Previous Article | Next Article 
Journal of Virology, August 2001, p. 7756-7762, Vol. 75, No. 16
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.16.7756-7762.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Use of Patient-Derived Human Immunodeficiency Virus Type 1 Integrases To Identify a Protein Residue That Affects Target
Site Selection
Amy L.
Harper,1
Lynn M.
Skinner,2
Malgorzata
Sudol,2 and
Michael
Katzman1,2,*
Department of Microbiology and
Immunology1 and Department of
Medicine,2 Pennsylvania State University
College of Medicine, The Milton S. Hershey Medical Center, Hershey,
Pennsylvania 17033
Received 13 December 2000/Accepted 21 May 2001
 |
ABSTRACT |
To identify parts of retroviral integrase that interact with
cellular DNA, we tested patient-derived human immunodeficiency virus
type 1 (HIV-1) integrases for alterations in the choice of nonviral
target DNA sites. This strategy took advantage of the genetic diversity
of HIV-1, which provided 75 integrase variants that differed by a small
number of amino acids. Moreover, our hypothesis that biological
pressures on the choice of nonviral sites would be minimal was
validated when most of the proteins that catalyzed DNA joining
exhibited altered target site preferences. Comparison of the sequences
of proteins with the same preferences then guided mutagenesis of a
laboratory integrase. The results showed that single amino acid
substitutions at one particular residue yielded the same target site
patterns as naturally occurring integrases that included these
substitutions. Similar results were found with DNA joining reactions
conducted with Mn2+ or with Mg2+ and were
confirmed with a nonspecific alcoholysis assay. Other amino acid
changes at this position also affected target site preferences. Thus,
this novel approach has identified a residue in the central domain of
HIV-1 integrase that interacts with or influences interactions with
cellular DNA. The data also support a model in which integrase has
distinct sites for viral and cellular DNA.
| |
TEXT |
Integration of a DNA copy of the
retroviral genome into cellular DNA is essential for retrovirus
replication. The retroviral integrase protein catalyzes two
endonuclease reactions that are necessary for integration. During
processing, integrase prepares the viral DNA for integration by
removing two or three nucleotides that follow highly conserved CA bases
near the 3' end of each DNA strand. During DNA joining (or strand
transfer), integrase inserts the processed viral DNA ends into cellular
DNA in a sequence-independent manner. These in vivo activities can be
modeled by using purified integrase proteins and oligonucleotides that
mimic either end of viral DNA (4, 16, 18). In particular,
human immunodeficiency virus type 1 (HIV-1) integrase processes
oligonucleotides derived from HIV-1 DNA by removing two nucleotides
that follow the conserved CA (Fig. 1A).
Integrase also inserts some of the processed oligonucleotides into
various sites on other oligonucleotides, which now act as cellular DNA,
to create a set of products that are longer than the substrate (Fig.
1B). As occurs in vivo, almost any site can be used as the target for
insertion even though certain sites are preferred (17).
Our studies with chimeric proteins between the integrases of HIV-1 and
visna virus demonstrated that the central domain of integrase plays a
major role in selecting the target sites for insertion of viral DNA
ends (22, 23). Moreover, the central region of integrase
was solely responsible for the selection of nonviral target sites when
the chimeric integrases catalyzed a reaction referred to as nonspecific
alcoholysis (20, 22). During this activity, which shares
certain characteristics with the DNA joining reaction
(23), integrase uses a variety of nucleophiles to nick
almost any site in nonviral DNA (Fig. 1C). In fact, the isolated
central fragment of HIV-1 integrase can catalyze nonspecific
alcoholysis and exhibits the same target site preferences as the
full-length protein (21, 23).
View larger version (12K):
[in this window]
[in a new window]
|
FIG. 1.
Integrase assays. The conserved CA bases near the 3'
ends of viral DNA are shown in boldface, the terminal two nucleotides
are indicated by NN, and asterisks denote 32P groups. (A)
During processing, integrase makes a site-specific nick to form a
labeled product two nucleotides shorter than the substrate. (B) During
DNA joining or strand transfer, integrase inserts the processed viral
DNA ends into any of various sites on either strand of target DNA (the
labeled strand is used in the schematic), yielding a set of labeled
products longer than the substrate. (C) During nonspecific alcoholysis,
integrase uses certain nucleophilic molecules (shown as ROH) to nick
nonviral DNA at any of multiple sites.
|
|
To identify amino acid residues of integrase that influence target site
preferences in nonviral DNA, we screened naturally occurring,
patient-derived HIV-1 integrase variants for alterations in the
selection of nonviral target sites. We reasoned that a set of natural
integrases, because of the genetic diversity of HIV-1, would provide
many integrases that differ by a small number of amino acids. We also
predicted that many of the proteins would be active for processing and
joining because of in vivo selection but hypothesized that biological
pressures on noncritical aspects of integrase function, such as the
choice of nonviral target sites, would be minimal. Although we realized
that some viral sequences recovered from clinical samples might not
come from infectious genomes, we had already established that 85% of
integrase sequences that we had amplified from HIV-infected patients
encoded full-length proteins (33). Moreover, substitutions
at any of the seven highly conserved amino acids known to be important
for integrase activity were rare. Testing these proteins in functional
assays has now validated these hypotheses. In particular, variations in
nonviral target site selection were found for many proteins that had
wild-type levels of processing and joining activity.
Target site preferences of patient-derived HIV-1 integrases.
