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Journal of Virology, April 2001, p. 3556-3567, Vol. 75, No. 8
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.8.3556-3567.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
DNase Protection Analysis of Retrovirus Integrase
at the Viral DNA Ends for Full-Site Integration In Vitro
Ajaykumar
Vora and
Duane P.
Grandgenett*
St. Louis University Health Sciences Center,
Institute for Molecular Virology, St. Louis, Missouri 63110
Received 2 November 2000/Accepted 16 January 2001
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ABSTRACT |
Retrovirus intasomes purified from virus-infected cells contain the
linear viral DNA genome and integrase (IN). Intasomes are capable of
integrating the DNA termini in a concerted fashion into exogenous
target DNA (full site), mimicking integration in vivo. Molecular
insights into the organization of avian myeloblastosis virus IN at the
viral DNA ends were gained by reconstituting nucleoprotein complexes
possessing intasome characteristics. Assembly of IN-4.5-kbp donor
complexes capable of efficient full-site integration appears cooperative and is dependent on time, temperature, and protein concentration. DNase I footprint analysis of assembled IN-donor complexes capable of full-site integration shows that wild-type U3 and
other donors containing gain-of-function attachment site sequences are
specifically protected by IN at low concentrations (<20 nM) with a
defined outer boundary mapping ~20 nucleotides from the ends. A donor
containing mutations in the attachment site simultaneously eliminated
full-site integration and DNase I protection by IN. Coupling of
wild-type U5 ends with wild-type U3 ends for full-site integration
shows binding by IN at low concentrations probably occurs only at the
very terminal nucleotides (<10 bp) on U5. The results suggest that
assembly requires a defined number of avian IN subunits at each viral
DNA end. Among several possibilities, IN may bind asymmetrically to the
U3 and U5 ends for full-site integration in vitro.
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INTRODUCTION |
Integration of the linear retrovirus
DNA genome (~10 kbp) into the host chromosome requires the viral
integrase (IN) (for a review, see reference 4). Viral DNA
and IN are found within cytoplasmic preintegration complexes (PIC) in
newly virus-infected cells. IN removes a dinucleotide from the
blunt-ended viral ends, producing 3'-OH recessed long terminal repeat
(LTR) termini (15). Purified PIC from cells infected with
murine leukemia virus (MLV) (3), Rous sarcoma virus
(25), and human immunodeficiency virus type 1 (HIV-1)
(13) can mediate the concerted insertion of viral DNA
termini into exogenous target DNA in vitro.
Concerted insertion of the viral DNA termini by IN into the host
chromosome (full-site integration) necessitates a close molecular interaction between the two termini. Molecular probing studies of the
HIV-1 PIC suggest the two termini are held together with a protein
bridge (30). The 3'-OH recessed LTR termini interact to
form a functional complex for integration in vitro, termed an intasome
(41, 42). The role cellular proteins have in PIC or
intasomes is undefined (12, 24, 26, 41). When
characterized by bacteriophage Mu-mediated PCR (MM-PCR) footprints,
intasomes exhibit protection and enhancements near the termini (~20
bp from the end) and an extended region of protection spanning several hundred base pairs from the ~20th nucleotide, both requiring a functional IN (2, 7, 42). The need of the relatively large MM-PCR footprints in relationship to only the ~15-bp terminal attachment (att) site sequence requirement for efficient
integration in vivo is unknown (7).
Early attempts to reproduce the full-site integration reaction in vitro
with linear DNA donors and IN required genetic selection or
amplification of the full-site integration products for measurement (10, 14, 22). Progress in producing nucleoprotein
complexes mediating more efficient full-site integration in vitro with
purified virion and recombinant IN is evident (1, 6, 16, 17, 21,
37, 38). The donor-target products produced by the full-site integration reactions are defined by restriction enzyme analysis and
agarose gel electrophoresis. Genetic isolation and DNA sequencing of
individual recombinants have verified the host duplications observed
upon concerted insertion of the viral DNA termini in vivo.
Assembly of nucleoprotein complexes generally requires a series of
discrete steps (31, 33, 35, 36). For full-site integration, the assembly time is rapid (~1 min) at 0°C using linear 480-bp LTR donors with avian myeloblastosis virus (AMV) IN (50 nM) (37). It was necessary to identify factors slowing the
assembly process to investigate events at the molecular level. The
480-bp donors (15 ng) were replaced with 4.5-kbp donors (10 ng)
containing the avian 330-bp LTRs at their termini (Fig.
1). With this modification, the number of
LTR termini in the assembly mixture was decreased by a factor of ~12,
and subsequently, the concentration of IN was lowered proportionally.
Different 4.5-kbp donors containing wild-type (wt) and att
site mutations were used to investigate the molecular processes
necessary for efficient and high-fidelity full-site integration. The
assembly of IN-LTR donor complexes appears cooperative and is dependent
on time, temperature, and protein concentration. DNase I footprint
analysis of assembled nucleoprotein complexes revealed that wt U3 or
gain-of-function ("G") LTR ends are specifically protected by IN
with the outer boundary mapping ~20 nucleotides on each terminus. IN
appears to bind only to the very terminal nucleotides (<10 bp) on wt
U5. The results suggest that assembly of nucleoprotein complexes
requires a defined number of IN subunits binding asymmetrically to the U3 and U5 ends for full-site integration in vitro.
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FIG. 1.
Schematic of the wt 4.5-kbp donor, the assembly and
integration assay protocols, the full-site integration product, and the
restriction enzyme fragments of full-site integration products. (A) The
linear 4.5-kbp donor containing the wt U3 and wt U5 LTR termini is
displayed. The att sites are identified at each end.
XhoI, SspI, NheI, and MluI
sites and the length of several fragments resulting from enzyme
digestions are shown. (B) Two donors are assembled by IN displayed as
four dimers. After assembly at 14°C, target (boldface type is added),
followed by integration. (C) The linear full-site product (11.9 kbp) is
the result of the concerted integration of two separate donors
(bimolecular reaction) into a 2.8-kbp circular target DNA (bold line).
