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INTRODUCTION |
The cyclophilins represent a ubiquitous family of proteins that
are abundant in nearly every cell type and are highly conserved through
evolution (1). The main representative of the cyclophilins in mammals
is the cytosolic 18-kDa
CyPA.1 The closely related
CyPB (21 kDa) contains a N-terminal signal sequence that directs this
isoform to the endoplasmic reticulum. A characteristic property of
cyclophilins is their peptidyl-prolyl cis-trans isomerase
(PPIase) activity and their ability to bind cyclosporin A (CsA) that
inhibits the PPIase activity. In T cells, the CsA·CyPA complex
associates with and inhibits the Ca2+-dependent
protein phosphatase calcineurin, resulting in immunosuppression. Natural endogenous ligands of cyclophilins with similar actions as CsA
are unknown, and the cellular functions of cyclophilins are still a
matter of investigation. A new important property of cyclophilins was
found in the human immunodeficiency virus life cycle (2, 3) when a
specific association of CyPA with the Gag polyprotein p55gag of
HIV-1 (4) was detected.
During or after budding of immature viral particles out of the host
cell, the noninfectious virions with a spherical core consisting of Gag
proteins are transformed to infectious virions and undergo a dramatic
structural rearrangement to an icosahedral shape (5). In the course of
this maturation, the pol-encoded HIV protease processes the
Gag polyprotein precursor into three major proteins and three smaller
polypeptides (6, 7). The capsid protein p24, consisting of a N-terminal
and a C-terminal domain, condenses to form the conical core structure
surrounding the viral genome. One CyPA molecule per 10 Gag molecules is
incorporated into nascent virions, amounting to a total of ~200
molecules/virion (2, 3). CyPB was shown to form in vitro
similar complexes with the Gag polyprotein as CyPA but was not found in
virions in vivo (2). The interaction with the Gag
polyprotein and p24 is mediated by the active site of CyPA and is
suppressed in the presence of CsA or SDZ NIM811 (3, 8), a natural
nonimmunosuppressive CsA analog that also binds to CyPA and inhibits
its PPIase activity. CsA prevents the incorporation of CyPA but
inhibits neither the assembly of viral components nor the production of
morphologically intact virions (9). However, virions lacking CyPA were
found to be considerably reduced in their propagation (10). Single point mutations in the p24 domain of the Gag polyprotein precursor revealed a region with an array of four proline residues to be involved
in binding to CyPA (2). Recently, the crystal structure of CyPA
complexed with a domain of p24 encompassing the N-terminal two-thirds
(residues 1-151) showed binding of CyPA to the proline-rich segment
that forms a flexible surface-exposed loop in the mainly
-helical
p24 fragment (11). The contact region on the p24 loop is restricted to
a relatively small area with Gly-Pro90 as the central
element buried in the active site of CyPA. This Gly-Pro motif seems to
be a prerequisite for stable association. Only if glycine precedes a
proline residue is the peptide stretch able to snuggle deeply into the
active site of CyPA.
So far, the role of CyPA in the interaction with HIV-1 p24 is not
clear. CyPA might act as PPIase and catalyze a cis-trans isomerization in the p24 peptide backbone or might function as chaperone by binding the capsid protein in a conformation prone for
propagation, i.e. by destabilizing the capsid cone for
postentry disassembly (10). Such an action of CyPA, however, could not be expected to result solely from binding to the site on the loop around Gly-Pro90 in the N-terminal domain. Very recently,
it was reported that CyPA disrupts high order p24 complexes, most
probably by interfering with p24-p24 interactions (12). As the assembly
and disassembly of p24 occur both in the same compartment of the host
cell, it remained a puzzle how this CyPA·p24 (Gly-Pro90)
interaction should initiate capsid cone disassembly without already
affecting viral assembly.
In the present investigation, we have identified two additional
CyPA-binding sites in the C-terminal domain of mature p24 capsid
protein. Both sites show higher affinities for CyPA and CyPB than the
Gly-Pro90 site of p24. These two high affinity binding
sites seem to be accessible for CyPA only in the mature p24 capsid
protein, i.e. after processing of immature p24 by the HIV-1
protease. A hypothetical model of the function of CyPA in the
disassembly of the capsid cone is proposed.
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EXPERIMENTAL PROCEDURES |
Materials--
Recombinant immature HIV-1 capsid protein was
obtained from D. Sizmann (Hoffmann-La Roche, Basel) in 6 M
guanidine HCl and was efficiently refolded by an established method
(13, 14) into a correctly folded soluble protein. Folded immature
capsid protein was analyzed by CD spectroscopy with regard to secondary structure composition. The obtained CD spectra were comparable ([
222]mre:
15,970
millidegrees·M
1·cm2) with that reported
for native p24 (14). The correct mass was verified by mass
spectrometry, and the protein concentration was determined by
quantitative amino acid analysis. The concentration of p24 stock
solutions was always kept below 1 mg/ml to avoid aggregation (15).
