From the Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces, New Mexico 88003
Received for publication, August 24, 2000, and in revised form, January 26, 2001
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ABSTRACT |
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Retroviral integrases (INs) interact with termini
of retroviral DNA in the conserved 5'-C(A/G)T. For most integrases,
modifications of critical moieties in the major and minor grooves of
these sequences decrease 3'-processing. However, for human
immunodeficiency virus type-2 (HTLV-2) IN, the replacement of the
guanine with 6-methylguanine or hypoxanthine not only reduced
3'-processing, but also promoted cleavage at a second site. This novel
cleavage activity required an upstream ACA, unique to the HTLV-2 U5
end. 3'-Processing assays with additional isosteric modifications at
Gua and filter binding experiments revealed that the mechanism of the
second site cleavage differed among the major groove, minor groove, and
mismatch modifications. Importantly, the decrease in 3'-processing
activity noted with the minor groove and mismatch modifications were
attributed to a decrease in binding. Major groove modifications,
however, decreased the level of 3'-processing, but did not affect
binding. This suggests that integrase binds the viral end through the
minor groove, but relies on major groove contacts for 3'-processing.
Several modifications were also examined in strand transfer and
disintegration substrates. HTLV-2 IN showed reduced activity with
strand transfer and disintegration substrates containing major groove,
but not minor groove modifications. This suggests major groove
interactions at guanine also provide an important role in these reactions.
The retroviral integrase
(IN)1 catalyzes the covalent
insertion of the retrovirus genome into the host chromosome following reverse transcription of the viral RNA genome to a DNA intermediate (1,
2). Successful integration of the viral DNA by IN requires three
distinct enzymatic events: 3'-processing, strand transfer, and gap
repair (3, 4). In the initial step of integration, IN catalyzes the
hydrolysis of the phosphodiester bond between the phylogenetically
conserved CA and the two or three terminal nucleotides from each 3' end
of the viral DNA. 3'-Processing of the newly synthesized viral DNA
intermediate occurs in the cytoplasm (5). The strand transfer reaction
follows in the nucleus where IN uses the 3'-hydroxyl group of each
viral end to catalyze a direct nucleophilic attack at staggered
5'-phosphates in the phosphodiester backbone of the host DNA (6). IN
can also catalyze the reverse of strand transfer, termed disintegration
(7). Each of these reactions has been characterized in vitro
with double-stranded oligonucleotide substrates that mimic the end of
the viral DNA molecule (Fig. 1A). The final step, gap
repair, requires host cellular enzymes and perhaps IN to remove the
unpaired nucleotides at the 5' ends of the viral DNA and complete the
filling-in and joining of the single-strand gaps between the viral and
host DNA.
Numerous studies support the contributions of the conserved CA as
necessary for recognition and catalysis by IN (5, 6-12). In addition,
IN relies on at least one additional nucleotide upstream of the CA to
distinguish its substrate from non-cognate viral long terminal repeat
ends. Although most INs have some level of activity with heterologous
viral ends (13). IN shows an equal binding affinity to both nonspecific
and specific (viral) DNA substrates (14-16), and therefore, precise
recognition of the CA may occur during catalysis. Previously we showed
the conserved C(A/G)T, particularly at the guanine and adenine
residues, share a common scaffold of molecular features which are
recognized by HIV-1, HTLV-2, and Moloney murine leukemia virus
INs during 3'-processing or dinucleotide cleavage (17). Isosteric
nucleotide analogue substitutions in these positions suggested both
major groove and minor groove interactions in this region with IN (17).
For example, insertion of an amino group into the minor groove at the
Ade or position 3 in the plus strand or its removal at Gua or position 4 in the minus strand decreases activity in each of these INs. In
contrast, these INs tolerate the insertion of a methyl group into the
major groove of their U5 substrates at positions 3 and 4 in the plus
strand and position 3 in the minus strand. However, the introduction of
a methyl group using the nucleotide analogue O6-methylguanine (6-MeGua) at position 4 in the
minus strand of the U5 substrate produces a dramatic decrease in the
level of 3'-processing activity.
In this work, we extended the use of analogue nucleotide substitutions
at Gua at position 4 in the minus strand to characterize the essential
components of viral DNA substrate recognition and catalysis by HTLV-2
IN. Clear distinctions were observed in the major and minor groove
contacts required for 3'-processing, strand transfer, and
disintegration. However, in the process of these studies, a novel
second site cleavage activity was uncovered in the HTLV-2 U5, but not
the U3 substrate. The cleavage followed an upstream ACA sequence unique
to the HTLV-2 U5 substrate. Cleavage at the upstream site required the
ACA sequence and a modification of the Gua at position 4. The level of
the upstream cleavage activity at this site was modulated by the type
of nucleotide substitutions or modifications and differed among the
major groove, minor groove, and mismatch modifications. Our results
support a model in which IN initiates binding of the viral end through
the minor groove, but relies on major groove contacts for
3'-processing, strand transfer, and distintegration.