MluI/XhoI-digestion produces a 4.2-kbp donor-target fragment
containing U3 and U5 ends. (D) Only the labeled donor-target
restriction fragments resulting from full-site integration are shown.
The frequency of assembling two paired ends (U3-U3, U3-U5, U5-U5) to
mediate bimolecular full-site integration is defined by Hardy-Weinberg
distribution frequency. For example, when both LTR ends have equivalent
integration activity, the distribution of the
XhoI/MluI-digested full-site fragments will be
1:2:1 (Fig. 2, lane 4).
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MATERIALS AND METHODS |
Linear DNA donors.
A linear 4.5-kbp donor was constructed
containing both the avian retrovirus wt U3 and wt U5 LTRs (each 330 bp
long) (Fig. 1) (14). An NdeI site was produced
at the circle junction of blunt-ended U3 and U5 ends by site-directed
mutagenesis (Stratagene's QuikChange). NdeI digestion
produces a linear 4.5-kbp donor containing 3'-OH recessed ends.
Numbering of nucleotides was from the blunt end. The 5th and 6th
nucleotides were modified at each terminus in several clones as
indicated in the text. Each donor sequence was verified by DNA
sequencing. The linear 3.4-kbp donor containing ~30 bp of wt U5 and
wt U3 sequences at its ends was described (14).
Labeling of donors.
The linear 4.5-kbp donors were 5' end
labeled using [
-32P]ATP and polynucleotide kinase at
the NdeI sites. The specific activities were ~2,500 cpm
(Cerenkov) per ng. For DNase I footprint analysis, the 5'-end-labeled
4.5-kbp donors were digested at either the SspI,
NheI, XhoI, or MluI sites to produce
different-size single-end-labeled LTR donors (Fig. 1). The fragments
were isolated by agarose gel electrophoresis, electroeluted, and
concentrated by a Centricon YM-30 filtering device. For nonspecific
DNA, pGEM-3 (2.8 kbp) was digested with EcoRI and labeled.
The labeled DNA was digested with NheI, and the
single-end-labeled 2.5-kbp DNA was isolated for DNase I footprint analysis.
Full-site integration assay.
Conditions for assembling
nucleoprotein complexes capable of producing full-site integration
products significantly better than half-site integration products at
low IN concentrations (<20 nM) were established. Assembly of IN with
the 4.5-kbp donors (10 ng) occurred in the presence of 330 mM NaCl, 10 mM MgCl2, 3 mM dithiothreitol, 8% polyethylene glycol
6000, and 20 mM HEPES (pH 7.5) at 14°C. Organic solvents used
previously in the integration assays were found to be inhibitory at low
concentrations of AMV IN (37). The standard volume was 20 µl or multiples thereof. The concentration of IN, the time, and the
temperature for assembly with donor were varied as indicated. The
integration reactions were initiated by the addition of 50 ng of
supercoiled DNA as target and immediately incubated at 37°C for only
5 or 10 min as indicated. Either pGEM-3 (2.8 kbp) or pUC19 (2.6 kbp)
was used as a target. The target-to-donor molar ratio was three. The
ratio of target (2.8 kbp) to donor (4.5 kbp) molecules was 8. The
integration reactions were stopped by addition of sodium dodecyl
sulfate and proteinase K, and the products were subjected to agarose
gel electrophoresis. The amount of donor incorporated into the target
was determined with a Molecular Dynamics PhosphorImager.
DNase I footprinting.
Double-end-labeled 4.5-kbp donors were
digested with either XhoI, NheI, or
SspI (Fig. 1A) to isolate single-end-labeled donors on
agarose gels for DNase I footprinting. Titration experiments using
DNase I with single-5'-end-labeled DNA were performed to determine the
concentration of DNase I necessary to produce approximately one nick
per DNA strand in assembly buffer. DNase I at 750 ng per ml was
optimal. For assembly and for the DNase I footprint analyses, IN and
the single-end-labeled 3.6-kbp donors were assembled at 14°C. An
aliquot was removed from the mixture for measuring integration activity
just prior to the addition of DNase I. The nucleoprotein complexes in
the assembly mixture were further incubated with DNase I for 90 s
at 14°C. The DNase I reactions were stopped by the addition of
phenol. The DNase I-treated samples were subjected to denaturing 8, 10, 13, or 20% polyacrylamide gel electrophoresis depending on the
required analysis. The dried gels (8 or 10%) were analyzed by a
PhosphorImager and exposure to X-ray film.
Restriction analysis and genetic selection of donor-target
recombinants.
The XhoI and MluI sites in the
4.5-kbp donors were used to characterize the donor-target products.
pGEM-3 (2.8 kbp) or pUC19 (2.6 kbp) served as target DNA. The
EcoRI site was destroyed in pUC19. The donor-target products
produced with pUC19 were digested with EcoRI to isolate a
unique 2.9-kbp fragment containing pUC19 with two flanking LTR termini
on agarose gels. The purified fragment was ligated and transformed into
Escherichia coli, and the plasmids from individual colonies
were isolated and sequenced. The viral DNA-host junctions were
sequenced as described (37).
Purification of AMV IN.
AMV IN was purified to near
homogeneity (19, 29).
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RESULTS |
Full-site integration using linear 4.5 kbp donors with AMV IN.
Linear 4.5-kbp donors containing the U3 and U5 LTR termini (Fig. 1A)
were used to investigate assembly conditions for full-site integration.
Three different linear 4.5-kbp donors were used. The wt donor contained
wt U3 (5'-ACTACAOH) and wt U5
(5'-GCTTCAOH) att site
sequences at its 3'-OH recessed termini. Another donor was modified at
both the U3 and U5 ends by changing the underlined 5th and 6th
nucleotide positions to 5'-ACAACAOH
and 5'-GCAACAOH, respectively.
These att site sequences are defined as having a ("G")
mutation for integration (28, 39, 44). Lastly, a donor with att site mutations lacking significant integration
activity contained 5'-ACCCCAOH and
5'-GCCCCAOH at its U3 and U5 ends, respectively.
