
reaction showed similar kinetics of appearance of the three types of
dinucleotide products. In addition, the relative amounts of the various
products were not affected by the concentration of substrate DNA when
initial rates of product formation were measured during the early,
linear phase of the time course. Analogous results were obtained for
the nonspecific alcoholysis reaction when the kinetics of appearance of
the hydrolysis and alcoholysis products and the initial rate of
formation of these products as a function of DNA concentration were
compared. Furthermore, the concentrations of integrase used in this
report were within the linear part of the reaction curve for both types of assays when initial rates of product formation were compared with
enzyme concentration (data not shown). Interestingly, the amount of
product formed in these experiments was always less than the amount of
HIV-1 and visna virus integrase present and only a low level of
turnover was detected for RSV integrase (and only during
Mn2+-dependent reactions). Although the
fraction of integrase that was catalytically active is unknown, these
results are consistent with the findings of others for HIV-1 and RSV
integrase during processing (32-35). Low or absent turnover may
reflect stable complex formation between integrase and substrate DNA
(36-41), which is mechanistically appealing because processing can
occur in the cytoplasm of a cell but DNA joining does not occur until
after the preintegration complex has entered the nucleus. Moreover, multiple turnovers are unnecessary, because integrase is only required
to nick two viral DNA ends and two strands of host DNA. In
vivo, the ratio of integrase molecules to viral DNA ends is about
75:2 (33), which is approximated by typical in vitro
conditions. Together, these additional experiments and observations
strengthen the validity of our conclusions regarding the choice of
nucleophile by the various integrases.
The most important new findings from this work are that the target DNA
influenced the choice of nucleophile during reactions catalyzed by
integrase, but the nucleophile did not affect the choice of target DNA
sites. These functional observations suggest that integrase achieves an
active conformation only after DNA becomes bound to the protein. A
similar suggestion has been made from recent structural information
(42, 43), although crystals of integrase with bound DNA have not been
described. In the context of retroviral integration, this conclusion
suggests that: 1) regardless of which nucleophile for processing has
been bound, integrase binds a viral DNA end and reaches an active
conformation poised to nick this target at the site 3' to the CA, and
2) whether or not the processed viral DNA end has been repositioned as
the attacking nucleophile for joining, integrase binds cellular DNA and
reaches an active conformation poised to nick this target at certain
sites. Although the data cannot order the integrase-nucleophile and
integrase-target DNA interactions, they suggest that interaction with
target DNA is the critical step before each catalytic event. Thus,
these results highlight an aspect of retroviral integration that should serve as the focus for efforts to develop antiretroviral agents.