PPIase-active recombinant human CyPA and CyPB were a gift from M. Zurini (Novartis, Basel). Synthetic peptides p(87-101), p(153-172),
and p(214-228) were purchased from Neosystem Laboratoire (Strasbourg).
Preparation of Mature p24--
In order to obtain mature p24,
folded immature p24 protein was incubated with 1/100 (w/w) of
recombinant HIV-1 protease in 30 mM MES, pH 5.5, 200 mM NaCl, 1% (w/w) glycerol, and 1 mM EDTA at
37 °C. The reaction was monitored by analyzing aliquots with SDS-polyacrylamide gel electrophoresis and silver staining (Phast gels,
Amersham Pharmacia Biotech). Usually, processing was complete after
4-6 h. The reaction was stopped by adjusting the pH to 8.0. Subsequently, 100 µM EDTA and 5 µM
phenylmethanesulfonyl fluoride was added, and the sample was dialyzed
against 3 mM Tris-HCl, pH 8.0, 20 mM NaCl to
remove cleaved peptides (2.8 and 2.3 kDa) and vacuum-concentrated up to
a concentration of
1 mg/ml. The concentration of p24 was determined
by quantitative amino acid analysis (AminoQuant, Hewlett-Packard) and
was in agreement with the concentrations determined by measuring the
absorbance at 280 nm (
280: 33.7 mM
1 cm
1; Ref. 14). An aliquot
(20 pmol) of the obtained product was analyzed by liquid
chromatography-mass spectrometry (C8-reversed phase HPLC
linked to an API III+ electrospray ionization mass
spectrometer; PE-Sciex), revealing a molecular mass of 25,578.3 ± 3 Da that was in agreement with the calculated molecular mass of
25,579.5 Da for mature p24. The CD spectrum of processed,
i.e. mature, p24 protein was comparable with that reported
for native HIV-1 capsid protein (14) and had a mean residue ellipticity
of
13,420 millidegrees·M
1·cm2 at 222 nm.
Acrylodan Labeling of p24 and Fluorescence
Measurements--
Folded immature p24 (10 µM) was
dialyzed against 10 mM HEPES, pH 7.0, and 10 µM dithiothreitol and then incubated with acrylodan (6-acryloyl-2-dimethylaminonaphthalene, 10-fold molar excess over sulfhydryl groups; Molecular Probes Europe BV) in 50 mM
HEPES, pH 7.0, 5% (v/v) acetonitrile, and 0.5 M guanidine
HCl (a concentration at which native p24 is still stable according to
Ref. 14) for 2 h at room temperature in the dark on a rotary
device. The reaction mixture subsequently was loaded onto a PD10 gel
filtration column (Amersham Pharmacia Biotech) and eluted with 50 mM HEPES, pH 7.0, 5% (v/v) acetonitrile, and 0.2 M guanidine HCl. Protein (p24)-containing fractions were
pooled; dialyzed against 2 mM sodium phosphate, pH 7.4, 6 mM NaCl, 6 mM KCl, and 1% (v/v) acetonitrile;
and vacuum-concentrated to a concentration still below 1 mg/ml. Mass
and UV spectroscopy of the product revealed only monolabeled p24 (most
probably at Cys218; see Ref. 16) with 0.3 mol of acrylodan
incorporated per mol of p24. In order to obtain acrylodan-labeled
mature p24, labeled immature p24 was treated with HIV-1 protease as
described before and proven by mass spectroscopy to be correctly processed.
Fluorescence measurements were performed at 25 °C with a
spectrofluorimeter (model A1010 equipped with FELIX fluorescence analysis software; Photon Technology International). Excitation was set
to 370 nm (3-nm bandwidth), and emission was recorded from 400 to 600 nm (3-nm bandwidth). In order to measure dissociation constants of the
complexes between the cyclophilins and mature or immature p24, CyPA or
CyPB was added at the indicated concentrations to labeled mature p24
(1.2 or 0.1 µM) or to labeled immature p24 (1.0 µM) in 20 mM sodium phosphate, pH 7.4, 60 mM NaCl, and 60 mM KCl. After each addition,
the mixture was incubated until equilibrium had been reached before
recording the spectra. In order to calculate Kd
values, the change in relative fluorescence was fitted (SigmaPlot,
Jandel Corp.) according to the following equation:
F = (
Fmax/2 × Lt) × [(Lt + Pt + Kd)
{(Lt + Pt + Kd)2
(4 × Lt × Pt)}0.5],
where
F is the relative fluorescence change,
Fmax is the maximal change,
Lt is the total concentration of p24, Pt is the total concentration of CyP, and
Kd is the dissociation equilibrium constant.