Two parameters established that IN produces the correct full-site
integration products with the new 4.5-kbp donors. The full-site
products contain two separate donor ends inserted in a concerted
fashion into a circular DNA target (Fig.
1C). Restriction enzyme
digestions of the 11.9-kbp full-site products by
XhoI or
MluI,
or both, produced the anticipated DNA fragments (Fig.
1A, C, and
D; Fig.
2). The donor contains
the "G" LTR mutations at both the
U3 and U5 termini.
XhoI digestion of the 11.9-kbp products (Fig.
2, lane 1)
produced the expected 10.2-, 7.4-, and 4.6-kbp fragments
(Fig.
2, lane
2) (Fig.
1D). The fastest migrating band (3.6 kbp)
in lane 2 is the
digested donor (Fig.
1A). The two slowest migrating
bands in lane 2 are
half-site products (circular target with one
inserted donor and
different-size donor tails) (
38).
MluI
digestion
of the full-site products also produced the anticipated
fragments
(Fig.
2, lane 3; Fig.
1D). The combined
XhoI/
MluI digestions produced
the expected 4.6-, 4.2-, and 3.7-kbp fragments (Fig.
2, lane 4).
With the "G" donor
containing nearly equivalent catalytic ends
(
39), the
three
XhoI/
MluI full-site digestion fragments
have
near-molar ratios of 1:2:1 (Fig.
2, lane 4). The appropriate
restriction
patterns were also obtained with the 4.5-kbp donor
containing
wt U3 and wt U5 ends (data not shown). In summary,
restriction
analysis of the full-site products produced the anticipated
fragments.
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FIG. 2.
Restriction enzyme analysis of "G" 4.5-kbp
donor-target full-site integration products. The circular half-site and
full-site products (lane 1) are indicated on the left (lettering not in
boldface type). The unincorporated donor on the 1.5% agarose gel is
the fastest migrating band in lane 1. The XhoI restriction
fragments in lane 2 resulting from the full-site products are indicated
in boldface lettering (10.2, 7.4, and 4.6 kbp) on the left under
XhoI (Fig. 1D). XhoI digestion of the circular
half-site products (lane 2) results in circles with two different sizes
of tails (X on right). MluI digestion (lane 3) produces the
anticipated half-site and full-site fragments (not marked) (Fig. 1D).
In lane 4, the combined XhoI/MluI digestion of
the full-site product produces the anticipated 1:2:1 ratio of fragments
migrating at 4.6, 4.2, and 3.7 kbp (Fig. 1D), respectively (at right,
in boldface type). The two slowest migrating fragments in lane 4 are
digested circular half-site products. Lane 5 contains molecular weight
markers varying in size by ~1 kbp (at right, not in boldface type).
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The second parameter to establish full-site integration required
sequencing of the donor-target junctions. The donor-target
products
from both wt and "G" donor reactions were digested with
EcoRI (unique sites located in both LTRs). A 2.9-kbp
donor-target
fragment was isolated by agarose gel electrophoresis,
ligated,
and transformed into
E. coli, and the plasmids from
20 colonies
were sequenced. The avian host site duplications (5 to 7 bp, with
the 6-bp duplication predominating) were present in all of the
plasmids at the donor-target junctions with both donors. The data
show
IN mediates efficient full-site integration with a high fidelity
with
either the 4.5-kbp donors or the 480-bp donors (
39).
Assembly of nucleoprotein complexes mediating full-site integration
is cooperative for protein concentration, is time-dependent, and is
partially temperature dependent.
The assembly of IN-"G" donor
complexes capable of full-site integration appears to be cooperative
with respect to protein concentration at 14°C (Fig.
3). No full-site integration activity is
observed at 14°C (data not shown). The assembly time was held constant at 10 min. After assembly, target was added and integration was allowed to proceed for only 5 min at 37°C, thus more closely reflecting the effects of the initial assembly events. A sigmoidal curve for incorporation of donor was apparent with respect to full-site
integration but not half-site integration (Fig. 3). Similar kinetic
data for both reactions were apparent upon assembly on ice for 10 min,
although the total quantities for both products were decreased ~25%
at the same concentrations of IN. Similar cooperative interactions for
assembly of complexes mediating full-site integration at 14°C were
observed using the 4.5-kbp donor with wt U3 and U5 termini and the
3.4-kbp donor having only ~30 bp of wt U3 and U5 LTR sequences at
their termini (data not shown).
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FIG. 3.
Assembly of IN-4.5 kbp donor complexes appears
cooperative for full-site integration. Various concentrations of AMV IN
(bottom) were assembled with the "G" 4.5-kbp donor at 14°C. The
integration products were subjected to electrophoresis on a 1% agarose
gel and quantified.
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To differentiate further the assembly events for full-site and
half-site integration, we investigated the effects of time
and
temperature. IN was added to the assembly mixture containing
the
"G" 4.5-kbp donor equilibrated at 14°C. The time of assembly
was
varied while the concentration of IN remained constant (6
nM) (Fig.
4A). The
integration assays were again limited to 5
min at 37°C. The rate for
forming nucleoprotein complexes capable
of mediating full-site
integration was time dependent, while half-site
integration was
essentially unchanged after 1 min (Fig.
4A). Varying
the concentration
of IN (4 to 15 nM) in a series of parallel assembly
experiments with
the 4.5-kbp "G" or wt donors also produced similar
time-dependent
assembly requirements for full-site integration
but not half-site
integration (data not shown). Similar assembly
rates for nucleoprotein
complexes were observed for both reactions
with the "G" donor at 6 nM IN if the assembly temperature was
at 0°C (Fig.
4B), although
half-site integration was always proportionally
higher at 0°C than at
14°C. Other studies revealed similar formation
rates for both types
of nucleoprotein complexes at 14°C in the
absence of Mg
2+
during assembly but more closely resembled the 0°C results observed
in Fig.
4B (data not shown). Similar assembly results were also
obtained if the 3.4-kbp donor containing ~30 bp of wt U3 and wt
U5
LTR sequences at its termini were assembled with 10 nM IN at
14°C
(Fig.
