Integration of a DNA copy of the retroviral RNA genome into a
host chromosome is a crucial step in viral
replication(1, 2, 3) . Two specific enzymatic
reactions are involved in integration. In the first reaction, 3`
processing, two nucleotides are removed from each 3` end of the linear
viral DNA made by reverse transcription. A subsequent DNA strand
transfer reaction then splices these 3` ends into the host chromosome.
The 5` ends of the viral DNA and the 3` ends of the host DNA remain
unjoined in the resulting integration
intermediate(4, 5) . Completion of integration
requires only removal of the unpaired bases at the 5` ends of the viral
DNA and repair of the single-strand gaps between the viral and host
DNA, steps which may be completed by cellular DNA repair enzymes.
HIV-1 (
)integrase catalyzes both the 3` processing and
DNA strand transfer reactions in vitro. Short oligonucleotides
that mimic the ends of HIV-1 DNA serve as the substrate for 3`
processing, and a second oligonucleotide acts as a target DNA for the
subsequent strand transfer
reaction(6, 7, 8) . Integrase will also
catalyze an apparent reversal of the strand transfer reaction, termed
disintegration(9) . In this reaction the viral DNA segment of a
branched substrate is liberated and the target DNA segment is resealed.
HIV integrase is comprised of three functional domains. Although the
central core domain alone can catalyze the disintegration reaction,
both the amino- and carboxyl-terminal domains are necessary for
catalysis of 3` processing and DNA strand
transfer(10, 11, 12) . Site-directed
mutagenesis has revealed that a triad of highly conserved acidic
residues within the core domain is essential for all three catalytic
activities of integrase(13, 14, 15) . These
residues, Asp-64, Asp-116, and Glu-152, comprise the D,D-35-E motif
that is conserved in all retroviral and retrotransposon integrase
proteins and is also found in some bacterial transposable
elements(13, 16, 17, 18) .
The
limited solubility of HIV-1 integrase has impeded structural studies.
However, the mutation of phenylalanine to lysine at amino acid 185
(F185K) within the catalytic core domain of HIV-1 integrase resulted in
a soluble protein (19) that enabled the core domain to be
crystallized and the structure solved to 2.5-Å
resolution(20) . We have now introduced the same mutation
(F185K) into full-length HIV-1 integrase, together with a cysteine to
serine substitution at position 280. The resulting protein is
dramatically more soluble than full-length wild-type HIV-1 integrase
and retains full activity for both the 3` processing and DNA strand
transfer reactions. The mutant protein exists in an equilibrium between
dimeric and tetrameric species in buffer containing 1 M NaCl.
Both the core and carboxyl-terminal domains are involved in
multimerization.
MATERIALS AND METHODS
Construction of Site-directed
Mutations
Site-directed mutagenesis was done by overlapping PCR (21) using a two-step procedure as described(19) .
Plasmid DNA encoding the mutations F185K/C280S within full-length
integrase, IN
/F185K/C280S, was prepared using
pINSD (13) as the PCR template DNA. After the second round of
PCR, the full-length fragment was digested with NdeI and BamHI and ligated with NdeI-BamHI-digested
pET-15b (Novagen, Madison, WI). This placed
IN
/F185K/C280S under the control of a T7 promoter (22) and also encoded a 20-amino acid histidine tag (HT) at the
amino terminus of the protein to facilitate rapid purification on a
nickel chelating column. The inclusion of a thrombin cleavage site
within the HT sequence allowed the removal of 17 amino-terminal
residues containing the polyhistidine motif following purification.
F185K was also introduced into the pol gene of pNL4-3,
an infectious plasmid DNA clone of HIV-1, using overlapping PCR.
Sequential PCR with pNL4-3 as the starting DNA template produced
a 1832-base pair fragment. The fragment was digested with AgeI
and PflMI and then ligated with AgeI-PflMI-digested pNL4-3 DNA. The sequences
of all PCR-generated regions were confirmed by DNA
sequencing(23) .
Expression and Solubility of F185K/C280S Integrase from
E. coli
Plasmid encoding IN
/F185K/C280S
was expressed in Escherichia coli strain BL21(DE3) (24) as described(6, 13) . The solubility of
the mutant protein was examined in a crude cell lysate, as follows.