Kd values of independent experiments were within the
indicated calculated S.D. values (Table I).
PPIase Activity Measurement--
PPIase activity was measured at
4 °C essentially as recently described (17) with the standard
chymotrypsin-coupled assay (18). The assay mixture (0.08 M
NaCl, 0.05 M Hepes, pH 8; final volume 200 µl) contained
50 µM of
succinyl-Ala-Leu-Pro-Phe-p-nitroanilide (Bachem) and was
incubated for at least 10 min at 4 °C. The reaction was started by
the addition of chymotrypsin (60 µM) and monitored at
390 nm with a Hewlett-Packard 8450A UV/VIS-spectrophotometer. For
competition experiments, recombinant human CyPA (1-5 nM)
or CyPB (8-21 nM) was preincubated with or without the
corresponding p24 component in the presence of 0.5 µM
bovine serum albumin for 1 h at room temperature prior to
chymotrypsin addition. In the case of peptide p(214-228), the assay
mixture additionally contained 250 µM dithiothreitol. The
apparent reaction rate constant kobs was
obtained by analyzing the exponential phase (50-500 s) of the curve
according to a pseudo-first order reaction. The rate of the
spontaneous, uncatalyzed isomerization kuncat
(0.0036 ± 0.0002 s
1, at 4 °C) was subtracted
from kobs. The percentage inhibition was
calculated in relation to the PPIase activity measured in the absence
of competitors. In general, each measurement was performed twice, and
the average was calculated. Obtained data were fitted according to the
equation for competitive inhibition: percentage inhibition = [(100 × It)/(It + IC50)], where It is the total
concentration of the competitor and IC50 corresponds to
[Ki × (1 + (S/Km))].
Ki is the inhibition constant of the competitor, S
is the concentration of the PPIase substrate used in the standard
assay, and Km is the Michaelis-Menten constant of
the substrate. Since S/Km was approximately
for the used PPIase substrate, the IC50 values
are reflecting Ki values.
Mapping of CyPA-binding Sites on p24--
Fragments of p24 were
obtained by incubating recombinant HIV-1 p24 protein with 1/50 (w/w) of
1-chloro-3-tosylamido-7-amino-2-heptanone-pretreated
-chymotrypsin
(Boehringer Mannheim) in 100 mM Tris-HCl, pH 8.0, 10 mM CaCl2 and 1 M guanidine HCl for
1 h at 37 °C. The reaction was stopped by the addition of
phenylmethanesulfonyl fluoride. An aliquot of the digested mixture
corresponding to 0.57 nmol (17.4 µg) of original p24 protein was
preincubated with or without 0.4 nmol (7.1 µg) of recombinant human
CyPA in binding buffer (20 mM Tris-HCl, pH 7.5, 60 mM NaCl, 60 mM KCl, 2 mM
CaCl2, and 2 mM MgCl2) for 1 h
at room temperature. Subsequently, the reaction mixture (100 µl) was
loaded onto a gel filtration column (G25 Superfine, Amersham Pharmacia
Biotech; 10 × 85 mm) and eluted with the same buffer containing
10 mM guanidine HCl at 2 ml/min. CyPA-containing fractions
(0.5 ml) were adjusted to pH 2 with trifluoroacetic acid and were
directly injected onto a C8-reversed phase HPLC column
(4.6 × 220 mm; Brownlee). Elution was performed with HPLC buffer
A (0.1% trifluoroacetic acid) and a gradient of buffer B (80%
acetonitrile, 0.1% trifluoroacetic acid) at 1 ml/min.
Alternatively, recombinant HIV-1 p24 was chemically fragmented by
cyanogen bromide in 1 M guanidine HCl and 70% (v/v)
trifluoroacetic acid for 20 h at room temperature in the dark. The
mixture was lyophilized, resuspended in 5% acetonitrile and 0.1%
trifluoroacetic acid, and desalted by reversed phase HPLC. Fractions
following the run-through were pooled, vacuum-concentrated, mixed with
binding buffer, and incubated with or without CyPA for 1 h at room
temperature prior to fast gel filtration chromatography. HPLC-purified
extracted CyPA-binding peptides were N-terminally sequenced by
automated Edman degradation.