4C).
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FIG. 4.
Different assembly rates for nucleoprotein complexes
mediating full-site and half-site integration. (A) IN was assembled
with the "G" 4.5-kbp donor at 14°C. At various time intervals
(bottom), aliquots were taken and added to tubes containing target DNA
at room temperature. Following strand transfer at 37°C, the
products were subjected to agarose gel electrophoresis and
quantified. (B) The same assembly experiment was repeated as described
in panel A, except assembly was on ice. (C) The same assembly
experiment was repeated as described in panel A at 14°C, except IN
was at 10 nM and the donor was the wt 3.4-kbp DNA containing ~30 bp
of wt U5 and wt U3 sequences at its ends.
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In summary, the assembly of IN-donor complexes capable of mediating
full-site integration appears to be cooperative for protein-DNA
interactions and is sensitive to time and temperature. The assembly
rates of nucleoprotein complexes for full-site events are significantly
different from those observed for half-site events. For full-site
integration, the assembly appears to be independent of LTR sequences
mapping >30 bp from the termini. The results also suggest some
or all
of the nucleoprotein complexes responsible for half-site
integration
under these assembly conditions may not be precursors
to nucleoprotein
complexes involved in full-site
integration.
IN-DNA complexes capable of full-site integration produce DNase I
footprints with an outer boundary mapping ~20 bp from the LTR
end.
The molecular probing of IN-donor complexes provided
information regarding the physical association of IN with the viral LTR termini. This association was examined under the same conditions as
required for assembly to investigate structure-functional relationships necessary for full-site integration. DNase I was chosen as the mildest
molecular probe. The physical interactions of IN with functional and
nonfunctional LTR donors for integration were analyzed.
The single 5'-end labeled 3.6 kbp DNA fragment (Fig.
1A) containing
"G" sequences on the U3 end was assembled with varying
concentrations of IN for 15 min at 14°C. After assembly of the
IN-DNA
complexes, aliquots were taken to measure integration activity
(10 min
at 37°C) (Fig.
5A),
and the rest of the samples were
subjected
to DNase I digestion for 90 s at 14°C prior to being
stopped with
phenol (Fig.
5B). Full-site integration dominates at low
concentrations,
while half-site integration is preferred at higher IN
concentrations
(Fig.
5A). At 5 nM IN, the full-site integration
reaction is approximately
fivefold more than the half-site reaction.
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FIG. 5.
Full-site integration is correlated to DNase I
protection by IN at the LTR termini. (A) The single-end-labeled 3.6-kbp
fragment containing "G" sequences on the U3 LTR end was assembled
with IN at different concentrations. (B) The rest of the assembly
mixtures in A were subjected to DNase I digestion. The DNase I-treated
samples were subjected to electrophoresis on a denaturing 8%
polyacrylamide gel. Equivalent counts were loaded per lane. The far
left lane contains the untreated 3.6-kbp DNA (Neg.). The two adjacent
lanes are G/A and C/T chemical reactions. The concentrations of IN in
lanes 1 to 7 were 0, 5, 10, 20, 30, 40, and 50 nM, respectively. The
nucleotide positions numbered from the blunt-end are marked (right) and
the area protected by IN (vertical rectangle) mapping ~20 bp from the
end is shown. Chemical markers have one less nucleotide than the
adjacent fragments produced by DNase I. The dried gel was exposed to
X-ray film for 10 days without an intensifying screen.
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The assembled complexes tested for integration activity (Fig.
5A) were
also subjected to parallel DNase I footprint analysis
to examine the
physical association of IN at the LTR ends. At
5 and 10 nM IN (Fig.
5B,
lanes 2 and 3), a specific region of
protection at the "G" donor
termini was observed whose outer boundary
mapped to ~20 nucleotides
from the end in comparison to the naked
"G" donor subjected to
DNase I without IN (Fig.
5B, lane 1). The
protection is from
approximately position 11 (T) to position 20
(C). DNase I is sensitive
to position relative to the ends of
the DNA and will not nick closer
than ~10 bp with high frequency.
Starting at 20 nM IN (Fig.
5B, lane
4) and particularly at higher
IN concentrations (lanes 5 to 7), an
extended pattern of partial
DNase I protection is observed which almost
encompasses the entire
length of visualized DNA fragments (~80
nucleotides past position
20). The results suggest that a specific size
of footprint, which
correlates to full-site integration activity, is
produced by IN
at lower concentrations (Fig.
5A).
Several other observations are apparent with the DNase I footprint
analysis. At the lower IN concentrations (Fig.
5B, lanes
2 to 4), there
were no observable adjacent or other internal regions
of protection
produced by IN after the 20th nucleotide relative
to the same DNA
treated with DNase I without IN (lane 1). Instead,
there appears to be
enhanced DNase I nicking at several nucleotides
within the next ~10
internal positions. PhosphorImager analysis
of the DNase I protection
patterns revealed IN protects DNA from
position 11 to 20 by a factor of
approximately five or more (Fig.
5B [compare lane 1 with lanes 2 and
3], 6, and 8). IN at concentrations
20 nM or more (Fig.
5B, lanes 4 to
7) did not alter this protection
pattern below position 20, suggesting
that this protected region
was also specific (see Fig.
7 for
nonfunctional donor). Lastly,
assembled IN-donor complexes treated with
DNase I were selected
by nitrocellulose filters to determine if the
purified complexes
possessed different footprints. After DNase I
treatment, the nucleoprotein
complexes were immediately filtered onto
nitrocellulose filters,
washed with assembly buffer, eluted with sodium
dodecyl sulfate
buffer, and examined on denaturing gels. The same
pattern of DNase
I protection (~20 bp from the end) by IN shown in
Fig.
5B (lanes
2 to 4) was observed (data not
shown).
In summary, comparing the full-site integration activity (Fig.
5A) to
the parallel DNase I footprint (Fig.