Cells were grown in 2 liters of Super broth (Biofluids) containing 100
µg of ampicillin per ml at 37 °C until the optical density of
the culture was between 0.8 and 1.0 at 600 nm. Protein expression was
induced by the addition of
isopropyl-1-thio-
-D-galactopyranoside to a final
concentration of 0.4 mM. After 3 h, the cells were harvested
and resuspended in 12 ml of 25 mM HEPES (pH 7.5), frozen in
liquid N
, and stored at -80 °C. Cells (100
µl) were lysed by the addition, to final concentration, of 0.15 M, 0.5 M, or 1 M NaCl, 2 mM dithiothreitol, and 0.3 mg/ml lysozyme, in a final volume of 170
µl. After 30 min at 4 °C, cells were frozen in liquid N
and then thawed at 4 °C. Following ultracentrifugation in a
Beckman TL-100 Ultracentrifuge for 45 min at 100,00
g,
10 µl of each supernatant was analyzed by SDS-PAGE.
Protein Purification
Frozen resuspended cells
expressing IN
/F185K/C280S from 24 liters of Super
broth were thawed and resuspended in lysis buffer (1 M NaCl,
20 mM HEPES, pH 7.5, 2 mM
-mercaptoethanol, 0.3
mg/ml lysozyme, 5 mM imidazole) to a final volume of 1 liter.
After a 30-min incubation at 4 °C, the lysed cells were homogenized
with a SDT-1810 Tissumizer (Tekmar), sonicated, and centrifuged in a
Beckman Ti-15 batch rotor for 1 h at 40,000
g. The
supernatant was filtered through a 0.25-µm filter and applied to a
nickel-affinity (Chelating Sepharose Fast Flow, Pharmacia Biotech Inc.)
column (5
7 cm). After loading, the column was sequentially
washed with 2 liters of 20 mM imidazole buffer containing 25
mM HEPES, pH 7.5, 2 M NaCl, and 2 mM
-ME, and 1 liter of 60 mM imidazole buffer
containing 25 mM HEPES, pH 7.5, 1 M NaCl, and 2
mM
-ME. Protein was eluted with a linear gradient of 60
mM to 1 M imidazole, containing 25 mM HEPES,
pH 7.5, 1 M NaCl, 2 mM
-ME, and 10% (w/v)
glycerol. Fractions containing integrase were pooled, and EDTA was
added to a final concentration of 5 mM. This protein was then
dialyzed against 25 mM HEPES, pH 7.5, 0.5 M NaCl, 0.3 M imidazole, 2 mM
-ME, 1 mM EDTA, and
10% (w/v) glycerol. To cleave the HT from integrase, thrombin (Sigma)
at 10 NIH units/mg of integrase was added and incubated at 26 °C.
After 30 min, additional thrombin at 10 NIH units/mg of integrase was
added and the incubation continued for another 30 min at 26 °C.
Thrombin was removed by adsorption to a benzamidine-Sepharose 6B column
(Pharmacia). Cleaved protein was dialyzed against 20 mM sodium
phosphate buffer, pH 6.2, 0.3 M NaCl, 1 M urea, 1
mM DTT, 1 mM EDTA, and 10% (w/v) glycerol. This
protein was loaded on to a Mono S HR 10/10 column (Pharmacia) and
eluted with a linear gradient of 0.25 M to 0.6 M NaCl, containing 1 M urea, 1 mM DTT, 1 mM EDTA, and 10% (w/v) glycerol. Fractions containing full-length
protein were pooled and diluted with an equal volume of 1 M urea, 1 mM DTT, 1 mM EDTA, and 10% (w/v)
glycerol and concentrated by readsorption to the Mono S column. Protein
was then eluted with 1 M NaCl, 1 M urea, 1 mM DTT, 1 mM EDTA, and 10% (w/v) glycerol, dialyzed against
storage buffer (1 M NaCl, 20 mM HEPES, pH 7.5, 1
mM EDTA, 1 mM DTT, and 10% (w/v) glycerol), and
frozen in liquid N
before storing at -80 °C. The
concentrations of purified IN
/F185K/C280S were
determined using a calculated extinction coefficient of 50,460 M
cm
at 280 nm, based on
the amino acid composition(25) . The corresponding extinction
coefficient at 296 nm was determined based on the experimental ratio of A
/A
. Wild-type integrase
was purified as described(13) .