We previously described 102 HIV-1 integrase sequences that were
amplified from viral DNA in blood cells or viral RNA in plasma that had
been obtained many years ago from 10 HIV-infected hemophiliacs (33). These sequences encode 87 full-length
proteins, of which 75 are unique. Each of these 75 proteins has
between 4 and 16 amino acid changes compared to the prototypic HIV-1
integrase used in our laboratory. We have now expressed and purified
the unique full-length proteins from a bacterial expression system and
screened them for enzymatic activity, with a particular focus on the
selection of target sites in the biologically relevant DNA joining
assay. Purifications were conducted from 10 ml of bacterial cultures,
using methods described previously (19, 20) but aided by
the use of magnetic Ni2+-nitrilotriacetic acid beads
(Qiagen, Chatsworth, Calif). Processing and joining reactions were
initially conducted under conditions known to optimize HIV-1 integrase
activity, including the use of oligonucleotides derived from the U5 end
of HIV-1 DNA and Mn2+ as the divalent cation cofactor
(34). Standard 10-µl reaction mixtures contained 0.5 pmol of double-stranded DNA, 25 mM Tris-HCl (pH 8.0), 10 mM
dithiothreitol, 1.0 µl of integrase or protein storage buffer, and 10 mM MnCl2; reactions were conducted for 90 min and then
analyzed by denaturing polyacrylamide gel electrophoresis and
autoradiography. As expected, some of the variant proteins did not
purify well and were inactive in these assays. However, 36 of the
purified proteins catalyzed DNA joining. Remarkably, most of these
proteins made patterns of strand-transfer products that differed from
that of the laboratory integrase, reflecting altered target site
preferences during DNA joining.
The novel sets of strand-transfer products created by the natural
integrases clearly separated into two major patterns. Reactions
with
representative patient-derived proteins are shown in Fig.
2. These proteins catalyzed processing at
levels comparable to
that of the laboratory HIV-1 integrase and
specifically removed
two nucleotides from the 18-mer viral DNA
substrate to create
a 16-mer product (Fig.
2A). Moreover, some of the
processed products
from these reactions were inserted into other
oligonucleotides
to create strand-transfer patterns that differed from
that of
the laboratory integrase (not shown). To highlight the longer
products, we performed joining assays with a preprocessed substrate
that was missing the two nucleotides 3' to the conserved CA. Under
these conditions, the laboratory integrase (referred to as wild-type
in
the figures) creates a distinct pattern of slower-migrating
products in
which certain bands are more prominent (Fig.
2B, lane
3). However, the
patterns created by many of the patient-derived
integrases reproducibly
had different relative intensities at
certain positions. For example,
the pattern referred to as type
1 included faint bands at positions
that were much darker in reactions
with the laboratory integrase (Fig.
2B, lanes 4 to 6, the bands
denoted by circles), even though other
bands in these lanes were
darker than bands in the wild-type pattern.
Similarly, the pattern
referred to as type 2 differed in intensity at
several positions
compared with the pattern of the laboratory integrase
(Fig.
2B,
lanes 7 to 9, e.g., the bands denoted by squares). These
patterns
were independent of the amount of joining catalyzed by the
enzymes
and were not affected by the duration of the reactions (data
not
shown).
View larger version (70K):
[in this window]
[in a new window]
|
FIG. 2.
Target site selection by patient-derived integrases.
Autoradiograms of a denaturing 20% polyacrylamide gel are shown. (A)
Processing assay. Double-stranded 18-mers derived from one end of HIV-1
DNA were 5' labeled on the strand that contains the conserved CA and
incubated with protein buffer (lane 1), a laboratory HIV-1 integrase
that contained an inactivating D116I mutation (lane 2), the wild-type
laboratory HIV-1 integrase (lane 3), or six patient-derived HIV-1
integrases that are grouped as type 1 (lanes 4 to 6) or type 2 (lanes 7 to 9) based on the results shown in panel B. The nomenclature for the
naturally occurring integrases is explained in Fig. 3. The 18-mer
substrate and the 16-mer product are indicated. The amount of
processing in lanes 3 to 9 ranged from 32 to 45%. (B) Joining assay.
Double-stranded 16-mers representing a preprocessed end of HIV-1 DNA
were 5' labeled on the strand that contains the conserved CA and
incubated with the same proteins as in panel A. The bands indicated by
circles or squares are reduced in intensity in the type 1 or type 2 pattern, respectively, compared to the wild-type pattern. The 16-mer
substrate and strand-transfer products are indicated. The amount of
joining in lanes 3 to 9 ranged from 8 to 23%. (C) Nonspecific
alcoholysis assay. Double-stranded 23-mers of nonviral sequence were 5'
labeled on one strand and incubated with the same proteins as in panel
A in reactions that included 20% glycerol. Sizes in nucleotides of
cleavage products are shown on the right; no bands were detected below
the position of 4-mers. The 23-mer substrate and prominent products
from the laboratory integrase are indicated on the left. The amount of
substrate nicked in lanes 3 to 9 ranged from 41 to 59%.
|
|
The proteins that created the type 1 pattern in Fig.
2B were derived
from different clinical samples from one patient, whereas
the selected
proteins that created the type 2 pattern were from
three different
patients. Overall, 10 of the 36 natural proteins
that catalyzed DNA
joining made the type 1 pattern, 10 made the
type 2 pattern, and 1 (not
discussed further) made a third novel
pattern. There was no correlation
between the target site patterns
and the rate of disease progression.
For example, the 15 proteins
that exhibited the same preferences as the
laboratory integrase
were derived from four patients who subsequently
died of AIDS
and three patients with slow or no progression, and the 10 proteins
grouped as type 2 were from two patients who died of AIDS and
two slow progressors. In two cases, different integrases from
the same
individual created a wild-type and a novel pattern (Fig.
3, code numbers 002 and 143). A detailed
analysis correlating
clinical features with the other activities of
these enzymes has
been completed (M. Katzman, A. L. Harper, M. Sudol, L. M. Skinner,
and M. E. Eyster, submitted for
publication).