5B) suggests a
specific set of IN
subunits binding cooperatively to LTR ends
is necessary for full-site
integration. The protected region maps
to ~20 nucleotides from the
"G" LTR end. The outer boundary is
specifically observed at lower
IN concentrations (<20 nM) and
is associated with full-site and not
half-site integration
activity.
Stability and specificity of assembled IN-donor complexes capable
of full-site integration are correlated to functional att
site sequences.
Additional time studies were undertaken to show
the stable physical association of IN with the 3.6-kbp "G" and wt
U3 LTR donor ends (Fig. 6A and B,
respectively). AMV IN was assembled at 14°C with each donor, and
aliquots were analyzed for full-site integration and by DNase I
footprint analysis. Both donors allowed IN to form a stable complex (15 min to 6 h) whose outer boundary maps ~20 bp from the end of the
DNA. For the "G" donor reaction, the percent donor incorporated for
full-site integration was 24, 27, 31, 32, 28, and 27, and for half-site
integration it was 6, 6, 8, 7, 6, and 7, respectively (Fig. 6A, lanes 2 to 7). For the wt U3 reaction, the percent donor incorporated for
full-site integration was 4, 6, 8, 13, 16, and 16, and for half-site
integration it was 1, 2, 2, 4, 3, and 4, respectively (Fig. 6B, lanes 2 to 7).
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FIG. 6.
Highly stable DNase I footprints associated with
IN-donor complexes for full-site integration. (A) IN (5 nM) was
assembled with the 3.6-kbp U3 donor containing "G" sequences.
Assembly times were 15, 30, 60, 120, 240, and 360 min in lanes 2 to 7, respectively. Lane 1 contains no IN. Labeling is the same as in Fig. 5.
(B) IN (10 nM) was assembled with the single-end-labeled 3.6-kbp
fragment containing wt U3 sequences for the same times. Labeling is the
same as in Fig. 5.
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The 5th, 6th, and 7th nucleotides from the ends of the avian U3 and U5
LTRs have a major influence on the ability of IN to
mediate half-site
and full-site integration (
9,
39). The
U5 LTR end
(5'-GC
TTCA
OH) of the 4.5-kbp donor
(Fig.
1A) was modified
to 5'-GC
CCCA
OH.
The single-end-labeled 3.6-kbp fragment containing
the
CC mutation was assembled with various concentrations of
IN.
Aliquots were taken for integration activity and DNase I protection
analysis. At 20 nM IN with the DNase I footprint (Fig.
7, lane
5), there was ~1%
incorporation of the U5 donor containing the
CC mutation
into each of the half-site and full-site integration
products (10-min
reaction) (data not shown). No specific protection
by IN with an outer
boundary mapping at ~20 nucleotides from the
end was evident with the
CC mutant donor (Fig.
7, lanes 1 to 5).
Increasing the
concentration of IN to 45 nM also resulted in no
distinct footprint on
the U5 end with the
CC mutation (data not
shown). The U3
end containing the
CC mutation at the fifth and
sixth
nucleotide also lacks significant integration activity.
Finally, IN was
also preincubated at 14°C with a 2.4-kbp nonspecific
DNA fragment
containing a 5'-end-labeled
EcoRI restriction site.
There
was neither integration activity nor evidence of a specific
DNase I
protection pattern by IN (10 to 30 nM) (data not shown).
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FIG. 7.
Lack of DNase I protection by IN with a nonfunctional
LTR donor. IN was assembled with a 3.6-kbp fragment containing a
mutation (5'-CCCCAOH) at the U5 LTR
end. Lane 1 contains naked DNA treated with DNase I. Lanes 2 to 5 contain 3, 5, 10, and 20 nM IN, respectively. Labeling is the same as
in Fig. 5.
|
|
In summary, IN is capable of forming a very stable complex having a
specific physical association within the first ~20 bp
on either the
wt U3 or "G" donor ends for full-site integration.
This specific
association appears to be related to
att site sequences
capable of efficiently mediating integration activity in
vitro.
Formation of the outer protected boundary at ~20 bp by IN on the
"G" donor end with target present.
Earlier attempts to
reconstitute full-site integration employed the preincubation of donor
and target together with IN (10, 14, 22). We investigated
whether AMV IN was capable of forming the same specific DNase I
protection patterns at the LTR ends with target present during assembly
and when assembled IN-donor complexes were challenged with target. The
single-end-labeled 3.6-kbp fragment with the U5 end containing the
"G" mutation (Fig. 1A) was used as the donor.
Assembly of AMV IN (5 to 20 nM) with the U5 end containing the "G"
mutation for 45 min at 14°C produced the usual DNase I
protection
pattern mapping ~20 bp from the end (Fig.
8A, lanes
1 to 4), and this protection
was correlated with full-site integration
activity (Fig.
8B, top).
These assembled IN-donor complexes were
also challenged with the normal
concentration of target used to
measure integration activity. The
target was further incubated
with the assembled complexes for 10 min at
14°C prior to DNase
I digestion. The DNase I protection pattern (Fig.
8A, lanes 5
to 7) and the full-site integration activity (Fig.
8B,
middle)
were slightly reduced relative to those of the unchallenged
controls
(Fig.
8A, lanes 2 to 4). Finally, IN was assembled on the
donor
ends in the presence of target. The donor and target were
preincubated
together with IN for 45 min at 14°C prior to DNase I
digestion.
Little DNase I protection (Fig.
8A, lane 8) and integration
activity
(Fig.
8B, bottom) were apparent at 5 nM IN. However at 20 nM
IN,
the ~20-bp DNase I protection pattern (Fig.
8A, lane 10) as well
as a significant increase in full-site integration activity was
observed (Fig.
8B, bottom).
View larger version (92K):
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|
FIG. 8.