Determination of the Multimeric State of
IN
/F185K/C280S
The multimeric state of
IN
/F185K/C280S was determined by both gel
filtration and analytical ultracentrifugation. For gel filtration,
purified protein was applied to a calibrated Superdex 200 PC 3.2/30
column (Pharmacia) on a Pharmacia Smart
System. The column
was equilibrated with 25 mM HEPES, pH 7.5, 1 M NaCl,
1 mM DTT, 1 mM EDTA, and 10% (w/v) glycerol, and
operated at a flow rate of 50 µl/min at 4 °C. Analytical
ultracentrifugation was performed using a Beckman XL-A analytical
ultracentrifuge. Data were acquired as an average of 25 absorbance
measurements at nominal wavelengths of 280 nm or 296 nm and a radial
spacing of 0.001 cm. All sedimentation equilibrium experiments were run
in 25 mM HEPES, pH 7.5, 1 M NaCl, 1 mM DTT,
1 mM EDTA, and 10% (w/v) glycerol, using double-sector
charcoal-filled epon centerpieces and column lengths of approximately 4
mm. The density of the solvent was measured at 20.00 °C (Anton Parr
DMA58 density meter) and corrected using standard tables. Sedimentation
equilibrium experiments were conducted at various rotor speeds, from
10,000 to 16,000 rpm, at 4.0 °C, using loading concentrations of
0.1 (280 nm), 0.3 (280 nm), 1.0 (296 nm), and 1.25 (296 nm) mg/ml. The
time required for the attainment of equilibrium was established by
running at a given rotor speed until successive scans, taken 12 h
apart, were invariant. Typical times taken to reach equilibrium varied
from 48 to 72 h when samples were loaded using a precooled rotor and
centrifuge. In the course of the experiment, no significant decrease in
the concentration of soluble protein was noted. Such losses were
observed only at loading concentrations above 5 mg/ml. Experiments were
performed at two different rotor speeds for each sample because it has
been demonstrated that the simultaneous fitting of data represents a
stringent criterion for establishing that the system indeed is
reversibly associating(26) . Data analyses by mathematical
modeling were performed using Sigma Plot 4.16 (Jandel Scientific).
Simultaneous weighted nonlinear least squares fitting of the data sets
at each concentration were performed using mathematical models of the
following form:

where A
is the absorbance of the protein
at a reference point r
, A
is
the absorbance at a given radial position r, H represents 
/2RT (
is the angular
speed in rads s
, R is the gas constant, T the absolute temperature), and E is a small
baseline correction term. M
represents the
calculated buoyant molecular mass M(1 - t;ex2html_html_special_mark_amp;ngr;
for the integrase dimer, using a
calculated partial specific volume, t;ex2html_html_special_mark_amp;ngr;,
based on the amino acid composition(27) . This model has the
equilibrium constant as a global fitting parameter and cell reference
concentrations and baseline corrections as local fitting parameters.
The equilibrium constant for this dimer-tetramer association, k, is on an absorbance concentration scale. The values of
lnk obtained experimentally were converted to lnK values, K now being the association constant on a molar
scale, using the extinction coefficients calculated for the integrase
dimer (
). Assuming that the extinction coefficient of the multimer
is the sum of the extinction coefficients of the components, it can be
shown that for a given path length l:

The model describing a reversible dimer-tetramer
self-association yielded excellent fits for both
IN
/F185K/C280S and
IN
/F185K/C280S. Data analysis in terms of a
single ideal solute gave poor data fits with residuals typical for
aggregating species and weight average molecular weights corresponding
to values between those expected for integrase dimers and tetramers.