View larger version (39K):
[in this window]
[in a new window]
|
FIG. 3.
Sequence alignment of central domains of patient-derived
integrases. The central region (residues 50 to 190) of the laboratory
HIV-1BH10 integrase, which is similar to the
HIV-1HXB2 integrase (30, 31), is shown above
the patient-derived proteins. The conserved acidic amino acids at
positions 64, 116, and 152 are indicated with exclamation points.
Identity to the laboratory integrase sequence is indicated by a dash.
Proteins are grouped according to the patterns exhibited in DNA joining
assays conducted with Mn2+. Confirmation in DNA joining
assays with Mg2+ is indicated as follows: a,
confirmed as wild type in reactions with Mg2+;
b, confirmed as type 1 in reactions with Mg2+;
c, created one type of novel pattern in reactions with
Mg2+; d, created another type of novel pattern
in reactions with Mg2+. Groupings also were confirmed by
nonspecific alcoholysis assays for all but the three proteins indicated
with asterisks. Residues 106 and 119, which were targeted by
site-directed mutagenesis, are indicated by triangles at the bottom.
Patient-derived proteins are named with an E or L (indicating whether
the samples were obtained early after infection or 5 years later), D or
R (indicating whether DNA or RNA was the source for amplification), a
three-digit patient code, and a single-digit number identifying the
clone (33). The sequences shown match those submitted to
GenBank but include three corrections compared to the previously
published sequences: S129 in LD061-3, K157 in LR061-1, and G119 in
ED002-7.
|
|
The altered target site preferences of the patient-derived proteins
were confirmed with the nonspecific alcoholysis assay.
Although
integrase can use glycerol or certain other alcohols
as the nucleophile
to nick nonviral DNA at any internal position,
certain sites are
reproducibly preferred by different integrases
(
21). For
example, the laboratory HIV-1 integrase prefers to
nick at positions
located 8, 11, 15, and 17 nucleotides from the
5' end of the
23-mer substrate used in this assay (Fig.
2C, lane
3). Significantly,
the novel alcoholysis patterns made by the
natural integrases shown in
Fig.
2 resulted in the same grouping
of the proteins as
occurred with the DNA joining assay. In particular,
the three natural
proteins that were grouped as type 1 for DNA
joining made an
alcoholysis pattern that included a very prominent
band at position 16 (Fig.
2C, lanes 4 to 6). In contrast, the
three proteins that were
grouped as type 2 for DNA joining made
a novel alcoholysis pattern that
included a strong band at position
11, as did the laboratory enzyme,
but very weak bands at positions
8, 15, and 17 (Fig.
2C, lanes 7 to 9).
The patterns in Fig.
2C
resulted from the unique alcoholysis activity
of integrase, and
not from contaminating nucleases, because attachment
of glycerol
to the appropriate sites was confirmed in assays that used
a 3'-labeled
substrate (data not shown) (
21). Overall, the
nonspecific alcoholysis
assay confirmed the categorization of 32 of the
35 proteins listed
in Fig.
3, whereas three proteins that were type 1 by the joining
assay were inactive or unclassifiable in the alcoholysis
assay
(Fig.
3,
asterisks).
Because of the importance of the central domain of integrase in the
selection of target sites in nonviral DNA (
13,
20-23,
32), we compared the sequences of this region between the
natural
proteins (Fig.
3). This alignment suggested amino acids that
might
be contributing to the observed patterns. For example, every
protein
grouped as type 1 has alanine at residue 106 and threonine at
residue 119, whereas proteins with the wild-type or type 2 pattern
do
not share these amino acids. Similarly, every protein grouped
as type 2 has glycine at residue 119, whereas no other protein
has this amino
acid. In contrast, isoleucine at residues 50 and
72 is common to the
type 1 proteins, but two proteins grouped
as wild type also have Ile at
one of these residues (i.e., ED127-1
and ED062-6). Similarly, although
all proteins grouped as type
2 have threonine at position 124, some
integrases with a wild-type
or type 1 pattern also have Thr at this
position (e.g., ED002-1
and ED061-5). The sequences of four other
proteins that would
have been classified as wild type (1 protein), type
1 (1 protein),
or type 2 (2 proteins) based solely on their patterns in
the alcoholysis
assay are not shown in Fig.
3 but are consistent with
these observations.
Thus, this analysis suggested that residues 106 and
119 contribute
to the selection of target sites in nonviral
DNA.
Single amino acid substitutions in the laboratory HIV-1
integrase.
To examine the role of particular protein residues in
the selection of target sites, we replaced single amino acids in the laboratory integrase by using the QuikChange site-directed mutagenesis system (Stratagene, La Jolla, Calif.). The entire integrase-coding region of all new proteins was confirmed by sequencing. Based on the
above discussion, we made single amino acid changes of Gly-106 to Ala
or of Ser-119 to Thr or Gly. We first established that each of the new
proteins specifically processed the HIV-1 viral DNA 18-mer substrate to
a 16-mer product (Fig. 4A). In addition, each of these enzymes catalyzed DNA joining (Fig. 4B). Moreover, the
S119T protein created a pattern that closely matched the type 1 pattern
made by a patient-derived integrase that included this amino acid among
13 differences from the laboratory enzyme (Fig. 4B, lanes 3 and 4).
Similarly, the S119G protein made a pattern that was identical to the
type 2 pattern made by a natural integrase that included this change
among eight differences (Fig. 4B, lanes 5 and 6). Thus, these
substitutions at residue 119 account for the novel patterns made by the
naturally occurring integrases. In contrast, substitution at residue
106 did not explain the type 1 pattern because the G106A protein made a
strand-transfer pattern indistinguishable from that of the laboratory
integrase (Fig. 4B, lanes 2 and 7).