Modification of Synthetic Peptides--
Biotinylation of
p(87-101) was carried out by incubating HPLC-purified peptide (in 100 mM Hepes, pH 8.0) with a 1.5-fold molar excess of
NHS-Biotin (Sigma), dissolved in Me2SO, for 1 h at
room temperature. Excess reagent was trapped by the addition of glycine (2-fold molar excess over NHS-Biotin). The product was purified on a
C18-reversed phase HPLC column (3.9 × 150 mm; Waters)
and proved to be correct by mass spectrometry and quantitative amino acid analysis.
For acetylation of p(153-172), acetic anhydride (3-fold molar excess,
dissolved in 20 mM acetic acid) was added dropwise to a
well stirred solution of HPLC-purified peptide in 200 mM
sodium phosphate, pH 7.0, on ice. After 30 min, the reaction was
stopped by the addition of 2 M hydroxylamine HCl in 0.5 M Tris-HCl, pH 9.6. The product was purified on a
C8-reversed phase HPLC column (4.6 × 220 mm;
Brownlee) and quantified by amino acid analysis. Fragment ion mass
spectrum of the triply charged peptide ion proved the peptide to be
exclusively N-terminally acetylated.
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RESULTS |
Interaction of CyPA and CyPB with p24--
The presence of a
reactive cysteine residue at position 218 of p24 (see Ref. 16 and Fig.
1B) was utilized to introduce
the sulfhydryl-specific, environmentally sensitive fluorophore
acrylodan (19) into immature p24 (see "Experimental Procedures").
Although formation of an intramolecular disulfide bond between
Cys198 and Cys218 has been observed in a
truncated p24 encompassing the C-terminal domain (20), such a disulfide
bond could not be shown under physiological conditions (16) and is not
expected, since the Gag precursor folds and oligomerizes in the
reducing environment of the host cell. Acrylodan-labeled immature p24
exhibited maximal fluorescence emission at 498 nm when excited at 370 nm (Fig. 2, C and
D). Upon the addition of CyPA, the fluorescence of labeled immature p24 increased in intensity and underwent a blue shift. A
dissociation equilibrium constant, Kd, of 8.2 µM (Table I) was calculated
from the binding curve (Fig. 2C). In contrast, the
fluorescence intensity of labeled immature p24 decreased upon the
addition of CyPB (Fig. 2D), and a Kd of
0.9 µM (Table I) was determined. The observed changes in
relative fluorescence of labeled immature p24 upon the addition of CyPA
or -B were abolished in the presence of CsA, yielding the same
fluorescence spectrum as that of unbound immature p24.

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Fig. 1.
Scheme of immature HIV-1 capsid protein p24
(A) and amino acid sequence of mature p24
(B). Immature p24 protein consists of the entire
p24 capsid protein (residues 1-231, B) flanked N-terminally
by a stretch of 25 amino acids (aa) including a
His6 tag and C-terminally by 20 amino acids. Similar to the
genuine Gag precursor, the N-terminal extension contained 13 specific
amino acid residues from the matrix protein p17, whereas the C-terminal
extension contained 12 specific residues from p2, a spacer peptide
linking the C terminus of p24 to the nucleocapsid protein p7 in the
original Gag polyprotein. The cleavage sites recognized by the HIV-1
protease are indicated by arrows. The numbering of the amino
acids (B) refers to the mature p24 capsid protein, and the
MHR is indicated by shading. The positions of the
CyPA-binding fragments I-V (Fig. 4F) within p24 are
indicated. The Gly-Pro sequence elements (boldface
type) are supposed to be the central CyPA-binding segments
of the peptides.
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Fig. 2.
Binding of CyPA and CyPB to acrylodan-labeled
mature and immature p24. Titration of acrylodan-labeled mature p24
(A and B) as well as labeled immature p24
(C and D) with recombinant human CyPA
(A and C) or CyPB (B and D)
is shown as the change in relative fluorescence at max.
The corresponding fluorescence emission spectra are shown in the
insets, and the emission spectra of unbound labeled p24 are
indicated by dashed lines. Experimental data were
fitted as described under "Experimental Procedures," and the
calculated Kd values are summarized in Table
I.
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In order to determine the binding characteristics of mature p24, folded
immature p24 was correctly processed to mature p24 by the HIV-1
protease, which is also responsible for in vivo processing of the Gag polyprotein. The fluorescence spectra of unliganded acrylodan-labeled mature p24 exhibited maximal emission at 508 nm (Fig.