Integrase Activity Assays
Double-stranded
oligonucleotides that mimic the sequence at the U5 end of HIV-1 DNA
were used as substrates for both the 3` processing and strand transfer
reactions. Oligonucleotides AE 117 (5`-ACTGCTAGAGATTTTCCACAC), AE 118
(5`-GTGTGGAAAATCTCTAGCAGT), and AE 150 (5`-GTGTGGAAAATCTCTAGCAG) were
gel-purified using denaturing PAGE. AE 117 and AE 150 were annealed to
generate a duplex oligonucleotide that was filled in with
[
-
P]TTP (3000 Ci/mmol; DuPont NEN) using
Sequenase version 2.0 T7 DNA polymerase (Amersham) as
described(28) . This labels the phosphate bridging the two
nucleotides that are cleaved by integrase and allows analysis of the
choice of the nucleophile utilized in the reaction. The substrate for
DNA strand transfer was prepared by labeling AE 118 at the 5` end with
[
-
P] ATP (3000 Ci/mmol; DuPont NEN) using
T4 polynucleotide kinase (Pharmacia). Unincorporated label was removed
by passage through a Bio-Spin 6 column (Bio-Rad) equilibrated with 10
mM Tris-HCl, pH 8.0, 20 mM NaCl, 0.1 mM EDTA. Labeled AE 118 was then annealed with AE 117. Assay
conditions were as described (29) with slight modifications.
Reaction mixtures contained 25 mM MOPS, pH 7.2, 10 mM DTT, 5% (v/v) polyethylene glycol-8000 (PEG-8000) (Fluka), 5%
(v/v) dimethyl sulfoxide (Aldrich), 0.05% Nonidet P-40 (Sigma), 30
mM NaCl, 10 mM MnCl
or MgCl
,
2.5 nM DNA substrate, and 0.08-0.64 µM integrase in a final 16-µl volume. Reaction products were
analyzed by electrophoresis in 15% polyacrylamide/urea gels as
described previously(19) .
Cells and Analysis of HIV-1 Proteins
HeLa cells
were grown in Dulbecco's modified Eagle's medium containing
5% fetal calf serum, 100 units of penicillin G sodium, and 0.1 mg/ml
streptomycin sulfate as described(30) . To prepare virus
stocks, 3.6
10
HeLa cells were transfected with 10
µg of plasmid (wild-type-pNL4-3 or F185K-pNL4-3) using the
Cell-Phect kit (Pharmacia). After 48 h, the culture supernatants were
assayed for the presence of Mg
-dependent
P-reverse transcriptase activity as described previously (31) . Filtered supernatants were equalized for
P-reverse transcriptase activity for the subsequent
infection of the human T-cell line, CEM-12D7(32) . CEM-12D7
cells were grown in RPMI 1640 containing 10% fetal calf serum, 100
units of penicillin G sodium, and 0.1 mg/ml streptomycin sulfate as
described(30) . 2.5
10
cells were infected
with 2.5
10
P-reverse transcriptase cpm in 0.5 ml
of RPMI 1640 for 1 h at 37 °C followed by the addition of 5 ml of
RPMI 1640 containing 10% fetal calf serum. Cultures were then monitored
for
P-reverse transcriptase activity. Cell- and
particle-associated HIV-1 proteins were analyzed by radiolabeling
transfected HeLa cells and immunoprecipitating with AIDS patient
antisera (donated by Dr. Alfred Prince through the NIH AIDS Research
and Reference Program) as described(30) . Radiolabeled proteins
were quantitated using a PhosphorImager and ImageQuant 3.3 (Molecular
Dynamics).
RESULTS
The F185K Mutation Increases the Solubility of HIV-1
Integrase
Expression of IN
/F185K and
subsequent purification by Ni
-affinity chromatography
yielded a protein that was considerably more soluble than wild-type
integrase in buffer containing 1 M NaCl (data not shown).
However, this protein displayed time-dependent aggregation in the
absence of a reducing agent. This aggregation was readily reversed in
the presence of 5 mM DTT. Preliminary studies with a
carboxyl-terminal deletion mutant and the soluble catalytic core domain
of integrase revealed that these proteins did not aggregate in the
absence of DTT. We therefore suspected that the aggregation was caused
by disulfide linkages involving cysteine 280, the only cysteine present
in the carboxyl-terminal region of the protein. Serine was substituted
for cysteine at this position. The resulting protein
IN
/F185K/C280S retained the markedly improved
solubility properties compared with wild-type integrase but, unlike
IN
/F185K, remained soluble even in the absence of
reducing agent.