Assembly of IN-donor complexes with target present and
challenge of assembled IN-donor complexes with target. (A) IN was
assembled with single-end-labeled 3.6-kbp fragment (10 ng) containing
"G" sequences on the U5 end. Lanes 2 to 4, 5 to 7, and 8 to 10 contain 5, 10, and 20 nM IN, respectively. In lanes 2 to 4, IN was
assembled on the donor DNA only prior to taking an aliquot for
integration (Fig. 8B, top) and DNase I treatment. In lanes 5 to 7, IN
was assembled with donor and then challenged with target as indicated
by the arrow (50 ng per 20 µl). An aliquot was taken for integration
activity (Fig. 8B, middle), and the remaining sample treated with DNase
I. In lanes 8 to 10, donor, target, and IN were assembled together
prior to assaying for integration activity (Fig. 8B, bottom) and DNase
I treatment. Labeling is the same as in Fig. 5. (B) Strand transfer
activity of samples shown in panel A.
|
|
The results suggest IN is capable of forming stable complexes with
donor ends for full-site integration in the presence of
target and the
assembled IN-DNA complexes are relatively stable
in the presence of
competitor DNA. The DNase I protection pattern
observed at the U5 end
containing the "G" mutation appears nearly
identical to the pattern
observed at the U3 end containing the
"G" mutation (Fig.
5 and
6).
Asymmetric binding at the wt U5 end by IN for full-site integration
in comparison to the "G" and wt U3 ends.
Asymmetric
recognition of DNA binding sites by proteins for DNA recombination
events is common. Two different approaches were used to address this
possibility for IN-LTR interactions. We investigated whether IN was
capable of producing the same DNase I protection pattern observed when
two wt U3 or two "G" LTR ends were coupled together (Fig. 5, 6, and
8) when two wt U5 ends were coupled together for full-site integration.
We also investigated the physical association of IN with wt U5 ends
coupled to either wt U3 or "G" donor ends for full-site integration
(28).
In the first approach, IN was assembled with the 3.6-kbp fragment
containing the wt U5 end for 45 min at 14°C prior to strand
transfer
for 10 min at 37°C. IN mediated efficient full-site integration
at
low concentrations (4 and 8 nM) (Fig.
9A,
top), with full-site
products being in the majority in comparison to
half-site products
(Fig.
9B, lane 1). At these low concentrations, no
DNase I protection
pattern with a distinct boundary at approximately
the 20th nucleotide
position with wt U5 was observed (Fig.
9C, lanes 1 to 5). Repeated
attempts to demonstrate a distinct DNase I footprint at
the wt
U5 end were negative. At higher IN concentrations (Fig.
9C,
lanes
4 and 5), only a general partial decrease of the entire DNase
I
footprint was evident relative to what was observed with IN
at lower
concentrations (lanes 2 and 3).
View larger version (56K):
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|
FIG. 9.
Outer boundary of ~20 bp DNase I protection by IN is
not observed at wt U5 end for full-site integration. (A) Top panel: IN
was assembled with the wt U5 end on the 3.6-kbp fragment, aliquots were
assayed for integration activity, and the percent donor incorporated
into target was determined. The concentrations of IN are indicated at
the bottom of each panel, and the donors are indicated on the right.
Middle panel: the U5 donor was mixed with unlabeled 2.9-kbp fragment
containing the wt U3 end prior to assembly with IN as described for
panel A. Bottom panel: same as the middle panel except the 2.9-kbp U3
fragment contained the "G" mutation. (B) Aliquots of each of the
reaction mixtures described for panel A produced at 4 and 8 nM IN were
pooled. The donor-target products were (+) or were not ( ) subjected
to XhoI digestion. Lanes 1 and 2 contain the U5-U5 products
(A, top), lanes 3 and 4 contain the U5-wt U3 products (A, middle), and
lanes 5 and 6 contain the U5-U3 with the "G" mutation products (A,
bottom). The full-site U5-U5 marker (left) identifies two U5 end
products while the U3-U5 marker identifies products of either the U5-wt
U3 (lane 4) or the U5-U3 with the "G" mutation (lane 6). Size
markers are in lane 7 (right). (C) DNase I footprinting of assembled
IN-donor complexes as indicated in panel A. Labeling is the same as in
Fig. 5 except lanes 2 to 5, 6 to 9, and 10 to 13 contain 4, 8, 16, and
32 nM IN, respectively. The combinations of donors are shown at the
top.
|
|
Possibly, the assembly of IN on wt U5 ends required the presence of
target to produce a DNase I protection pattern mapping
to approximately
the 20th nucleotide. No distinct DNase I footprints
were obtained at wt
U5 donor ends if assembly occurred in the
presence of target (data not
shown). As shown previously with
the "G" donor (Fig.
8), IN (20 nM)
was capable of assembling with
the 3.6-kbp wt U5 donor in the presence
of target (45 min at 14°C)
in an efficient manner (22 and 21%
full-site and half-site products
in 10 min at 37°C), even though no
distinct DNase I footprint
was evident at the wt U5
end.
In the second approach, we investigated whether a distinct DNase I
protection pattern could be produced by IN when a wt U5
end was coupled
to either wt U3 or "G" donor ends for full-site
integration. Equal
amounts (5 ng each) of the single-end-labeled
3.6-kbp wt U5 fragment
were assembled together with either unlabeled
2.9-kbp wt U3 fragment or
the same U3 fragment containing the
"G" mutation. The 3.6-kbp U5
fragment was produced by
XhoI digestion
while
SspI digestion produced the 2.9-kbp U3 fragments containing
the
XhoI site (Fig.
1A). From PhosphorImager analysis,
approximately
73% of the full-site integration product produced with
the labeled
wt U5 end was coupled to either unlabeled U3 or "G"
donor ends
(Fig.
9A, middle and bottom, respectively). This conclusion
was
reached because the full-site products produced with only the
labeled wt U5 donor in comparison to the wt U5 donor coupled with
either unlabeled wt U3 or "G" donor results in different size
restriction fragments after
XhoI digestion (Fig.
9B, compare
lane
2 with lanes 4 and 6, respectively). Coupling of the labeled wt
U5
end with either the wt U3 donor or the U3 donor with the "G"
mutation did not appear to significantly modify the DNase I footprints
on wt U5 (Fig.
9C, lanes 6 to 9 and lanes 10 to 13, respectively)
in
comparison to the footprint obtained with only two wt U5 ends
(Fig.