2, A and B). The fluorescence decreased in
intensity upon the addition of CyPA or CyPB without a perceivable shift in
max. The Kd values for the
CyPA·p24 complex and for the CyPB·p24 were calculated from their
respective binding curves (Fig. 2, A and B; Table
I). The contribution of the acrylodan label to the binding of p24 to
CyP was negligible, as judged by acrylodan-labeled
-mercaptoethanol
that did not show any change in fluorescence intensity upon CyP
addition. Again, upon the addition of CsA to the mixture of CyP and
p24, the fluorescence spectrum was the same as that of unbound p24.
Thus, CsA prevented also the interactions of CyPA and CyPB with labeled
mature p24, demonstrating specific binding of labeled p24 in the active
site of the cyclophilins.
PPIase Inhibition of CyP by Immature and Mature p24--
To verify
the binding properties obtained with the fluorimetric measurements, the
inhibitory effect of immature p24 on the PPIase activity of CyPA and
CyPB was determined. Immature p24 inhibited the PPIase activity of CyPB
(Ki ~ 1.2 µM; Fig. 3) more efficiently than that of CyPA
(Ki ~ 8.5 µM). Both
Ki values were in excellent agreement with the
fluorimetrically determined Kd values.

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Fig. 3.
Inhibition of the PPIase activity of CyPA and
CyPB each by immature (circles) or mature
(squares) p24. PPIase activity was measured and
calculated as described under "Experimental Procedures." The
Ki values of immature p24 are summarized in Table
I.
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Due to the presence of a His6 tag in immature p24 (Fig. 1),
preformed complexes between CyPA and immature p24 could be precipitated with Ni2+-agarose and quantified by densitometric analysis
of silver-stained protein bands after SDS-polyacrylamide gel
electrophoresis. The calculated apparent dissociation equilibrium
constant Kd' of ~19 µM obtained from
experiments in which relative low concentrations have been used (0.25 µM p24 and 25 nM to 2.5 µM
CyPA) is, as expected (due to a certain dissociation during the
procedure), higher (~2-fold) than the Kd and
Ki values (Table I). CsA efficiently disrupted the
complex (i.e. no CyPA was co-precipitated with immature p24
in the presence of CsA).
Mature p24 was, in contrast to immature p24, susceptible to cleavage by
chymotrypsin (used in high concentrations in the standard PPIase
activity assay; see "Experimental Procedures"). Most of it was
found to be degraded upon analysis by SDS-polyacrylamide gel
electrophoresis at the end of the assay. Nevertheless, the PPIase
activity of CyPA and CyPB was effectively inhibited at lower
concentrations of mature p24 than of immature p24 (Fig. 3). In the case
of CyPB, nanomolar concentrations of mature p24 already inhibited part
of the PPIase activity of CyPB, but complete inhibition was achieved
only at higher concentrations (>10 µM). Therefore, the
fluorimetrically determined Kd values seem to
reflect the true binding affinities of mature p24 (Table I).
Mapping the CyPA-binding Sites on the p24 Capsid Protein--
The
higher affinity of mature p24 as compared with immature p24 with
cyclophilins indicates that additional binding sites on p24 become
accessible after the precursor is processed by the HIV-1 protease. To
map potential CyPA-binding sites on HIV-1 p24, chymotryptic fragments
of recombinant p24 were incubated with human CyPA and subjected to a
fast size exclusion chromatography (Fig.
4). CyPA-bound peptide segments of p24
co-eluted together with CyPA in the void volume. CyPA-containing
fractions were analyzed by reversed-phase HPLC (Fig. 4,
D-F). Five CyPA-associated p24 peptides (I-V) were found
and identified by mass spectrometry. Peptide I corresponded to the four
proline residue-containing sequence 81-117, which encompasses the
known Gly-Pro90 site (Fig. 1B). The molecular
mass of peptide II mapped to C-terminal fragment 187-227 of mature p24
containing a Gly-Pro sequence element. Peptides III, IV, and V,
consisting of residues 152-185, 130-164, and 153-187, respectively,
overlap in the major homology region (MHR) of p24 (21), all having two
proline residues and a Gly-Pro sequence element.

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Fig. 4.
Extraction of CyPA-bound p24 peptide
fragments by differential gel filtration chromatography. CyPA
(A), chymotrypsin-digested p24 (B), and a
preincubated mixture (C) of the same amounts of CyPA and
chymotrypsin-digested p24 were subjected to a fast gel filtration
chromatography, according to "Experimental Procedures."
Panels D-F show the corresponding HPLC chromatograms of
fraction 5 of the preceding gel filtration. HPLC fractions containing
CyPA-bound p24 fragments (F) were subjected to mass
spectrometry. The p24 fragments had the following molecular masses:
3980.67 ± 0.65 (I), 4208.60 ± 0.52 (II), 4188.97 ± 0.20 (III), 4239.72 ± 0.22 (IV), and 4303.72 ± 0.35 (V).