Purification of
IN
/F185K/C280S
HIV-1
IN
/F185K/C280S was purified from the soluble
fraction following lysis of cells in the presence of 1 M NaCl (Fig. 1). The HT at the amino terminus of the protein permitted
rapid purification by nickel-affinity chromatography; 280 mg of protein
were obtained from 24 liters of cells expressing
IN
/F185K/C280S. The integrase peak was pooled and
the HT was removed by cleavage with thrombin; the purity of the pooled
integrase was analyzed by SDS-PAGE and staining with Coomassie Blue (Fig. 2). Minor contaminants were successfully removed by
subsequent chromatography on Mono S.
Figure 1:
SDS-PAGE of whole cell extracts and
soluble fractions of cells expressing wild-type IN and
IN
/F185K/C280S. Whole cell extracts of E.
coli cells expressing wild-type and
IN
/F185K/C280S show induced proteins of the
predicted size that migrate just above the 31-kDa molecular mass marker
(see arrow). IN
/F185K/C280S migrates
slightly slower as this protein contains a HT, whereas wild-type IN
does not. Supernatants, from cells lysed in 0.15 M, 0.5 M, or 1.0 M NaCl and subsequently ultracentrifuged,
are shown in adjacent lanes.
Figure 2:
Purification of
IN
/F185K/C280S. Purified protein fractions were
analyzed by SDS-PAGE and stained with Coomassie Blue. A 5-µg
loading of IN
/F185K/C280S eluted from the
Ni
-affinity column revealed minor contaminating
species. These were removed by chromatography on a Mono S cation ion
exchange column. Loading of 2-12 µg of the pooled Mono S
fractions demonstrates the purity of the
protein.
IN
/F185K/C280S Forms Both Dimers
and Tetramers in Solution
We used both gel filtration and
sedimentation equilibrium studies to determine the multimeric state of
IN
/F185K/C280S in solution. Gel filtration using
a Superdex 200 column, with the protein loaded at high concentration
(>4 mg/ml), resulted in a single eluted peak that migrated at the
predicted position of a tetramer relative to globular protein standards (Fig. 3). When IN
/F185K/C280S was loaded
at a lower concentration (<0.4 mg/ml), a single peak was again
eluted; however, this corresponded to the position expected for a dimer
of integrase. Nevertheless, both the dimer and tetramer peaks contained
elements of tetramer and dimer, respectively (Fig. 3).
Figure 3:
Gel
filtration profiles of IN
/F185K/C280S. Two
separate gel filtration chromatographs are superimposed. 30 µl of
purified protein was loaded onto the column at either 0.4 mg/ml (solid line) or at 4 mg/ml (dashed line). The
multimeric state, estimated from the mobility relative to globular
protein standards, is indicated. The A
scale on
the left is for the 4 mg/ml injection, and the A
scale on the right is for the 0.4
mg/ml injection.
Although qualitatively informative, the gel filtration data
represent the average behavior of reassociating molecules as they
migrate through the column. To more rigorously determine the multimeric
state of the protein, a series of protein dilutions were examined by
sedimentation equilibrium at 4.0 °C and different rotor speeds. A
single ideal solute fit gave molecular masses which indicated the
presence of at least a dimer, even at integrase concentrations as low
as 0.1 mg/ml. At higher concentrations, evidence for the
self-association of the integrase dimers was observed. The
sedimentation equilibrium data for each loading concentration were
fitted using a dimer-tetramer association model (see ``Materials
and Methods,'' ). Protein concentrations of 0.3, 1.0,
and 1.25 mg/ml yielded an excellent fit to this model (Fig. 4A) with residuals normally distributed about
zero. For all three concentrations, similar values of lnK were
obtained, averaged at 10.7 ± 0.2, leading to an apparent
association constant, K
, of 44,000 M
. This corresponds to an effective
dissociation constant, K
, of 2.2
10
M. The experimental data are thus
consistent with a reversible dimer-tetramer self-association (Fig. 4B).
Figure 4:
Analytical ultracentrifugation of
IN
/F185K/C280S. A, sedimentation
equilibrium profiles and the corresponding residuals at 296 nm. The
symbols correspond to the experiments carried out at the following
rotor speeds: 1 (squares), 10,000 rpm; 2 (circles), 16,000 rpm. Initial protein concentrations
were 1.25 mg/ml. The lines through the data points are the
best fit for a reversible dimer-tetramer self-association with a
lnK value of 10.7 ± 0.2, leading to an apparent
association constant, K
, of 44,000 M
. B, predicted proportion of
dimer (solid line) and tetramer (dashed line) as a
function of the total protein concentration based on the K
value of 2.2
10
M.