View larger version (57K):
[in this window]
[in a new window]
|
FIG. 4.
Site-directed mutations explain the novel target site
patterns. Details are as in the legend to Fig. 2. For each panel, the
substrates were incubated with protein buffer (lane 1), the laboratory
integrase (lane 2), a patient-derived integrase that makes the type 1 pattern (ED061-7, lane 3), the laboratory integrase with an S119T
substitution (lane 4), a patient-derived integrase that makes the type
2 pattern (ED171-1, lane 5), the laboratory integrase with an S119G
substitution (lane 6), or the laboratory integrase with a G106A
substitution (lane 7). The amount of products formed in lanes 2 to 7 ranged from 25 to 52% in panel A, 8 to 27% in panel B, and 40 to 52%
in panel C.
|
|
The above results were confirmed when the proteins were tested in the
nonspecific alcoholysis assay. In particular, the S119T
protein made a
pattern that closely matched the type 1 alcoholysis
pattern made by a
patient-derived integrase that included this
amino acid (Fig.
4C, lanes
3 and 4). Similarly, the S119G protein
made a pattern that was very
similar to the type 2 pattern made
by a natural integrase that included
this amino acid (Fig.
4C,
lanes 5 and 6). These results were confirmed
by using a 3'-labeled
substrate and by using a different nonviral DNA
substrate that
was 45 bp long (data not shown). In contrast, residue
106 did
not contribute to the type 1 pattern because the G106A protein
made an alcoholysis pattern identical to that of the laboratory
integrase (Fig.
4C, lanes 2 and 7). Although the relevance of
nonspecific alcoholysis to the retrovirus life cycle is unknown,
our
conclusions are strengthened by finding the same results with
two
assays that use very different nucleophilic donor molecules.
Thus, we
conclude that residue 119 either interacts with or influences
interactions with nonviral DNA during
catalysis.
The above assays used Mn
2+ as the divalent cation cofactor;
however, Mg
2+ may be more relevant within cells. Although
HIV-1 integrase is
usually inactive with Mg
2+ in standard
assays, our preparations of the laboratory HIV-1
integrase catalyze
Mg
2+-dependent DNA joining in the presence of dimethyl
sulfoxide (
28).
Interestingly, the strand-transfer pattern
created under these
conditions (Fig.
5,
lane 2) differs slightly from that seen with
Mn
2+ (Fig.
4B,
lane 2). Thus, all of the patient-derived proteins
that were active
with Mn
2+ were tested in joining reactions using
Mg
2+. The results, as indicated in the legend to Fig.
3,
confirmed
the groupings obtained previously. In particular, 12 of the
15
proteins that were classified as wild type in joining reactions
conducted with Mn
2+ created the same pattern as the
laboratory integrase in reactions
with Mg
2+ (the other
three proteins were inactive with Mg
2+). Similarly, 8 of
the 10 proteins classified as type 1 in joining
reactions with
Mn
2+ made a novel but similar pattern with Mg
2+
(e.g., Fig.
5, lane 3; the other two proteins were inactive with
Mg
2+). Finally, 9 of the 10 proteins classified as type 2 in joining
reactions with Mn
2+ were active with
Mg
2+, and each made a new pattern distinct from the
wild-type or type
1 pattern. Interestingly, these nine proteins
made two different
new patterns (Fig.
5, lanes 5 and 8),
suggesting that other amino
acids worked in conjunction with
residue 119 to influence the
selection of target sites (see below).
More importantly, even
under Mg
2+-dependent conditions, the
S119T and S119G proteins made strand-transfer
patterns that
matched those of natural integrases that included
these changes
(Fig.
5, lanes 3 and 4 and lanes 5 and 6, respectively),
whereas the
G106A protein made a pattern similar to that of the
laboratory enzyme
(Fig.
5, lanes 2 and 7). Thus, the findings
with Mg
2+
paralleled those with Mn
2+, even though the sets of
preferred target sites differed as a
function of the divalent metal.
View larger version (80K):
[in this window]
[in a new window]
|
FIG. 5.
DNA joining reactions conducted with Mg2+.
Reactions were performed as described in the legend to Fig. 2B except
that Mn2+ was replaced by 3 to 10 mM Mg2+ plus
30% dimethyl sulfoxide. Lanes 1 to 7 tested the same enzymes as in the
legend to Fig. 4 except that the patient-derived protein for the type 2 pattern in lane 5 was ED002-7. Lane 8 shows the other novel pattern
created by some of the type 2 proteins in reactions with
Mg2+ (type 2'; the reaction shown used the LR157-5
protein); only one intervening gel lane was removed between lanes 7 and
8. Bands a to e distinguish the patterns as follows: the most intense
bands in the upper portions of the lanes for the wild-type, type 1, type 2, and type 2' patterns are a/c, c/e, a/c/d/e, and a/d,
respectively. Unequal amounts of counts per minute were loaded into the
various lanes to display the patterns at comparable intensities. The
amount of joining catalyzed by the proteins in lanes 2 to 8 ranged from
0.5 to 8%.
|
|
Given the different strand-transfer and alcoholysis patterns that
resulted from the S119T and S119G substitutions, we made
other changes
at residue 119 in the laboratory integrase. Substitution
of alanine or
lysine at this position resulted in proteins that
efficiently catalyzed
Mn
2+-dependent processing, joining, and nonspecific
alcoholysis (lanes
5 and 6 in Fig.
6A, B, and
C, respectively). In addition, the
new
proteins exhibited target site preferences in the latter two
assays
that differed from those of the wild-type or other point-substituted
proteins (lanes 2 to 4 in Fig.