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To confirm the binding of the proteolytic p24 fragments to CyPA, the
same procedure was performed with cyanogen bromide-fragmented p24. Two
CyPA-bound peptides were subjected to N-terminal amino acid sequence
analysis. The obtained N-terminal sequences YXPTSILDIKQ and
TAXQGVG, where X denotes an unidentified amino
acid residue, corresponded to cyanogen bromide-generated fragments
145-185 and 216-235, respectively (Fig. 1). Both peptides thus
matched the two new binding sites on p24 found with the chymotryptic fragments.
PPIase Inhibition of CyP by p24 Peptide Segments--
Synthetic
peptides, corresponding to the chymotryptic fragments with Gly-Pro as
the central binding motif, were used to confirm and to define the new
CyP-binding sites on p24. The binding properties of these p24 peptides
were examined by means of their inhibitory effect on the PPIase
activity of CyPA and CyPB. Peptide p(87-101), corresponding to a
segment within peptide I, inhibited the PPIase activity of CyPA with a
Ki of 170 µM (Table I). The elimination of the positive charge by biotinylation of the N terminus improved the binding 6-fold (Table I), whereas biotin itself did not
affect the PPIase activity in concentrations of up to 0.2 mM. Additional amino acid residues preceding
His87 contributed to better binding of peptide I
(Ki of 8.3 µM), which encompasses the
entire sequence of the surface-exposed loop between helices 4 and 5 of
p24 (Fig. 5A; Table I).
Peptide I was purified from the chymotrypsin digest of p24 in amounts sufficient for the inhibition studies.

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Fig. 5.
Inhibition of PPIase activity by p24 peptides
corresponding to the deduced CyPA-binding sites. Either CyPA
(filled circles) or CyPB (hollow
circles) was preincubated with the indicated concentrations
of peptide I (A) representing the entire surface-exposed
loop between helices 4 and 5 of p24 (Figs. 1B and
4F), N-acetylated peptide p(153-172)
(B) representing the major homology region, or peptide
p(214-228) (C) from the C terminus of p24. PPIase activity
was measured and calculated as described under "Experimental
Procedures." The determined Ki values are
summarized in Table I.
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The N-acetylated synthetic MHR peptide p(153-172) was shown
to inhibit the PPIase activity of CyPA and CyPB already at lower concentrations (Fig. 5B) compared with peptide I (Fig.
5A). The experimental data displayed a complex inhibition
behavior due to the fragmentation of the Phe-containing MHR peptide
during the time of activity measurement by chymotrypsin, which was
present in high concentrations in the standard PPIase assay.
Apparently, the inhibition curves reflect a heterogeneous and dynamic
inhibition of the PPIase activities by a mixture of truncated MHR
peptides, whose individual affinities will be lower than that of the
original MHR peptide. Thus, only IC50 values of 12 and 27 µM for CyPA and CyPB, respectively, could be determined.
The effective concentrations of intact MHR peptide must be much lower
than the indicated concentrations (Fig. 5B). The observed
partial inhibition at initial concentrations of the peptide below 0.1 µM, however, shows that intact MHR peptides are able to
bind to the active site of both cyclophilins at very low
concentrations. This suggests the MHR peptide to be a high affinity
ligand for CyPA (Kd
1 µM) if
residues following Phe161 are not cleaved off by
chymotrypsin. The fact that complexes of CyPA with chymotryptic or
cyanogen bromide fragments of p24 encompassing the MHR could be
isolated (Fig. 4F) corroborates this conclusion.
Peptide p(214-228), corresponding to the C-terminal binding site on
p24, displayed a homogeneous competitive inhibition of PPIase activity
and seems to have the highest affinity toward CyPA
(Ki of 0.35 µM) and CyPB
(Ki of 0.29 µM) among the three
identified p24 binding sites (Fig. 5C; Table I). The
presence of a cysteine residue (Cys218) allowed us to label
this peptide with acrylodan (for details, see Ref. 22) and to measure
its interactions with CyPA and CyPB. The calculated
Kd values 0.40 and 0.29 µM,
respectively, corresponded excellently with the Ki
values determined by the competitive PPIase assay (Table I),
emphasizing the equivalence of the different methods applied.