Oligonucleotide Substrates--
Oligonucleotide sequences
corresponding to the wild type (WT) U5 and/or U3 ends of the HTLV-2
genomes (Fig. 1C) were used as substrates in enzymatic
assays as described previously (17). Oligonucleotide synthesis,
introduction of 2'-deoxynucleoside phosphoramidite analogues, and high
performance liquid chromatography purification of oligonucleotides were
performed at Integrated DNA Technologies. Oligonucleotide size
standards (8-32) were purchased from Amersham Pharmacia Biotech.
Protected 2'-deoxynucleoside phosphoramidites were purchased from Glen
Research. Oligonucleotides were further purified on 20% denaturing
polyacrylamide gels, 5'-end-labeled with [ Protein Purification--
HTLV-2 IN was expressed in
Escherichia coli BL21(DE3) cells and purified as
hexahistidine-tagged fusion proteins from the insoluble fraction as
described previously (17, 18). For native preparation of HTLV-2 IN,
cultures were prepared as previously described (17) with the following
modifications in the cell lysis. Pellets were resuspended in 10 ml of
extraction buffer (50 mM Tris, pH 8.0, 500 mM
NaCl, 10 mM imidazole) supplemented with 1 mg/ml lysozyme
(Sigma) and EDTA-free protease inhibitor tablets (Roche Molecular
Biochemicals). The resuspended pellet was incubated on ice for 30 min.
Triton X-100 was added to a 1% (v/v) concentration and the resulting
solution was homogenized by douncing. The viscous cell lysate was then
cleared by sonication, 5 × 30 s with 1 min rest in between
pulses on ice. Clearing of lysates and native His-Tag IN purifications
were performed as described (17). Protein concentrations were measured
by the Bradford method (19) using the Bio-Rad Micro-Assay.
Integration and Disintegration Assays--
Reaction buffer for
HTLV-2 IN contained 25 mM MOPS (pH 7.2), 10 mM
Filter Binding Assays--
U5 end substrates were purified,
32P-labeled as described, and hybridized at a molar ratio
of 1:1. Binding reactions were assembled on ice with 1.0 pmol of
substrate and 10 nM to 2.1 µM HTLV-2 IN in
reaction buffer (25 mM MOPS, pH 7.2, 10 mM
Substitution of Hyp or 6-MeGua at for Gua at Position 4 Blocks
Precise Recognition and Catalysis of the U5 End Substrates by HTLV-2
IN--
Previously, we noted that the replacement of guanine (Gua) at
position 4 in the minus strand of the U5 substrate with 6-MeGua (4/6-MG) or Hyp (4-Hyp) decrease 3'-processing activity as compared with wild type U5 end substrate for several INs, HTLV-2, Moloney murine
leukemia virus, and HIV-1 (17). 6-MeGua and Hyp introduce changes in
the major and minor groove, respectively (Fig.
1D). Further analysis of the
HTLV-2 IN reactions catalyzed with these two substrates, 4-Hyp and
4/6-MG, revealed a novel pattern of cleavage (Fig.
2, lanes 4 and 6)
as compared with the wild type (WT) U5 end substrate (Fig. 2,
lane 2). In each modified substrate, a novel cleavage site
occurred 3' to position 7 (Fig. 1C), and, of interest, this
site follows a second internal CA in the HTLV-2 U5 end substrate. A low
amount of additional heterogenous cleavages were also noted adjacent to
the major novel cleavage site.
To further explore the mechanism involved in promoting cleavage at the
internal site in the HTLV-2 U5 substrate, 6-MeGua was introduced in the
minus strand at position 8 in addition to the original analogue
substitution at position 4. It was expected that IN's cleavage activity
would be blocked at the conserved CA and the second internal CA.
Surprisingly, we observed an additional distinct pattern of cleavage
following catalysis with IN. Imprecise cleavage occurred at two sites
in the substrate, following positions 5 and 9; both of which were 5' to
the conserved CA and the internal CA (Fig. 2, lane 8). A
time course of this reaction revealed no relationship between the
cleavage of these two sites; i.e. the two sites were cleaved
independently.2 The unique
activities displayed by IN with the single and double modifications
suggested that the effect of 6-MeGua may not cause a simple block in
the hydrogen bonding between IN and Gua in the major groove as
interpreted for other protein-DNA interactions (20). We hypothesized
that 6-MeGua may generate a change in the geometry or conformation of
van der Waals and hydrogen bonding interactions at the ACA through a
structural alteration in the G-C base pair that affects the manner in
which IN recognizes, binds, and/or cleaves the DNA substrate. In the
following, we first address whether the upstream CA or ACA was required
for cleavage.