6B and C). In some cases, the patterns
differed only slightly in the joining assay. For example, the
relative
intensities of the bands indicated by the circle and
square in Fig.
6B
are the only differences between the laboratory
and S119A proteins
(lanes 2 and 5) or between the S119T and S119K
proteins (lanes 3 and
6), but these differences were reproducibly
observed in multiple
experiments. Similarities also were evident
between the laboratory and
S119A proteins and between the S119T
and S119K proteins in joining
reactions conducted with Mg
2+ (not shown). However, the
distinct target site preferences of
these proteins are readily apparent
in the alcoholysis assay.
In particular, the S119A protein differed
from the laboratory
integrase by nicking inefficiently at positions 8 and 17 despite
similar preferences for positions 11 and 15 (Fig.
6C,
lanes 2
and 5). Similarly, the S119T and S119K proteins differed by
their
relative usage of positions 4 and 11 (Fig.
6C, lanes 3 and 6).
These differences were confirmed in alcoholysis assays that used
a
3'-labeled substrate (not shown). Thus, multiple substitutions
at
residue 119 influenced the interactions between HIV-1 integrase
and
nonviral DNA. These data, which were obtained with point-substituted
proteins, are consistent with our previous studies that used protein
fragments or chimeric integrases to map nonviral target site selection
to the central domain of integrase (
20-23). Together, the
results
suggest that the central domain of integrase interacts with
cellular
DNA during integration.
View larger version (47K):
[in this window]
[in a new window]
|
FIG. 6.
Other substitutions at position 119 in HIV-1 integrase.
Details are as in the legend to Fig. 2. For each panel, substrates were
incubated with the laboratory integrase that contained an inactivating
D116I mutation (lane 1), the wild-type laboratory integrase (lane 2),
or the laboratory integrase with the indicated amino acid substitutions
(lanes 3 to 6). The circle and square in panel B identify bands that
are discussed in the text. The amount of products formed in lanes 2 to
6 ranged from 49 to 67% in panel A, 4 to 15% in panel B, and 38 to
46% in panel C.
|
|
Implications.
Although other regions in HIV-1 integrase may
influence interactions with cellular DNA (13, 20, 22, 23,
32), our data prove that changes at one particular residue in
the central region were responsible for the novel patterns observed in
this study. Residue 119 is a serine in the consensus sequence for North American subtype B of HIV-1 and in the consensus sequence or isolates of other HIV-1 subtypes (24). However, many of the natural
integrases recovered from the hemophiliacs in our study had Thr or Gly
at position 119 (33). Other amino acids found at this
position in some HIV-1 integrases include Pro and Arg
(24). Our data show that HIV-1 integrase tolerated several
amino acids at this position, including Ser, Thr, Gly, Ala, and Lys,
without a significant effect on the efficiency of its catalytic
activity. However, as we had hypothesized, certain changes affected
noncritical aspects of integrase function. The finding that changes at
residue 119 do not affect specificity for viral DNA ends during
processing but do affect selectivity for nonviral DNA sites during
joining suggests that integrase has two sites for binding DNA (i.e.,
one for viral DNA and one for cellular DNA). We previously made a similar suggestion based on the finding that preferred target sites
during DNA joining differ when Mg2+ substitutes for
Mn2+, even though specific processing of viral DNA is
supported by both metals (28). Competition studies also
are consistent with two DNA sites on the enzyme (7, 9,
29).
Whether residue 119 affects target site selection by directly binding
to nonviral DNA or by indirectly affecting protein conformation
is
unknown. However, other data suggest that amino acids near
residue 119 also interact with cellular DNA. For example, the
proteins classified
as type 2 in the joining assay with Mn
2+ created two novel
strand-transfer patterns with Mg
2+. Although all of these
proteins have glycine at residue 119,
the two
Mg
2+-dependent patterns segregated with the presence of
isoleucine
at residue 122 (Fig.
3). Substitutions at residues 117 and
120
in the related HIV-2 integrase also were shown to affect target
site preferences during DNA joining reactions conducted with
Mn
2+ (
36). Moreover, contact between residue
117 of HIV-1 integrase
and cellular DNA has been suggested by
cross-linking and structural
data (
14). In contrast, our
data show that certain amino acid
substitutions at 22 other residues in
the central domain of integrase
did not affect target site selection
during DNA joining (i.e.,
residues 50, 59, 72, 79, 101, 106, 112, 124, 125, 128, 129, 131,
135, 147, 154, 157, 160, 161, 162, 166, 169, and
172, as deduced
from Fig.
3 and
4). Thus, merely substituting any amino
acid within
the catalytic domain of integrase is not sufficient to
alter the
preferences of the enzyme for certain target sites in
nonviral
DNA.
Residue 119 is the second of four amino acids in
-helix 2 near the
middle of the central domain of HIV-1 integrase. It is
close to Asp-64,
Asp-116, and Glu-152, the highly conserved acidic
residues that form
the D,D-35-E motif of the active site. These
four residues (i.e., 64, 116, 119, and 152) are on the surface
of the protein, which is involved
in many critical functions.
For example, the first two conserved
residues of the D,D-35-E
motif coordinate the required Mn
2+
or Mg
2+ cofactor in crystals of HIV-1 and Rous sarcoma
virus integrase
(
1,
2,
12,
26), and some data suggest that
all three
conserved active-site residues may coordinate two metal ions
(
1,
25). In addition, data from mutational, chemical, or
genetic
studies suggest that residues 136, 143, 148, 152, and 159 interact
with the ends of viral DNA (
5,
10,
11,
15,
27).