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DISCUSSION |
The results presented in this study indicate that HIV-1 capsid
protein p24 contains, in total, three sites that are able to interact
specifically with CyPA and CyPB. One binding site, the low affinity
site, matches to the loop around Gly-Pro90, which has been
previously proposed on the basis of mutation experiments (2) and
verified by crystallographic analysis of the complex of CyPA with
CA151, a truncated p24 encompassing the N-terminal domain
(11). The reported Kd of ~16 µM for the CA151·CypA complex (23) is comparable with the
present value of 8 µM for peptide I (Table I). The two
additional binding sites, one within the MHR and another at the very C
terminus of mature p24, show higher affinities and were not known so
far. Both of them contain the Gly-Pro motif, which was shown to be the
central element and a prerequisite for binding to CyPA (11, 24). A homology among the Gly-Pro adjacent amino acid residues of the three
binding sites is not evident. The more than 20-fold higher affinity of
CyPA for the C-terminal peptide than for the Gly-Pro90
peptides might be attributed to the presence of an additional Gly and
of positive charged amino acid residues in the segment following
Gly-Pro224 (Table I). A homologous peptide containing the
sequence SIHIGPGRAF and which fulfills the above criteria was shown to
bind to CyPA with a Kd of 0.33 µM and
to CyPB with a Kd of 0.19 µM (22).
Basic residues, C-terminal of Gly-Pro, are also found in the MHR
peptide p(153-172), which binds more tightly to the cyclophilins than
the Gly-Pro90 peptides if the residues following
Phe161 are still present (see "Results"). The
crystallographic studies of the low affinity binding site revealed that
the Gly-Pro90 adjacent residues predominantly interact with
CyPA by their backbone atoms (11, 25). The present results with the two
high affinity binding sites, however, demonstrate significant
contributions of GP-flanking side chains to the binding to cyclophilins.
Whereas complex formation of CyPA and CyPB with the p24 peptides was
comparable, substantial differences in affinity were found with mature
and immature p24. The immature protein form has, like the unprocessed
Gag polyprotein precursor, an N- and C-terminal extension (Fig. 1) that
seems to be important for proper assembly of the initial virion core
shell (26-29) and thus probably also for its proper conformation. The
binding affinity of CyPA for immature p24 (Kd ~ 8.3 µM) is identical with that for the
Gly-Pro90 site. These results suggest that the binding of
CyPA to immature p24 occurs through the Gly-Pro90 site, as
proposed previously (2, 11, 23, 30). In contrast to CyPA, the affinity
of CyPB for immature p24 is 8-fold higher (Kd ~1
µM) indicating a complex formation via a
different binding site on p24, most likely the MHR. Evidence for a
different binding site for CyPB on immature (i.e.
unprocessed) p24, compared with CyPA, is given by the different
fluorescence changes upon the addition of the cyclophilins (Fig. 2,
C and D) but also by the observation that two Gag
mutants, which had either Gly89 or Pro90
exchanged for Ala, lost their ability to bind CyPA but were still able
to associate with CyPB (2, 9). The stronger interaction of the Gag
polyprotein with CyPB than with CyPA explains why 10-fold higher
concentrations of CsA are needed to disrupt this complex (4).
Processing the immature p24 protein to the mature p24 form of the
capsid protein by HIV-1 protease increased the affinity 13- and 4-fold
for CyPA and CyPB, respectively. The agreement of the
Kd values for mature p24 with that for peptide p(214-228) suggests the C-terminal binding site to be responsible for
the high affinity interaction of the mature p24 form with the
cyclophilins and not the Gly-Pro90 site. The fluorescence
intensity of acrylodan-labeled immature p24 increased upon CyPA
binding, resulting in a Kd of 8.2 µM,
whereas that of labeled mature p24, carrying the environmentally sensitive label at the same site, decreased upon the addition of
already low concentrations of CyPA. This observation indicates different primary interaction sites of immature and mature p24 with
CyPA and thus different conformations of the two p24 forms. The induced
accessibility of the C-terminal binding site(s) on maturation has to be
caused by a conformational rearrangement in the C-terminal domain of
the capsid protein or by the loss of N-terminal and C-terminal
extensions of immature p24. The observed processing-induced change in
the fluorescence spectrum of unliganded p24 (Fig. 2, A and
B versus C and D) indicates
a structural rearrangement, particularly in the C-terminal domain where
the label is attached (see "Experimental Procedures"). A
maturation-induced accessibility of the MHR-located binding site for
CyPA is also indicated by the comparable estimated
Kd for the MHR peptide p(153-172) (see discussion
under "Results") and for the mature p24 protein. The recently
reported structure of the C-terminal domain of p24 (20) suggests that
both high affinity binding sites are accessible for CyPA. Structural
data of the C-terminal 11 amino acids, including Gly223-Pro224, however, could not be collected,
suggesting a rather flexible structure. Thus, this part of p24 is
very likely to be as well suited as the free peptide to fit into the
active site of CyPA and CyPB as predicted from the present study.