ACA Is Sufficient for Catalysis by the HTLV-2 IN, But Not
Sufficient for Cleavage at the Internal Site--
To determine whether
the ACA context was required for the observed internal cleavage
activity, we examined HTLV-2 IN activity with the HTLV-2 U3 substrate
and the U3 substrate with the 6-MeGua substituted opposite of the C in
the conserved CA (U3-4/6-MG). The WT HTLV-2 U3 sequence was chosen
because it does not have an internal CA at this position, although it
does have a CA further upstream (Fig. 1C). The expected
dinucleotide cleavage pattern was observed with the unmodified, WT U3
end substrate (Fig. 3A, lane
2), while the U3-4/6-MG substrate showed a 5-fold decrease in
this product (Fig. 3A, lane 4) as compared with the WT U3
substrate. Neither the WT U3 nor U3-4/6-MG substrates showed internal
cleavage when incubated with HTLV-2 IN. Previously, we showed that the ACA sequence was important for recognition and 3'-processing of the U5
substrate by HTLV-2 IN by comparison with its activity on heterologous
retroviral substrates from HIV-1, HTLV-1, and Moloney murine leukemia
virus (13). Therefore, we hypothesized that mutation of the U3
substrate sequence from 5'-GGT to 5'-ACA in positions 7, 8, and 9 coupled with the 6-MeGua substitution at position 4 in the terminal
conserved C(A/G)T were required for cleavage of the ACA engineered into
the U3 substrate. As predicted, the modified substrate, U3-4/6-MG-ACA,
was processed by the HTLV-2 IN at the internal CA (Fig. 3A, lane
6). These experiments support the hypothesis that the
modifications created at position 4 in the minus strand were required
for 3'-processing at the upstream ACA.
The above results suggested that the ACA was necessary and sufficient
for recognition and catalysis of the viral end substrates by HTLV-2 IN,
and that such a sequence may be recognized internally in a non-U3 or U5
context. To determine whether the ACA was alone sufficient for
3'-processing, non-U3 or U5 end
substrates (NUS) with ACA (3-ACA NUS) and without (NUS)
were designed and tested (Fig. 1C). 3'-Processing of the
3-ACA NUS was observed (Fig. 3B, lane 7). A random
substrate, included as a control (NUS), was recognized, but was
processed at much lower level compared with WT (Fig. 3B,
compare lanes 5 and 3). Three additional random
substrates, 5-ACA NUS, 6-ACA NUS, and 7-ACA NUS (Fig. 1B),
were designed to test whether HTLV-2 IN could recognize and process the
ACA when placed internally in a random substrate context. These
substrates were examined in standard reactions along with the WT U5 end
substrate (Fig. 3B, lane 2). HTLV-2 IN mediated catalysis
was noted in reactions in which the substrate, ACA-NUS, had ACA placed
at positions 3 through 5 (Fig. 3B, lane 7), positions 5 through 7 (Fig. 3B, lane 9), or positions 6 through 8 (Fig.
3B, lane 11). These reactions produced products that were
chiefly composed of internal cleavages 3' to the internal ACA. Thus,
the 3-ACA reaction produced an 18-mer, the 5-ACA reaction resulted in a
16-mer, and the 6-ACA reaction produced a 15-mer. NUS substrates that
contained ACA at positions 7 through 9 were not 3'-processed by the
HTLV-2 IN (Fig. 3B, lane 13). This suggests that IN can only
gain access to internal ACA sequences if they are placed within the
first 6 bases of the 3' end. Strand transfer was evident with both the
5-ACA NUS and 6-ACA NUS substrates (Fig. 3B, lanes 9 and
11). This serves as further confirmation that the HTLV-2 IN
relies solely on the ACA for complete processing of these substrates.
Major Groove Modifications or a Base Pair Mismatch at Position 4 Promote Internal Cleavage--
To explore whether a simple lack in the
hydrogen bonding contact in the major groove in Gua at position 4 was
promoting catalysis at the second site, three additional modifications
were made. 6-Thio-Gua, 2-aminopurine (2-AP), or Cyt (Fig.
1D) were substituted for Gua in the U5 substrate (Fig.
1C). The 6-thio-Gua is a conservative substitution in that
the sulfur atom of the thioketo group can act as a weak hydrogen bond
acceptor, and unlike the 6-MeGua substitution, 6-thio-Gua maintains
Watson-Crick base pairing with Cyt (21, 22). However, the hydrogen bond
length for N-H···S distance is about 0.4 Å greater than
the N-H···O distance (21, 22). Furthermore, the van der Waals
radius of sulfur is 0.45 Å greater than oxygen and this may introduce
propellar distortion. 2-AP simply removes the carbonyl functional group
from Gua, and the Cyt substitution created a mismatch at this position.
Dinucleotide cleavage of the 4/6-thio-Gua substrate by HTLV-2 IN showed
levels similar to the WT substrate (Fig.