9C,
lanes 2 to 5). At 4 or 8 nM IN, no distinct protection
of DNA from
nucleotides ~11 to 20 was evident even though full-site
integration
was the predominant reaction (Fig.
9C, lanes 2 and
3, 6 and 7, and 10 and 11, respectively). Partial protection of
this DNA region and the
adjacent DNA was evident at higher IN
concentrations. Similar results
were also obtained if the ratio
of the labeled wt U5 (3.5 ng) to
unlabeled wt U3 and "G" donors
(6.5 ng) was used during assembly
(data not
shown).
The second approach was further modified to determine the physical
association of IN upon coupling U3 and U5 termini for full-site
integration. As a positive control for this approach, the labeled
donor
was now the 2.9-kbp U3 fragment containing the "G" mutation
(1.5 ng) while the unlabeled donor was the 3.6-kbp fragment with
the wt U5
end (8.5 ng). Assembly was with IN (5 to 15 nM) for
45 min at 14°C.
As shown previously by restriction analysis (Fig.
9B), 90% of the
observed full-site integration reaction at 5 nM
IN was the result of
coupling U3 and U5 ends together (data not
shown). As anticipated,
DNase I footprint analysis showed IN protected
the labeled U3 end with
a defined outer boundary ~20 nucleotides
for the end (data not shown)
as previously observed upon coupling
of two U3 ends together for
full-site integration (Fig.
5,
6,
and
8).
In summary, the results suggest IN binds within the first ~10
nucleotides on the wt U5 ends (beyond the detection level of
the DNase
I probe) for full-site integration. The lack of DNase
I protection by
IN at lower concentrations with a clear boundary
at approximately the
20th nucleotide on the wt U5 ends coupled
to either the wt U3 ends or
the "G" ends suggests IN binds asymmetrically
at the ends of the
viral DNA to mediate full-site integration
(Fig.
10).
View larger version (11K):
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|
FIG. 10.
Models for multimerization of IN. (Left) IN is
represented as a dimer, and a dimer is required for the 3'-OH
processing step and half-site integration (4). The
location of IN is only an approximation in relationship to the 20-bp
marker. (Middle) A tetramer per LTR end is predicted for full-site
integration (5, 20). (Right) The model predicts an unknown
number of avian IN subunits that forms multimers on the U3 end with a
predicted tetramer on the U5 end.
|
|
| |
DISCUSSION |
The reconstitution of nucleoprotein complexes with AMV IN
and linear 4.5-kbp donors capable of mediating full-site integration in
vitro has revealed novel insights into assembly requirements and the
physical association of IN at the donor LTR ends. The apparent
cooperative binding of IN dimers at the LTR ends results in the
formation of multimers producing a region of DNase I protection whose
outer boundary maps to approximately the 20th nucleotide position on wt
U3 and LTR ends containing the "G" mutation. Interestingly, the
binding of IN to the wt U5 end appears to be only near the terminal
att sequences (<10 bp) for full-site integration. The asymmetric binding of AMV IN to the U3 and U5 LTR ends suggests a model
in which IN has multiple roles for assembly of nucleoprotein complexes capable of full-site integration in vitro (Fig. 10).
The assembly of nucleoprotein complexes for full-site integration at
14°C is significantly different from those for half-site integration
with respect to time and protein concentrations (Fig. 3 to 5). At 5 nM
IN, the ratio of IN dimer to 4.5-kbp donor ends is 15. The requirements
for full-site integration suggest an ordered set of events being
performed by IN. These events may be related to formation of a specific
number of multimers at the LTR ends as well as to holding together the
two donors in a stable structure necessary for the concerted insertion
of the termini into the target (Fig. 10). Both events may require
different activation energies for the protein-protein and protein-DNA
interactions envisioned necessary for assembly. The assembly process
appears to be independent of internal LTR sequences (>30 bp from the
end Fig. 4C) and can occur in the presence of target (Fig. 8). The time-dependent assembly process of avian intasome-like complexes in
vitro mirrors the slow formation of MLV intasomes after virus infection
(41) and some of the assembly processes associated with
the phage transposase-Mu DNA complexes in vitro (31, 36). Further studies will be needed to determine the number of subunits, the
role of the individual subunits, and the role of the three functional
domains of IN (4) in assembly of intasome-like complexes. Similar in vitro studies are actively being investigated for assembling Tn5 and Mu transposase-DNA complexes (43).
For full-site integration, purified retrovirus intasomes and
nucleoprotein complexes assembled at low AMV IN concentrations catalyze
equivalent incorporation of viral DNA into target (percent incorporated) in vitro. DNase I footprints of the reconstituted AMV
IN-donor complexes have revealed new insights into the physical association of IN at the viral ends with some limitations. DNase I is
not capable of efficiently nicking DNA closer than ~10 bp from the
end. As with the MM-PCR footprinting technique used to investigate
retrovirus intasomes, DNase I footprinting is also restricted to
observations obtained with the 5'-labeled end of the donor
(nonprocessed strand). The most striking similarity between the two
integration systems is that the protection of sequences by AMV IN
possesses a clear boundary ~20 bp from the end (Fig. 5, 6, and 8)
while the boundary for MM-PCR protection and enhancements for both MLV
and HIV-1 intasomes is also ~20 bp from the end (7, 41,
42). Both the reconstituted intasome-like complexes and the
purified retrovirus intasomes require active att site
sequences for assembly, protection, and full-site integration activity.
The retrovirus intasomes appear to have near-symmetrical MM-PCR
footprints, while the reconstituted AMV IN-donor complexes have
asymmetrical DNase I footprints at their DNA ends. Another dissimilarity between the systems is the lack of DNase I enhancements within the AMV IN protected sequences (~11 to 20 nucleotides from the
end on wt U3 and LTR ends with the "G" mutation) while the HIV-1
intasome MM-PCR major enhancements mapped to two regions (at
nucleotides 8, 9, and 13 on wt U3 and at nucleotides 9, 11, and 16 on
wt U5) (2). On only three random occasions with numerous analyses, there were additional fragments produced by DNase I digestion
migrating near position 9 or 10, or both, when AMV IN was bound to
either the "G" or U3 donor ends which were not observed with the
naked DNA treated with DNase I (Fig. 5 to 9). The significance of the
occasionally observed enhanced DNase I cleavage near these positions
and its relationship to a possible transient assembly intermediate is
under investigation. Further studies are needed to unravel the
organization of IN at the viral DNA ends in both systems.