The Carboxyl-terminal Domain of Integrase Is Required for
Tetramerization
To further investigate the requirements for
tetramerization, two deletion mutant proteins were constructed. The
first contained the core and carboxyl-terminal domains,
IN
/F185K/C280S, and was purified as described
for the full-length counterpart. The second comprised the core and
amino-terminal domains, IN
/F185K, and was
purified as described for IN
/F185K(19) .
Gel filtration experiments revealed that IN
/F185K
is exclusively a dimer in solution (data not shown). In contrast,
sedimentation equilibrium data showed that
IN
/F185K/C280S, like
IN
/F185K/C280S, exists in an equilibrium between
dimers and tetramers. Data were collected at 0.4 mg/ml and fitted using
a dimer-tetramer association model (). The data yielded an
excellent fit to this model (Fig. 5) with residuals normally
distributed about zero. A lnK value of 10.8 ± 0.04 was
obtained, leading to an apparent association constant, K
, of 51,400 M
. This
corresponds to an effective dissociation constant, K
, of 2.0
10
M. (Fig. 5), a value very similar to that
obtained for IN
/F185K/C280S.
Figure 5:
Analytical ultracentrifugation of
IN
/F185K/C280S. Sedimentation equilibrium
profiles and the corresponding residuals at 292 nm. The symbols
correspond to the experiments carried out at the following rotor
speeds: 1 (squares), 12,000 rpm; 2 (circles), 16,000 rpm. Initial concentrations were 0.40
mg/ml. The lines through the data points are the best fit for
a reversible dimer-tetramer self-association with a lnK value
of 10.8 ± 0.04, leading to an apparent association constant, K
, of 51,400 M
. This corresponds to an effective
dissociation constant, K
, of 2.0
10
M.
Catalytic Activities of
IN
/F185K/C280S
Integrase activity was
analyzed using double-stranded oligonucleotide substrates that mimic
the sequences found at the U5 end of HIV-1 DNA(6, 7) .
It has been shown that 3` processing by wild-type integrase in the
presence of Mn
generates three specific products of
nucleophilic attack on the phosphodiester bond at the site of
cleavage(28, 33) . When water acts as the nucleophile,
a simple dinucleotide (D) is generated, attack by glycerol produces a
glycerol adduct (G), and attack by the 3`-OH end of the DNA strand
yields a cyclic dinucleotide product (C). However, in the presence of
Mg
, the simple dinucleotide (D) is the sole
product(33) . IN
/F185K/C280S was compared
with wild-type integrase for the preference of nucleophile utilization
in the cleavage reaction with either Mn
or
Mg
. No difference could be observed between the two
enzymes with regard to the selection of nucleophile or the extent of
cleavage under either assay condition (Fig. 6). The strand
transfer activities of IN
/F185K/C280S and
wild-type integrase were also compared, and no significant differences
were observed (Fig. 7). Disintegration activities were also
found to be identical. We conclude that the combined mutations of F185K
and C280S do not significantly alter in vitro activities of
integrase.
Figure 6:
3` processing activities of wild-type IN
and IN
F185K/C280S. 3` processing of a duplex
oligonucleotide substrate labeled at the 3` end of the cleaved strand
generated three distinct dinucleotide products in the presence of
Mn
. The migration positions of the 21-mer substrate (S), simple dinucleotide product (D), cyclic
dinucleotide product (C), and glycerol adduct product (G) are indicated on the left.
Figure 7:
DNA strand transfer activities of
wild-type IN and IN
/F185K/C280S. Strand transfer
activity was assayed using a duplex oligonucleotide substrate labeled
at the 5` end of the cleaved strand. Migration positions of the 21-mer
substrate (S) and strand transfer products (P) are
indicated. The proteins were assayed at 37 °C for 1 h in the
presence of Mg
.