These
sites are within or near a disordered, flexible surface loop that
extends from residue 141 approximately to residue 150 (
6).
Recently, computerized docking of an 18-bp viral DNA end to two-domain
crystals of HIV-1 integrase placed the viral DNA end near residue
159 and more-internal viral DNA positions near residues 186 to
219 (
3). Other data implicate residues 114, 117, 120, 121,
143, 146, 147, and 148 in interactions with the attacking nucleophile
(
8,
35,
37). Thus, residue 119 may be appropriately
situated
to interact with cellular DNA during insertion of viral DNA
ends.
Although it would not be surprising if some residues of integrase
participate in more than one function, especially within the context
of
the multimeric enzyme complex, current data (including those
presented
in this report) support a model that includes the following:
the metal
cofactor is coordinated by residues 64 and 116, the
attacking
nucleophilic group interacts with residues near 116
and 152, the viral
DNA end is bound near residue 159, and cellular
DNA is bound near
residue 119. Thus, future studies that explore
how amino acids near
residue 119 affect the selection of nonviral
target DNA sites have the
potential to define the binding site
for cellular DNA and would
identify a new target for antiviral
drug design. The effects of changes
at these positions on the
preintegration complexes that mediate
integration in vivo also
await further
study.
| |
ACKNOWLEDGMENTS |
This work was supported by Public Health Service grant R21 AI47216
from the National Institute of Allergy and Infectious Diseases, by
W. W. Smith Charitable Trust Research grant A9804, by the Julius H. Caplan Foundation (in honor of Helen Caplan), and by a Dean's Feasibility Grant from the Pennsylvania State University College of Medicine.
We thank M. Elaine Eyster for providing the clinical samples from which
the HIV-1 integrases were derived.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Medicine, Division of Infectious Diseases, Pennsylvania State
University College of Medicine, The Milton S. Hershey Medical Center,
P.O. Box 850, Mail Services H036, Hershey, PA 17033-0850. Phone: (717) 531-8881. Fax: (717) 531-4633. E-mail: mkatzman{at}psu.edu.
| |
REFERENCES |
| 1.
|
Bujacz, G.,
J. Alexandratos,
A. Wlodawer,
G. Merkel,
M. Andrake,
R. A. Katz, and A. M. Skalka.
1997.
Binding of different divalent cations to the active site of avian sarcoma virus integrase and their effects on enzymatic activity.
J. Biol. Chem.
272:18161-18168[Abstract/Free Full Text].
|
| 2.
|
Bujacz, G.,
M. Jaskolski,
J. Alexandratos,
A. Wlodawer,
G. Merkel,
R. A. Katz, and A. M. Skalka.
1996.
The catalytic domain of avian sarcoma virus integrase: conformation of the active-site residues in the presence of divalent cations.
Structure
4:89-96[Medline].
|
| 3.
|
Chen, J. C. H.,
J. Krucinski,
L. J. W. Miercke,
J. S. Finer-Moore,
A. H. Tang,
A. D. Leavitt, and R. M. Stroud.
2000.
Crystal structure of the HIV-1 integrase catalytic core and C-terminal domains: a model for viral DNA binding: Proc.
Natl. Acad. Sci. USA
97:8233-8238[Abstract/Free Full Text].
|
| 4.
|
Craigie, R.,
T. Fujiwara, and F. Bushman.
1990.
The IN protein of Moloney murine leukemia virus processes the viral DNA ends and accomplishes their integration in vitro.
Cell
62:829-837[CrossRef][Medline].
|
| 5.
|
Du, Z.,
P. O. Ilyinskii,
K. Lally,
R. C. Desrosiers, and A. Engelman.
1997.
A mutation in integrase can compensate for mutations in the simian immunodeficiency virus att site.
J. Virol.
71:8124-8132[Abstract/Free Full Text].
|
| 6.
|
Dyda, F.,
A. B. Hickman,
T. M. Jenkins,
A. Engelman,
R. Craigie, and D. R. Davies.
1994.
Crystal structure of the catalytic domain of HIV-1 integrase: similarity to other polynucleotidyl transferases.
Science
266:1981-1986[Abstract/Free Full Text].
|
| 7.
|
Ellison, V., and P. O. Brown.
1994.
A stable complex between integrase and viral DNA ends mediates human immunodeficiency virus integration in vitro.
Proc. Natl. Acad. Sci. USA
91:7316-7320[Abstract/Free Full Text].
|
| 8.
|
Engelman, A., and R. Craigie.
1992.
Identification of conserved amino acid residues critical for human immunodeficiency virus type 1 integrase function in vitro.
J. Virol.
66:6361-6369[Abstract/Free Full Text].
|
| 9.
|
Espeseth, A. S.,
P. Felock,
A. Wolfe,
M. Witmer,
J. Grobler,
N. Anthony,
M. Egbertson,
J. Y. Melamed,
S. Young,
T. Hamill,
J. L. Cole, and D. J. Hazuda.
2000.
HIV-1 integrase inhibitors that compete with the target DNA substrate define a unique strand transfer conformation for integrase.
Proc. Natl. Acad. Sci. USA
97:11244-11249[Abstract/Free Full Text].
|
| 10.
|
Esposito, D., and R. Craigie.
1998.
Sequence specificity of viral end DNA binding by HIV-1 integrase reveals critical regions for protein-DNA interaction.
EMBO J.
17:5832-5843[CrossRef][Medline].
|
| 11.
|
Gerton, J. L.,
S. Ohgi,
M. Olsen,
J. Derisi, and P. O. Brown.
1998.
Effects of mutations in residues near the active site of human immunodeficiency virus type 1 integrase on specific enzyme-substrate interactions.