4A, compare lanes 2 and 4). This suggested that carbonyl oxygen in the major groove might not be an important hydrogen bonding acceptor during recognition or catalysis of the U5 end substrate by IN. A smaller amount of cleavage, 1.8-fold decreased as compared with WT dinucleotide level, was also noted at the second site (Fig. 4A, lane 4).
From this result, one might predict that complete removal of the
carbonyl group would not effect 3'-processing. However, catalysis of
the substrate with the 2-AP substitution showed a 4-fold decrease in
3'-processing (Fig. 4B, lane 4). This argues for
interactions at the major groove in this position. Furthermore, this
suggests that we are observing two distinct activities with these
substitutions. One activity results from the absence of a major groove
acceptor, which causes a decrease in dinucleotide cleavage. The other
results from the presence of the isosteric modification, which promotes cleavage at the upstream ACA.
Previously, Scottoline et al. (23) has shown that the
insertion of 2-base pair mismatches immediately 3' to an internalized CA end will promote internal cleavage of viral DNA substrates. In
contrast to this result, we observed internal cleavage at a distance of
3 base pairs when we introduced 6-MeGua in the position 4 of the minus
strand, i.e. the third base pair downstream from the
internal CA. To further probe the influence of distortion at the
position 4, a mismatch was made by substitution of Cyt for Gua (4C/C,
Fig. 1C). A mismatch can create much more distortion in a
B-DNA than isosteric nucleotide modifications. 3'-Processing of this
substrate by HTLV-2 IN produced cleavage at the internal CA (Fig.
4A, lane 6). Similar results were also observed for a G-G
base pair substitution at this
site.3 A summary of
activities of several of the modified substrates used in the cleavage
assays is presented in Fig. 5. The result obtained with the 4 C/C substrate, combined with the isosteric substitutions presented above, suggests that the greater the local structural distortions at position 4, the greater the decrease in the
dinucleotide product and increase in catalysis at the upstream site. In
summary, these results reflect two modes by which IN may interact with
the viral end: one which relies on interactions at the major groove
contact at this position for 3'-processing, and second, a mechanism
that directs recognition and cleavage to the upstream site in the
absence of a "correct end."
Effect of Minus Strand Substitutions on Strand Transfer and
Disintegration Activity of HTLV-2 IN--
We were also interested in
what effect both major and minor groove alterations had on recognition
and catalysis of the strand transfer and disintegration substrates.
Previous work with the disintegration substrates has strongly argued in
favor of an indirect as opposed to direct sequence readout of the
substrate (13, 24-26). 6-MeGua and Hyp were substituted at position 4 in the minus strand of precleaved and disintegration substrates of
HTLV-2 (Fig. 1, A and B). In contrast to our
results with the blunt end substrates in the 3'-processing reaction
which showed a 5-fold decrease with Hyp (Fig. 2), the precleaved
substrate containing the Hyp substitution, 4/Hyp, showed levels of
activity similar to the WT substrate in HTLV-2 IN mediated reactions
(Fig. 6A, compare lanes
2 and 4). Moreover, strand transfer activity was not
detected when the 6-MeGua substitution was introduced into position 4 (Fig. 6A, lane 6) or positions 4 and 8 (Fig. 6A, lane
8). Comparison of the strand transfer reaction products generated
by HTLV-2 IN with WT, 4/6-MG, and the 4,8/6-MG blunt substrates showed
cleavage at the internal CA in the 4/6-MG, but not the 4,8/6-MG or WT
substrate (see Fig. 2). This suggests the internally processed 4/6-MG
substrate was not viable for subsequent strand transfer events. The
major groove substitution, 2-AP, was also introduced into precleaved
substrate. This substrate showed no strand transfer products when
incubated with HTLV2 IN as compared with the WT substrate (Fig.
6C, lane 4).
Reactions with HTLV-2 IN and modified disintegration substrates were
also examined. Similar to IN catalyzed strand transfer reactions with
the precleaved substrate containing the Hyp substitution, the Hyp
replacement at position 4 in the disintegration substrate had little
effect on IN catalysis as compared with activity with the WT
disintegration substrates. This substrate yielded a 1.3-fold decrease
in disintegration products as compared with the WT substrate (Fig.
6B, compare lanes 2 and 4).
PhosphorImager analysis of the products generated from the 4/6-MG and
the 4,8/6-MG disintegration substrates showed a greatly reduced level
of activity, an 8-10-fold decrease, as compared with the WT
disintegration substrate (Fig. 6B, lanes 6 and
8). The introduction of 2-AP into position 4 reduced disintegration activity 10-fold (Fig. 6D, lane 4) as
compared with WT (Fig. 6D, lane 2).