Mutation F185K Is Deleterious to Virion Assembly in
Vivo
Following our in vitro characterization of the
soluble mutant IN/F185K, but before introduction of the additional
mutation C280S, we investigated the in vivo effects of the
F185K mutation on the replication of HIV-1 in cell culture. The
mutation was introduced into the infectious molecular clone
HIV-1
, and virus particles were generated by
transfection of HeLa cells. Supernatants from cells transfected with
wild-type or F185K clones were normalized for reverse transcriptase
activity and used to infect the human T-cell line, CEM-12D7. To assess
infection kinetics, CEM-12D7 cell supernatants were monitored for
reverse transcriptase activity for 60 days. Wild-type virus production,
as measured by reverse transcriptase activity, peaked at day 8. No
replication of the mutant (F185K)-HIV-1
was
detected (Fig. 8A). After failure of the mutant virus
to infect CEM-12D7 cells, the virus generated by transfection of the
HeLa cells was examined. Transfected cells were radiolabeled, and cell-
and virus-associated viral proteins were analyzed by
immunoprecipitation and SDS-PAGE. Lysates of cells transfected with the
wild-type clone contained prominent precursor and mature forms of HIV-1
envelope and GAG proteins (Fig. 8B), whereas the levels
of these proteins were reduced in cells transfected with the mutant
clone. Analysis of lysates of virions from cells transfected with the
wild-type clone revealed two mature products of the pol gene,
the p66 subunit of reverse transcriptase and integrase (p32). However,
when normalized for gp120 and p24 content, both p66 and p32 were
dramatically reduced in lysates of virions from cells transfected with
the F185K mutant clone. Electron microscopy of cross-sections of
transfected cells revealed that the mutant virus appeared to consist of
immature rings devoid of nucleoid material or aberrant particles that
contained nucleoid material located near the membrane of the particle
(data not shown). The F185K change is apparently another example of an
integrase mutation which can affect virion protein composition and
morphology(30, 34, 35) .
Figure 8:
Effects of mutation F185K on viral
replication. A, replication kinetics of wild-type (WT)-HIV-1
and mutant
(F185K)-HIV-1
clones in CEM-12D7 cells. Cells
were infected with filtered supernatants equalized for
P-reverse transcriptase activity. CEM-12D7 culture
supernatants were monitored for
P-reverse transcriptase
activity as indicated. B, SDS-PAGE analysis of cell- and
particle-associated HIV-1 proteins from HeLa cells transfected with
(WT)-HIV-1
and mutant
(F185K)-HIV-1
clones. Migration positions of
precursor and mature forms of envelope, gp160 and gp120(SU),
respectively, and capsid, Pr55gag, and p24(CA), respectively, are
indicated on the left. The migration position of the p66
subunit of reverse transcriptase and p32(IN) are included on the right. Gels for analyzing particle-associated HIV-1 proteins
were exposed for approximately 5 weeks. The migration positions of
molecular mass standards are indicated in kilodaltons in the center.
DISCUSSION
Structural and biophysical studies of HIV integrase have been
impeded by the poor solubility of the protein. We find that a single
amino acid substitution that dramatically improves the solubility of
the core domain (19) has a similar effect on the solubility of
the full-length protein. This protein required the presence of reducing
agent to prevent slow time-dependent aggregation, a need which was
alleviated by introducing the additional mutation C280S. The double
mutant provides a protein that should facilitate structural studies of
full-length integrase.
Solubility Studies
Wild-type HIV-1 integrase is poorly
soluble except in the presence of reagents that are likely to cause at
least partial denaturation. Maximum solubility is approximately 1
mg/ml, even under the most favorable conditions of high ionic strength.
The F185K mutation considerably increases the solubility of the
full-length integrase, provided high ionic strength is maintained.
However, like the wild-type integrase, the solubility is greatly
reduced at low ionic strength. The requirement for the presence of a
reducing agent to maintain the protein in a nonaggregated state is
abrogated by the additional mutation C280S. We speculate that
intermolecular disulfide cross-links involving Cys-280 may also
contribute to the propensity of wild-type integrase to aggregate, but
the presence of reducing agents does not significantly reduce
aggregation because other intermolecular interactions dominate the
aggregation process.