J. Virol.
72:5046-5055[Abstract/Free Full Text].
|
| 12.
|
Goldgur, Y.,
F. Dyda,
A. B. Hickman,
T. M. Jenkins,
R. Craigie, and D. R. Davies.
1998.
Three new structures of the core domain of HIV-1 integrase: an active site that binds magnesium.
Proc. Natl. Acad. Sci. USA
95:9150-9154[Abstract/Free Full Text].
|
| 13.
|
Goulaouic, H., and S. A. Chow.
1996.
Directed integration of viral DNA mediated by fusion proteins consisting of human immunodeficiency virus type 1 integrase and Escherichia coli LexA protein.
J. Virol.
70:37-46[Abstract/Free Full Text].
|
| 14.
|
Heuer, T. S., and P. O. Brown.
1998.
Photo-cross-linking studies suggest a model for the architecture of an active human immunodeficiency virus type 1 integrase-DNA complex.
Biochemistry
37:6667-6678[CrossRef][Medline].
|
| 15.
|
Jenkins, T. M.,
D. Esposito,
A. Engelman, and R. Craigie.
1997.
Critical contacts between HIV-1 integrase and viral DNA identified by structure-based analysis and photo-crosslinking.
EMBO J.
16:6849-6859[CrossRef][Medline].
|
| 16.
|
Katz, R. A.,
G. Merkel,
J. Kulkosky,
J. Leis, and A. M. Skalka.
1990.
The avian retroviral IN protein is both necessary and sufficient for integrative recombination in vitro.
Cell
63:87-95[CrossRef][Medline].
|
| 17.
|
Katzman, M., and R. A. Katz.
1999.
Substrate recognition by retroviral integrases.
Adv. Virus Res.
52:371-395[Medline].
|
| 18.
|
Katzman, M.,
R. A. Katz,
A. M. Skalka, and J. Leis.
1989.
The avian retroviral integration protein cleaves the terminal sequences of linear viral DNA at the in vivo sites of integration.
J. Virol.
63:5319-5327[Abstract/Free Full Text].
|
| 19.
|
Katzman, M., and M. Sudol.
1994.
In vitro activities of purified visna virus integrase.
J. Virol.
68:3558-3569[Abstract/Free Full Text].
|
| 20.
|
Katzman, M., and M. Sudol.
1995.
Mapping domains of retroviral integrase responsible for viral DNA specificity and target site selection by analysis of chimeras between human immunodeficiency virus type 1 and visna virus integrases.
J. Virol.
69:5687-5696[Abstract/Free Full Text].
|
| 21.
|
Katzman, M., and M. Sudol.
1996.
Nonspecific alcoholysis, a novel endonuclease activity of human immunodeficiency virus type 1 and other retroviral integrases.
J. Virol.
70:2598-2604[Abstract/Free Full Text].
|
| 22.
|
Katzman, M., and M. Sudol.
1998.
Mapping viral DNA specificity to the central region of integrase by using functional human immunodeficiency virus type 1/visna virus chimeric proteins.
J. Virol.
72:1744-1753[Abstract/Free Full Text].
|
| 23.
|
Katzman, M.,
M. Sudol,
J. S. Pufnock,
S. Zeto, and L. M. Skinner.
2000.
Mapping target site selection for the non-specific nuclease activities of retroviral integrase.
Virus Res.
66:87-100[CrossRef][Medline].
|
| 24.
|
Kuiken, C.,
F. McCutchan,
B. Foley,
J. W. Mellors,
B. Hahn,
J. Mullins,
P. Marx,
S. Wolinsky, and B. Korber.
1999.
Human retroviruses and AIDS 1999: a compilation and analysis of nucleic acid and amino acid sequences.
Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, N.Mex.
|
| 25.
|
Lins, R. D.,
A. Adesokan,
T. A. Soares, and J. M. Briggs.
2000.
Investigations on human immunodeficiency virus type 1 integrase/DNA binding interactions via molecular dynamics and electrostatics calculations.
Pharmacol. Ther.
85:123-131[CrossRef][Medline].
|
| 26.
|
Maignan, S.,
J.-P. Guilloteau,
Q. Zhou-Liu,
C. Clément-Mella, and V. Mikol.
1998.
Crystal structures of the catalytic domain of HIV-1 integrase free and complexed with its metal cofactor: high level of similarity of the active site with other viral integrases.
J. Mol. Biol.
282:359-368[CrossRef][Medline].
|
| 27.
|
Mazumder, A.,
N. Neamati,
A. A. Pilon,
S. Sunder, and Y. Pommier.
1996.
Chemical trapping of ternary complexes of human immunodeficiency virus type 1 integrase, divalent metal, and DNA substrates containing an abasic site: implications for the role of lysine 136 in DNA binding.
J. Biol. Chem.
271:27330-27338[Abstract/Free Full Text].
|
| 28.
|
Morgan, A. L., and M. Katzman.
2000.
Subterminal viral DNA nucleotides as specific recognition signals for human immunodeficiency virus type 1 and visna virus integrases under magnesium-dependent conditions.
J. Gen. Virol.
81:839-849[Abstract/Free Full Text].
|
| 29.
|
Pemberton, I. K.,
H. Buc, and M. Buckle.
1998.
Displacement of viral DNA termini from stable HIV-1 integrase nucleoprotein complexes induced by secondary DNA-binding interactions.
Biochemistry
37:2682-2690[CrossRef][Medline].
|
| 30.
|
Ratner, L.,
A. Fisher,
L. L. Jagodzinski,
H. Mitsuya,
R.-S. Liou,
R. C. Gallo, and F. Wong-Staal.
1987.
Complete nucleotide sequence of functional clones of the AIDS virus.