Mutagenesis studies of retrovirus att sequences have
established that the first ~7 to 12 nucleotides from the ends are
necessary and sufficient for near maximum integration activity in vivo
and for 3'-OH processing and half-site integration in vitro (2, 4, 7, 27). These terminal nucleotides interact with specific residues on IN (11, 20). Mutagenesis of avian retrovirus
LTR sequences has shown the 5th, 6th, and 7th nucleotides play critical roles for controlling half-site and full-site integration activities and for coupled communications between two LTR termini by IN in trans for full-site integration (9, 39).
The DNase I protection experiments in this report define the physical
association of AMV IN within the wt U3 and wt U5 LTR termini for
assembly and full-site integration. The minimum physical association of
IN at low concentrations (<10 nM) with wt U5 appears to be limited to
fewer than 10 nucleotides from the U5 end (Fig. 10, middle) even when
coupled to the wt U3 end for full-site integration (Fig. 10, right). We
cannot rule out a different model wherein a different array of
multimers on the U5 end occurs when coupled to wt U3, particularly at
higher IN concentrations at which partial protection is observed (Fig.
9C, lanes 6 to 9 and 10 to 13). However, binding of IN at the very end
of wt U5 (<10 nucleotides) appears sufficient for assembly and
efficient full-site integration because no DNase I protection is
observed between nucleotides ~10 to 30 (Fig. 9C, lanes 2 to 5, and A,
top) (data not shown). Other molecular probes may reveal IN
interactions at the very end (<10 bp) of U5. AMV IN at low
concentrations is capable of forming an extended nucleoprotein complex
at the wt U3 ends or U3 and U5 ends containing the "G" mutation
with a well-defined outer boundary of DNase I protection mapping ~20
nucleotides from the LTR end (Fig. 5, 6, and 8). The reasons for the
apparent asymmetry of IN binding at the wt U3 and wt U5 ends for
assembly and full-site integration is unknown. Interestingly, HIV-1 IN
possibly recognizes the U3 and U5 att sites independently in
vivo (27).
The extended association of IN with several LTR termini appears to be
directly related to at least the 5th and 6th nucleotides (underlined
below), because wt U5 lacks the extended protection (Fig. 9) while the
U5 end containing the "G" mutation has the extended protection
(Fig. 8). It is interesting to speculate that these nucleotides (<8
nucleotides from the end) allow IN at lower concentrations to nucleate
at the very terminus and that this initial event mediates further
recruitment of IN at higher concentrations (Fig. 5B). The possible
requirement effect requires wt U3
(5'-CTACAOH) or "G"
(5'-CAACAOH) ends, while the effect
is not apparent with wt U5 (5'-CTTCAOH)
or CC mutant
(5'-CCCCAOH) ends. The catalytic strand specificity by IN (39) suggests IN has the capacity
to distinguish A · T from T · A possibly, in a minor DNA
groove capacity (23, 40).
At higher IN concentrations (>20 nM), the gradually increasing partial
DNase I footprints observed past the 20th nucleotide position may
represent either specific or nonspecific multimer formation (Fig. 5B).
IN may possess different DNA binding modes depending upon protein
concentration and solution conditions, possibly similar to the
single-stranded DNA binding protein from E. coli
(34). Direct visualization of assembled IN-donor complexes and IN-donor-target complexes (Fig. 8) may reveal insights into the
physical structure of these complexes, the role of the asymmetric binding of IN at the LTR ends, and the ability of IN to loop DNA (8, 18). IN is involved in forming and maintaining
critical protein-protein and protein-DNA contacts within the retrovirus PIC or intasome structures (7, 30, 41). Efforts to
identify other internal viral sequences (up to ~150 bp from the end)
protected by AMV IN (<20 nM) by DNase I footprinting were
unsuccessful. At either low or moderate IN concentrations,
intramolecular looping of DNA by IN (transient or stable) may
facilitate physical interactions between two donors, thereby enhancing
interactions of donor ends for full-site integration in vitro. In
contrast, at high IN concentrations, a large percentage of IN-donor
complexes are nonspecific in nature, thereby decreasing proper pairing
of LTR ends necessary for full-site integration but not single LTR ends
for half-site integration (Fig. 5).
Studies with the MLV and HIV-1 PIC and their respective intasomes
suggest IN has multiple functions in these structures (7, 42). The minimum IN structure for 3'-OH processing and half-site integration is a dimer (4) (Fig. 10, left), with the
predicted minimum structure of two tetramers required for full-site
integration (5, 20) (Fig. 10, middle). IN bound to two LTR
appears to communicate in trans for integration in vivo
(32) and for full-site integration in vitro (1, 28,
38). AMV IN apparently forms a multimeric structure encompassing
~20 nucleotides on the wt U3 LTR (Fig. 10, right) whose function in
assembly and full-site integration has yet to be defined. The
simultaneous use of reconstituted intasome-like complexes with
site-directed mutagenesis of IN may provide insights into the multiple
structural-functional relationships of IN for assembly and full-site integration.
| |
ACKNOWLEDGMENTS |
This work was supported by a National Cancer Institute grant (CA16312).
We thank Roger Chiu and Sapna Sinha for reviewing the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: St. Louis Health
Sciences Center, Institute for Molecular Virology, 3681 Park Ave., St.
Louis, MO 63110. Phone: (314) 577-8411. Fax: (314) 577-8406. E-mail:
Grandgdp{at}SLU.EDU.
| |
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Journal of Virology, April 2001, p. 3556-3567, Vol. 75, No. 8
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.8.3556-3567.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