That the high affinity interaction of CypA with p24 was not detected in
previous isothermal titration experiments with directly expressed
mature recombinant p24 (23) might well be due to the different
experimental conditions. The fluorescence method applied in the present
study allowed measurements at 50-500-fold lower concentrations of p24,
thus avoiding aggregations of p24 that occur at higher concentrations
(15) and that could have disturbed the accessibility of the high
affinity sites in isothermal titration experiments. Differences in
conformations due to different manufacturing methods of the mature p24
might be another reason. In the present study, mature p24 was prepared,
similar as in vivo, from a folded precursor that was
processed as in vivo by the HIV-1 protease.
Role of CyPA-p24 Interaction--
Growing evidence emphasizes the
role of different cyclophilin-binding sites on a single p24 molecule.
During viral assembly of the Gag polyprotein precursors, the essential
incorporation of CyPA into the virion is mediated by its binding to the
Gly-Pro90 site, which, at this stage, is according to the
present results the only accessible CyPA binding site. The ratio of
only one incorporated CyPA to 10 Gag polyproteins (2, 3) might reflect
the moderate binding affinity, as has been already discussed (23). A
function of virion-incorporated CyPA in the viral assembly process has been ruled out (9); rather, a role in capsid cone disassembly after
viral infection, possibly by weakening the capsid cone already prior to
viral host penetration, has been postulated (10). The present results
suggest that virus-incorporated CyPA bound to the Gly-Pro90
site might be attracted in the course of the maturation process by the
newly generated higher affinity binding sites in the C-terminal domain
of p24, which is important for p24 oligomerization (Fig. 6). Indeed, CyPA disrupts high order p24
complexes (12) but obviously allows assembly of the C-terminal
domain-missing capsid protein (CA151) into continuous
planar strips even if it is bound to the Gly-Pro90 site
(11). The function of C-terminal domain-bound CyPA could thus be to
promote destabilization of the capsid cone structure. This hypothesis
would be in agreement with the observation that propagation of virions
generated in the presence of CsA, which abolishes CyPA incorporation,
is blocked after infection (9). Obviously, the absence of CyPA in the
virus cannot be compensated by abundant cytosolic CyPA after infection
of a target cell (3, 9). If the Gly-Pro90 site would indeed
remain the target of CyPA action, cytosolic CyPA should be able to
substitute CyPA deficiency after viral entry, due to the outside
exposure and accessibility of this loop on the capsid cone. In
contrast, the C-terminal binding sites in the mature capsid cone might
be accessible to cellular cyclophilins after infection only, if the
regularity and stability of the structure of the capsid cone is
impaired, by virus-intrinsic CyPA molecules. In some cases,
destabilizing mutations in p24 (31, 32) might dispense the virus from
incorporation of CyPA. Intracellular cyclophilins and possibly other
components might then facilitate complete disassembly. If the capsid
cone is not destabilized, initiation of the disassembly process will be
difficult but might be possible in cells with high concentrations of
intracellular cyclophilins, as was recently observed (33).

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Fig. 6.
Model of CyPA action on the capsid cone
architecture. The capsid protein p24 of HIV-1 is supposed to
dimerize by interactions between the N-terminal domains (N)
of two p24 molecules and to oligomerize to higher order structures by
aggregation of p24 dimers through their C-terminal domains
(C) containing the MHR (20, 34). After processing of the Gag
polyprotein and due to conformational changes, high affinity binding
sites for cyclophilins are generated in the C-terminal domain of p24
additionally to the low affinity Gly-Pro90 site in the
N-terminal domain. Cyclophilin interaction in the C-terminal domain is
expected to destabilize the capsid cone for postinfection
disassembly.
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In conclusion, the present finding of two high affinity binding sites
for CyPA and CyPB within the C-terminal domain of HIV-1 p24 revealed
not only sequence motifs of Gly-Pro flanking regions that are important
for tight binding of peptide ligands to the cyclophilins but also a
feasible model for the cyclophilin-capsid protein interactions in the
HIV-1 life cycle (Fig. 6). CyPA is incorporated into the virions by
binding to the Gly-Pro90 site (N-terminal domain of p24) of
the Gag polyprotein precursor. Processing of Gag proteins by the viral
protease induces a conformational change and generates high affinity
binding sites in the C-terminal domain of p24 that will attract CyPA,
and this complex could destabilize the capsid cone for disassembly. In
view of the observed binding of CyPA to a site in the MHR of p24, which
is well conserved in HIV-1, HIV-2, and other lentiviruses as well as in
oncoviruses, intracellular cyclophilins might well be the postulated
highly conserved binding partner for this region (20).