Major Groove, not Minor Groove or Mismatch Substitutions in the U5
End Substrate, Enhance the Binding Affinity of HTLV-2 IN--
The
activities observed in the previous experiments could be due to binding
and/or catalytic interactions with the various substrates. To define
the contribution of the initial DNA binding interactions, we have
employed a nitrocellulose filter binding assay to measure the DNA
binding affinity of IN for several of the U5 substrates used herein. It
has been demonstrated that IN displays increased specificity for viral
DNA ends in the presence of divalent metal ions (27). Therefore,
binding reactions were performed in the presence of metal under
standard reaction conditions using a natively purified HTLV-2 IN and
HTLV-2 U5 and modified substrates.
Four types of modified U5 substrates were analyzed; those with major
groove and minor groove modifications and those with mismatches and
substitutions in the conserved C/G base pair at position 4. An
additional random substrate, Ran, was also included. The minor groove
substitution Hyp, located at position 4 in the minus strand, bound
significantly less than the WT HTLV-2 U5 substrate at all
concentrations (Fig. 6). Its level of binding was similar to the random
substrate, Ran. Substitutions in the major groove of the conserved G
included 4/6-MeGua and 2-AP (Fig. 7).
Both of these substrates behaved equivalently in the filter binding assay. They bound more than 10 to 15%, respectively, higher than the
WT substrate at 3.6 × 10 Similar to other INs, the 3'-processing activity of HTLV-2 IN
tolerates the insertion of a methyl group into the major groove of the
HTLV-2 U5 substrate at positions 3, 4, 5, and 7 in the plus strand and
position 3, 5, and 7 in the minus strand, but not at position 4 in the
minus strand (17). Introduction of a methyl group at the carbonyl
oxygen at position 6 in the carbonyl ring decreased 3'-processing
activity and promoted cleavage activity at an upstream CA. The sequence
ACA was shown to be required for this novel activity. Two plausible
hypotheses for the effect of the 6-MeGua on the 3'-processing reaction
were explored. Briefly, our first hypothesis suggested the
O6-methyl group might interfere with
3'-processing of the U5 substrate because it blocks the potential
hydrogen bonding acceptor site in the major groove (20, 28). However,
it has also been reported that 6-MeGua can change the local shape of a
base pair (29). Therefore, we alternatively proposed that this
substitution could distort the O-P torsion angle, and thereby,
interfere with 3'-processing of the U5 substrate through a change in
the local structure of the DNA. Several lines of evidence suggest the
loss of the 3'-processing activity was due to a loss in the hydrogen
bonding in the major groove by the introduction of the methyl group
with the 6-MeGua substitution, and not due to a decreased affinity of
IN for the substrate. First, U5 substrates substituted with 2-AP for
Gua showed a decrease in 3'-processing, and showed WT levels of
binding. Second, while the Cyt mismatch at this position resulted in a loss of activity, which indicated that the local structure was not
recognized, filter binding experiments showed that the ability of
HTLV-2 IN to bind the substrate was poor. In contrast, filter binding
analysis of IN with U5 substrates containing 6-MeGua or 2-AP showed a
very high binding affinity as compared with the WT U5 substrate.
Therefore, the data suggest that the loss of 3'-processing activity by
the 2-AP or 6-MeGua substitutions were due to a loss in hydrogen
bonding and not disruption of the local structure. A summary of these
results is shown in Table I.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP
(PerkinElmer Life Sciences) and T4 DNA kinase (New England Biolabs),
and hybridized to complementary strands as described previously
(13).
-mercaptoethanol, 10% (v/v) glycerol, 0.75 mM CHAPS,
and 7.5 mM MnCl2. Reactions were assembled in
reaction buffer with 1 pmol of substrate and 0.013 µg/µl HTLV-2 in
a final volume of 15 µl at 37 °C for 1 h. Reactions were
terminated by addition of 10 µl of loading dye (95% formamide, 20 mM EDTA, 0.05% bromphenol blue, 0.5% xylene cyanol).
Reaction products were separated on 20% polyacrylamide denaturing
gels, subjected to autoradiography or PhosphorImager screens (Molecular
Dynamics). Products were quantified with ImageQuant software (Molecular
Dynamics). The amount of product was calculated from a minimum of three
separate trials for each experiment performed in duplicate or triplicate.
-mercaptoethanol, 10% (v/v) glycerol, 7.5 mM
MnCl2, and 0.75 mM CHAPS) in a final volume of
20 µl. The ratio of enzyme to substrate was varied from 1:5 to 40:1.
Reactions were conducted at room temperature for 20 min for complex
formation. Each reaction was slot blotted onto a nitrocellulose, washed
with 200 µl of the reaction buffer without metal and detergent. The
blot was air-dried, and exposed to a phosphorscreen. Reactions were
quantified from PhosphorImager data as described above, and results
quantified from a minimum of two experiments with duplicate data sets.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Illustration of retroviral integrase
activities, viral end substrates, and modified nucleobases used in this
study. A, schematic representation of the enzymatic
activities catalyzed by the retroviral IN in vitro for a
single U5 end: 1) 3'-processing; 2) strand
transfer; B, schematic representation of the enzymatic
activities catalyzed by the retroviral IN in vitro for
disintegration. Symbols: (+), plus strand, ( ), minus strand.