AIDS Res. Hum. Retroviruses
3:57-69[Medline].
|
| 31.
|
Ratner, L.,
W. Haseltine,
R. Patarea,
K. J. Livak,
B. Starcich,
S. F. Josephs,
E. R. Doran,
J. A. Rafalski,
E. A. Whitehorn,
K. Baumeister,
L. Ivanoff,
S. R. Petteway, Jr,
M. L. Pearson,
J. A. Lautenberger,
T. S. Papas,
J. Ghrayeb,
N. T. Chang,
R. C. Gallo, and F. Wong-Staal.
1985.
Complete nucleotide sequence of the AIDS virus, HTLV-III.
Nature
313:277-283[CrossRef][Medline].
|
| 32.
|
Shibagaki, Y., and S. A. Chow.
1997.
Central core domain of retroviral integrase is responsible for target site selection.
J. Biol. Chem.
272:8361-8369[Abstract/Free Full Text].
|
| 33.
|
Skinner, L. M.,
S. L. Lamers,
J. C. Sanders,
M. E. Eyster,
M. M. Goodenow, and M. Katzman.
1998.
Analysis of a large collection of natural HIV-1 integrase sequences, including those from long-term nonprogressors.
J. Acquir. Immune Defic. Syndr. Hum. Retrovirol.
19:99-110[Medline].
|
| 34.
|
Skinner, L. M.,
M. Sudol,
A. L. Harper, and M. Katzman.
2001.
Nucleophile selection for the endonuclease activities of human, ovine, and avian retroviral integrases.
J. Biol. Chem.
276:114-124[Abstract/Free Full Text].
|
| 35.
|
van den Ent, F. M. I.,
A. Vos, and R. H. A. Plasterk.
1998.
Mutational scan of the human immunodeficiency virus type 2 integrase protein.
J. Virol.
72:3916-3924[Abstract/Free Full Text].
|
| 36.
|
van Gent, D. C.,
A. A. M. Oude Groeneger, and R. H. A. Plasterk.
1992.
Mutational analysis of the integrase protein of human immunodeficiency virus type 2.
Proc. Natl. Acad. Sci. USA
89:9598-9602[Abstract/Free Full Text].
|
| 37.
|
van Gent, D. C.,
A. A. M. Oude Groeneger, and R. H. A. Plasterk.
1993.
Identification of amino acids in HIV-2 integrase involved in site-specific hydrolysis and alcoholysis of viral DNA termini.
Nucleic Acids Res.
21:3373-3377[Abstract/Free Full Text].
|
Journal of Virology, August 2001, p. 7756-7762, Vol. 75, No. 16
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.16.7756-7762.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Hobaika, Z., Zargarian, L., Boulard, Y., Maroun, R. G., Mauffret, O., Fermandjian, S.
(2009). Specificity of LTR DNA recognition by a peptide mimicking the HIV-1 integrase {alpha}4 helix. Nucleic Acids Res
37: 7691-7700
[Abstract]
[Full Text]
-
Diamond, T. L., Bushman, F. D.
(2005). Division of Labor within Human Immunodeficiency Virus Integrase Complexes: Determinants of Catalysis and Target DNA Capture. J. Virol.
79: 15376-15387
[Abstract]
[Full Text]
-
Lu, R., Ghory, H. Z., Engelman, A.
(2005). Genetic Analyses of Conserved Residues in the Carboxyl-Terminal Domain of Human Immunodeficiency Virus Type 1 Integrase. J. Virol.
79: 10356-10368
[Abstract]
[Full Text]
-
Konsavage, W. M. Jr., Burkholder, S., Sudol, M., Harper, A. L., Katzman, M.
(2005). A Substitution in Rous Sarcoma Virus Integrase That Separates Its Two Biologically Relevant Enzymatic Activities. J. Virol.
79: 4691-4699
[Abstract]
[Full Text]
-
Lu, R., Limon, A., Ghory, H. Z., Engelman, A.
(2005). Genetic Analyses of DNA-Binding Mutants in the Catalytic Core Domain of Human Immunodeficiency Virus Type 1 Integrase. J. Virol.
79: 2493-2505
[Abstract]
[Full Text]
-
Gao, K., Wong, S., Bushman, F.
(2004). Metal Binding by the D,DX35E Motif of Human Immunodeficiency Virus Type 1 Integrase: Selective Rescue of Cys Substitutions by Mn2+ In Vitro. J. Virol.
78: 6715-6722
[Abstract]
[Full Text]
-
Deprez, E., Barbe, S., Kolaski, M., Leh, H., Zouhiri, F., Auclair, C., Brochon, J.-C., Le Bret, M., Mouscadet, J.-F.
(2004). Mechanism of HIV-1 Integrase Inhibition by Styrylquinoline Derivatives in Vitro. Mol. Pharmacol.
65: 85-98
[Abstract]
[Full Text]
-
Chiu, R., Grandgenett, D. P.
(2003). Molecular and Genetic Determinants of Rous Sarcoma Virus Integrase for Concerted DNA Integration. J. Virol.
77: 6482-6492
[Abstract]
[Full Text]
-
Harper, A. L., Sudol, M., Katzman, M.
(2003). An Amino Acid in the Central Catalytic Domain of Three Retroviral Integrases That Affects Target Site Selection in Nonviral DNA. J. Virol.
77: 3838-3845
[Abstract]
[Full Text]
-
Appa, R. S., Shin, C.-G., Lee, P., Chow, S. A.
(2001). Role of the Nonspecific DNA-binding Region and alpha Helices within the Core Domain of Retroviral Integrase in Selecting Target DNA Sites for Integration. J. Biol. Chem.
276: 45848-45855
[Abstract]
[Full Text]
Top
Abstract
Text