Multimerization of Integrase
Our results
demonstrate that IN
/F185K/C280S exists in a
reversible dimer-tetramer self-association in solution ( Fig. 3and Fig. 4). Gel filtration results demonstrate the
absence of higher molecular weight aggregates, and that at protein
concentrations approaching 10 mg/ml the predominant form of the protein
is tetrameric. Dimers, but not tetramers, are observed with either the
HIV-1 integrase core domain alone, IN
/F185K; a
carboxyl-terminal truncation mutant, IN
/F185K; or
with the carboxyl-terminal domain, IN
. In fact,
NMR studies of a truncated carboxyl-terminal domain of HIV-1 integrase,
IN
, show a dimer in
solution(36, 37) . However, a deletion mutant
containing the core and carboxyl-terminal domains,
IN
/F185K/C280S, exists in a dimer-tetramer
self-association in solution (Fig. 5). Thus, multimerization
interfaces appear to be located in both the core and carboxyl-terminal
domains of HIV-1 integrase.Complementation studies with HIV-1 and
HIV-2 integrases (12, 38) have demonstrated that
multimerization is required for the 3` processing and strand transfer
activities, but these experiments cannot discriminate whether the
active multimer is dimeric, tetrameric, or higher order. Several
retroviral integrases have been reported to exist as
dimers(6, 39, 40, 41) . Rous sarcoma
virus integrase has been reported to form a reversible
monomer-dimer-tetramer association in solution, and it was also
suggested that the protein functioned as a multimer(41) .
Collectively, these studies point to a propensity of integrases to form
both dimers and tetramers, but do not directly address which multimer
is the active species for catalysis.
Effects of the F185K/C280S Mutations on Enzymatic
Activity and Multimerization
A major concern when using
mutagenesis as a tool to improve the behavior of a protein for
structural studies is fortuitous alteration of other properties,
especially catalytic activity. We therefore examined
IN
/F185K/C280S to determine if there were any
measurable perturbations in catalytic activity. Previous studies with
F185K in the context of the catalytic core domain revealed that the
mutant protein was in fact slightly more active than the unmutagenized
core(19) . However, no significant differences could be
observed between the unmutagenized and mutant full-length proteins in
assays for 3`-processing, strand transfer, or disintegration
activities. We conclude that the combined F185K and C280S mutations do
not adversely affect the catalytic properties of HIV-1 integrase in
vitro.We were unable to detect any difference in the
multimerization properties between IN
/F185K and
IN
/F185K/C280S (data not shown) and conclude that
this amino acid substitution has little, if any, effect on
multimerization. It was not possible to compare directly the
multimerization properties of IN
/F185K/C280S with
wild-type protein because the latter exhibits extensive aggregation,
except at relatively low concentrations in the presence of detergent.
The F185K mutation was previously reported to stabilize dimerization of
the isolated core domain(19) , and this mutation appears to
have the similar effect of stabilizing dimerization of the full-length
integrase; at a protein concentration of 0.2 mg/ml, wild-type protein
exhibits a monomer-dimer equilibrium in the presence of the detergent
CHAPS, whereas IN
/F185K/C280S was exclusively
dimeric (data not shown).
We also wished to determine the effect of
the F185K mutation on HIV replication in cell culture. Unfortunately,
this mutation disrupted proper virion assembly and blocked replication
at a step prior to reverse transcription; its potential effect on
integration, therefore, could not be determined. This phenotype has
been observed with several other mutations in integrase (30, 34, 35) and probably reflects effects on
protein-protein interactions involving integrase at earlier stages of
the replication cycle.
Utility for Structural Studies
The soluble
integrase mutant IN
/F185K/C280S is an attractive
candidate for structural studies since it is well behaved and soluble
up to at least 20 mg/ml in a buffer containing 1 M NaCl.
Soluble derivatives of HIV-1 integrase are now available that include
all three domains. Structures of the core (20) and
carboxyl-terminal domains (36, 37) have already been
determined for HIV-1 integrase, and the structure of the core domain of
the closely related Rous sarcoma virus integrase has also been
solved(42) . These structures should eventually prove
invaluable in determining the mechanism of action of inhibitors to HIV
integrase and ultimately to guiding the design of therapeutically
useful compounds. A major remaining objective is determination of the
structure of the intact HIV integrase in a complex with its DNA
substrates. The soluble mutant described here may help accomplish of
this goal.