C, sequences of HTLV-2 viral long terminal repeat end
substrates. The positions substituted with analogues or mutations are
in boldface and/or underlined. The numbering
system used for nucleotide identification is indicated above
the sequence. For the U5 substrate, the top strand in each substrate
represents the plus strand and the bottom strand represents the minus
strand. This order is reversed for the U3 substrate. D,
chemical structures of the base analogues used in this study.
Numbers within the structure denote the conventional
numbering scheme for nucleobases. Arrows denote chemical
moieties that have been changed from the natural purine
nucleobases.
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Fig. 2.
3'-Processing activity of HTLV-2 IN with
HTLV-2 WT and modified U5 substrates. HTLV-2 IN reactions with the
WT U5 substrate (lane 2), and U5 substrates with
substitutions at position 4 in the minus strand including Hyp (4/Hyp,
lane 4), 6-MeGua (4/6-MG, lane 6), and 6-MeGua at
strand positions 4 and 8 (4,8/6-MG, lane 8). Odd
lanes represent reactions performed with substrate in the absence
of protein. Symbols: Prd, products; sub,
substrate. Lane numbers are located below the
panel.
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Fig. 3.
3'-Processing activity of HTLV-2 IN with
HTLV-2 WT U3, modified U3, and NUS substrates. A and
B, HTLV-2 IN reactions with the WT U5 substrates are shown
in lane 2 of each panel. A, HTLV-2 IN reactions
are shown for the WT U3 substrate (lane 2), the modified U3
substrates with a 6-MeGua substitution at position 4 in the minus
strand (4/6-MG, lane 4), and the 6-MeGua at position 4 in
the minus strand and ACA substitutions in positions 7-9 of the U3 plus
strand (4/6-MG ACA, lane 6). B, reactions with
either the NUS substrate or NUS substrates with substitutions of ACA at
varying positions in the plus strand are shown for: lane 5,
NUS; lane 7, positions 3 through 5 (3-ACA NUS);
lane 9, positions 5 through 7 (5-ACA NUS);
lane 11, positions 6 through 8 (NUS 6-ACA);
lane 13, positions 7 through 9 (NUS 7-ACA).
Even lanes 2-12 show reactions done in the presence of
substrate and the absence of protein. Symbols: Prd,
products; sub, substrate. Lanes 1 and
14 contain a labeled oligonucleotide size marker.
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Fig. 4.
3'-Processing activity of HTLV-2 IN with
HTLV-2 WT and modified U5 substrates. In panels A and
B, HTLV-2 IN reactions are shown with the WT U5 substrate
(lane 2), and modified U5 substrates at position 4 in the
minus strand. Modifications included substitution of Gua for:
A, lane 3, 6-thio-Gua (4/6-thio); and
lane 6, Cyt (4C/C); B, lane
3, 2-AP. Odd lane numbers in each panel show reactions
performed with substrate in the absence of protein.
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Fig. 5.
Summary of HTLV-2 IN 3'-processing activity
with U5 WT, modified U5, and modified U3 substrates. HTLV-2 IN
reactions are shown with the WT U5 substrate (lane 3), an
oligonucleotide size marker (lanes 1 and 16), and
modified U5 and U3 substrates at positions 4 and/or 8 in the minus
strand. Modifications included substitution of Gua for:
lane 5, Cyt (4C/C); lane 7, 2-AP; lane
9, 6-thio-Gua (4/6-thio); lane 11, 6-MeGua
(4/6-MG); lane 13, 6-MeGua substituted at both
positions 4 and 8 (4,8/6-MG); and lane 15,
6-MeGua substituted at position 4 of the U3 minus strand and containing
ACA substituted at positions 7-9 of the U3 plus strand (U3
4/6-MG ACA). Even lane numbers 2-14 show reactions
performed with substrate in the absence of protein. Lanes 1 and 16 contain a labeled oligonucleotide size marker. The
asterisk denotes the cleavage product of the U3 4/6-MG ACA
substrate. Due to the different composition of this substrate, both the
substrate and the product do not run true to the size marker.
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Fig. 6.
Strand transfer and disintegration activity
of HTLV-2 IN with modified U5 substrates. HTLV-2 IN reactions are
shown with the WT U5 substrate, and modified strand transfer
(panels A and C) and disintegration U5 substrates
(panels B and D). Modifications in substrates
were made at Gua in position 4 in the minus strand. In panels
A and C, HTLV-2 IN strand transfer reactions are shown
for: A, lane 2, the WT U5 precleaved substrate
(Wt); and modified strand transfer substrates: lane 4, Hyp
(4/Hyp); lane 6, 6-MeGua (4/6-MG); lane 8,
6-MeGua substituted at positions 4 and 8 in the minus strand (4, 8/6-MG). C, lane 2, the WT U5 precleaved
substrate (Wt); and modified strand transfer substrates:
lane 4, 2-AP. In panels B and D,
HTLV-2 IN disintegration reactions are shown for: B,
lane 2, the WT U5 disintegration substrate (Wt);
and modified disintegration substrates: lane 4, Hyp
(4/Hyp): lane 6, 6-MeGua (4/6MG); and lane
8, 6-MeGua substituted at positions 4 and 8 of the Y substrate
(4,8/6-MG); D, lane 2, the WT U5
disintegration substrate (Wt) and modified disintegration
substrates: lane 4, 2-AP. Odd lane numbers in
each panel show reactions performed with substrate in the absence of
protein.
2 and 7.2 × 10
2 µg/µl, and modestly higher at lower
concentrations of IN. The concentration, 3.6 × 10
2
µg/µl, was that used in all previous integration and disintegration experiments shown herein. The 4/6-MeGua and 2-AP substrates also bound
at significantly higher levels than the Ran substrate. The mismatch
substrate, 4C/C, bound nearly the same as the Hyp and Ran substrates,
but significantly less than the WT HTLV-2 U5 substrate (Fig. 7).
Substrates containing the context of a viral U5 end with a substitution
at the conserved 4 position in the minus strand, 4G/C and 4T/A, showed
the lowest binding of all the substrates examined; more than 2-fold
less than the WT substrate at the two higher concentrations studied.
These substrates have no 3'-processing activity.4 These substrates
were also bound significantly less than the Ran substrate, 2-3-fold.
Complexes were not observed when IN was not added to the
reaction.5 In summary, the
major groove modifications showed an enhanced binding of the U5
substrate to IN, while the minor groove modification had a decreased
binding affinity. The mismatch substrate did not show the enhanced
binding, and this suggests that the interaction of IN with this
substrate is through a mechanism distinct from that of the 4/6-MeGua
and 2-AP substrates. The low binding affinity of IN to the 4G/C, and
4T/A, also indicates an interaction with IN that is unique to these
substrates. These data suggest that IN was able to recognize some, but
not all, of the key nucleotide functional groups in position 4. Since
some determinants were present, it is possible that these substrates
were not recognized as targets, but as faulty viral ends.
View larger version (38K):
[in a new window]
Fig. 7.
Binding affinity of HTLV-II IN for HTLV-2 U5
and modified substrates. HTLV-2 IN was incubated with the HTLV-2
U5 and modified substrates and examined for their binding affinity with
a filter binding assay as described under "Experimental
Procedures." Shown is the mean and S.E. of a minimum of two assays
done in duplicate of the fraction of substrate retained on the
membrane. Legend: HTLV-2 U5 substrate (Wt, ), random 20-mer
duplex substrate (Ran,
), minor groove modification Hyp replacing
the conserved G in the HTLV-2 U5 substrate (4/Hyp,
), major groove
modifications 6-MeGua and 2-aminopurine located in the 4 position of
the HTLV-2 U5 substrate (4/6-MG,
, and 2-AP,
), Cyt mismatch in
the 4 position of the HTLV-2 U5 substrate (4C/C,
). The double
mutants in the 4 position of the HTLV-2 U5 substrates (4G/C,
, and
4T/A, ).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Summary of activities of HTLV-2 IN with modified U5 and random
substrates
The available data suggest that the major groove at position 4 does
contribute to hydrogen bonding interactions, however, the data also
suggest that the distortion in the structure of the DNA molecule
promotes cleavage of the upstream ACA. We propose the following model
for the retroviral integrase reaction pathway based on these
observations (Fig. 8). IN first binds to
the retroviral DNA end (Fig. 8, step 1). Our data suggests that minor
groove contact at the amine at position 2 of the Gua was a major
determinant for a stable IN/substrate interaction. Several groups have
shown that certain proteins, for example, SRY and LEF-1, can alter DNA structure through the minor groove. These proteins target certain sequences that may have an intrinsic tendency to underwind and roll one
or more base steps. SRY and LEF use an intercalative wedge to pry open
a single base step and distort the DNA (30). There is ample evidence
that this type of DNA distortion occurs in retroviral integration (23,
31). Interestingly, the target of SRY (AACAAA) and LEF (TTCAAA) have a
high similarity to the viral termini recognized by retroviral IN.
Nevertheless, herein we show the importance of the minor groove in
binding of the viral end by the HTLV-2 IN. However, this was not
observed for the strand transfer and disintegration substrates. We
propose that only WT substrates that contain a conserved C(A/G)T
sequence will orient the IN complex in a way as to ensure the ends are
processed correctly. The reduced binding affinity IN displayed with the
mutations in the 4 position of the HTLV-2 U5 long terminal repeat, the
4G/C and 4T/A substrates supports this hypothesis; i.e. IN
does not recognize the key nucleotides at position 4, and therefore
releases the substrate. Yi et al. (27) has reported that
flipping of the C(A/G)T causes a decrease in binding in the presence of
metal ion. We have reported herein that this reduction in binding can be traced, at least in part, to minor groove interactions at position 4. This mechanism apparently does not hold true for the random substrate, Ran, which showed only a slight reduction in binding. This
in turn implies a different set of contacts for the target DNA
(sequences without a CA) as proposed by recent modeling studies by
Yang et al. (32).
|
A correct orientation in the binding step proceeds to unwinding of substrate (Fig. 7, step 2), and correct cleavage at the 3-2 phosphodiester bond, if there is a major groove O6 contact available at position 4 (Fig. 8, step 4). A transition state that could be present at step 3 would most probably reflect the melting of the three terminal base pairs as proposed by Chen et al. (31). Those distortions caused by our modified substrates may be viewed by IN as such a transition state described by step 2 in this model for the integration reaction mechanism. In effect, these modified substrates may orient the complex further upstream on the viral DNA, and thus, promote aberrant catalysis of the substrate at an upstream ACA. Interestingly, the greater the distortion at the Cyt/Gua, the greater the increase in the percentage of cleavage at the internal ACA motif. In the case of HIV-1 IN, when a 2-base pair mismatch was placed immediately downstream of an internalized CA, precise cleavage of the substrate occurred (23). Herein, we have shown that a single mismatch located at the third base pair, downstream of the CA, could promote internal cleavage. Finally, the major groove O6 contact at Gua may also allow the IN·substrate complex to change conformation, and achieve the transition state of the complex, which leads to strand transfer as shown in step 4 of our model. Conformational changes initiated by divalent metal binding have been mapped to all three domains of IN (33, 34). Although a metal binding step is not depicted in our model, these conformational changes may allow IN to productively interact with substrate. We hypothesize that other substrate-induced conformational changes may occur immediately before or concurrent to 3'-processing as shown in step 4 of our model, although as of yet this has not been shown. These substrate induced conformational changes could also include reorganization of the IN·substrate complex as well as intramolecular changes in the tetrameric or higher ordered IN structure in preparation for the strand transfer event.
Interestingly, when Gua in the conserved and internal AC(A/T)GT
sequences was substituted with 6-MeGua or 4,8/6-MG, IN no longer
made a precise cleavage at either of the ACA sequences. Cleavages were
noted 5' to the conserved and upstream CA sequences. Imprecise cleavage
of a substrate by IN has not been previously documented. However,
nonspecific alcoholysis of DNA has been reported, and differs from the
cleavage noted with the 4,8/6-MG substrate in that it occurs at most
positions in nonviral DNA substrates (35). Based on the activity of the
4,8/6-MG substrate we hypothesize that HTLV-2 IN does not recognize the
sequence of this substrate, but instead has read the substrate by its
structure or rather the distortion introduced by the base modification.
The conformation of the substrate may reposition IN on the substrate in
such a way as to promote cleavage at the alternative sites. It is clear that conformational changes play an essential role in retroviral integrase catalysis. Additional experiments will further map the chronological and spatial locations of these structural changes as they
relate to substrate binding and catalysis by IN.
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ACKNOWLEDGEMENT |
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We thank Dr. Monica Roth, Department of Biochemistry, University of Medicine and Dentistry of New Jersey, for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant R15CA74398-01 (to C. B. J.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Contributed equally to the results of this work.
§ Present address: 1650 Lincoln, number 1106, Montreal, QC, H3H 1H1 Canada.
¶ Present address: Dept. of Chemistry and Biochemistry, Box 30001, MSC 3C, New Mexico State University, Las Cruces, NM 88003.
To whom correspondence should be addressed: Dept. of Chemistry
and Biochemistry, New Mexico State University, Box 30001, MSC 3C, Las
Cruces, NM 88003. Tel.: 505-646-3346; Fax: 505-646-2649; E-mail:
cjonsson@nmsu.edu.
Published, JBC Papers in Press, February 2, 2001, DOI 10.1074/jbc.M007754200
2 T. Wang and C. B. Jonsson, unpublished observations.
3 T. Wang, A. J. Piefer, and C. B. Jonsson, unpublished results.
4 T. Wang and C. B. Jonsson, unpublished information.
5 A. J. Piefer and C. B. Jonsson, unpublished observations.
![]() |
ABBREVIATIONS |
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The abbreviations used are: IN, integrase; HIV-1, human immunodeficiency virus type-1, HTLV-2, human T-cell leukemia virus type 2; 6-MeGua, O6-methylguanine; Hyp, hypoxanthine; 2-AP, 2-aminopurine; 6-thio-Gua, O6-thioguanine; CHAPS, 3[(3-chloramidopropyl)dimethylammonio]-1-propanesulfonate; MOPS, 4-morpholinepropanesulfonic acid; NUS, non U substrate.
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