From the Département de Pharmacochimie Moléculaire et Structurale U 266 INSERM-UMR 8600 CNRS, UFR des Sciences Pharmaceutiques et Biologiques 4, 75270 Paris Cedex 06, France
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ABSTRACT |
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The human immunodeficiency virus (HIV-1)
nucleocapsid protein NCp7 containing two
CX2CX4HX4C-type
zinc fingers was proposed to be involved in reverse transcriptase
(RT)-catalyzed proviral DNA synthesis through promotion of
tRNA3Lys annealing to the RNA primer binding site,
improvement of DNA strand transfers, and enhancement of RT
processivity. The NCp7 structural characteristics are crucial because
mutations altering the finger domain conformation led to noninfectious
viruses characterized by defects in provirus integration. These
findings prompted us to study a putative RT/NCp7 protein-protein
interaction. Binding assays using far Western analysis or RT
immobilized on beads clearly showed the formation of a complex between
NCp7 and RT. The affinity of NCp7 for p66/p51RT was 0.60 µM with a 1:1 stoechiometry. This interaction was
confirmed by chemical cross-linking and co-immunoprecipitation of the
two proteins in a viral environment. Competition experiments using
different NCp7 mutants showed that alteration of the finger structure
disrupted RT recognition, giving insights into the loss of infectivity
of corresponding HIV-1 mutants. Together with structural data on RT,
these results suggest that the role of NCp7 could be to enhance RT
processivity through stabilization of a p51-induced active form of the
p66 subunit and open the way for designing new antiviral agents.
The HIV-11 nucleocapsid
protein NCp7 is a highly basic protein containing two zinc fingers of
the
CX2CX4HX4C
type (Fig. 1) found in tight association with the dimeric RNA genome in
the retrovirus core (1). In vivo, NCp7 is required for the
protection of the genome against cellular nucleases and is involved in
genomic RNA packaging and morphogenesis of virus particles (review in Ref. 2). Most of these functions are related to its well demonstrated high affinity for single-stranded nucleic acids (3). NMR studies have
demonstrated that the folded CCHC boxes of NCp7 are in spatial proximity, whereas the N- and C-terminal sequences remain flexible (4,
5). Mutations inducing modifications in the general conformation of the
protein, such as the replacement of His23 by Cys,
Pro31 by Leu, or Trp37 by a nonaromatic residue
led to more or less important defects in RNA packaging and virus core
morphology (6-11), which seem hardly reconcilable with the complete
loss of infectivity of the mutated viruses. One possible explanation
could be that changes in NCp7 structure hinders one essential
NCp7-dependent step of virus life cycle such as reverse
transcription and provirus synthesis (12-14). This process is
catalyzed by the p66/p51 heterodimeric virion-associated reverse
transcriptase (RT). This enzyme exhibits RNA- and
DNA-dependent DNA polymerase activities and an RNase H
activity, which are achieved by the p66 polypeptide chain (review in
Ref. 15). In vitro, HIV-RT shows an unusual low processivity for a replicative enzyme, suggesting that additional factors are required for efficient viral DNA synthesis in vivo.
In vitro, NCp7 has been shown to activate the annealing of
the replication primer tRNA3Lys at the
initiation site of reverse transcription (16, 17) and the 5'-3' viral
DNA strand transfer, leading to provirus formation (18-21). NCp7 was
shown to reduce nonspecific reverse transcription (18, 21, 22) and to
enhance the efficiency and processivity of RT (19, 23-26), suggesting
that it could activate reverse transcription through direct interaction
with the enzyme (18, 20, 22-24, 26, 27). Accordingly, NCp7 was shown
to be capable of re-establishing strand transfer efficiency and RNase H
activity of a defective RT mutant (14).
In this study, RT and NCp7 have been shown for the first time to form a
1:1 complex characterized by an affinity of 6.0 × 10 Chemicals--
NCp7, (12-53)NCp7,
W16F37(12-53)NCp7,
L37(12-53)NCp7, C23(12-53)NCp7, (a-D)NCp7,
which corresponds to a peptide in which the zinc finger domains have
been replaced by two Gly-Gly linkers (see Fig. 1), and Vpr were
synthesized on a 433 automated peptide synthesizer (Applied Biosystem)
using the procedure already described (30, 31). Anti-NCp7 mouse
monoclonal antibodies (mAb) were either HH3 (32) or
2B10,2 which are directed
against the C-terminal part (52-67) of NCp7 and the first zinc finger
of NCp7, respectively. Rabbit polyclonal antibody against RT was a
generous gift from S. Litvak. Monoclonal antibody against Vpr was
obtained from the synthetic peptide (31).
Virus Production--
HIV-1 (NL43 strain) was produced by
transfecting plasmid pNL43 into 293T cells using the calcium phosphate
precipitation standard procedure. This virus was pseudotyped with the
vesicular stomatotitis virus G glycoprotein by co-transfection of
vesicular stomatotitis virus G glycoprotein-encoding plasmid.
Enzyme-linked immunosorbent assay (kit from E. I. du Pont de
Nemours & Co.) was carried out to quantify p24 content of the viral
stock. The infectivity of the virus was determined on HeLa cells as
described (33). After ultracentrifugation (50,000 × g), the virus was resuspended in 200 µl of phosphate
buffer, pH 7.5, containing 150 mM NaCl and 0.5% Triton.
The concentrations of viral proteins in the sample were 30 µg of
p24/ml, 10 µg of NCp7/ml, and 3.5 µg of RT/ml.
Far Western Blot Analysis--
2 µg of purified HIV-1 p66/p51
RT (Worthington, Freehold, New Jersey) were immobilized onto a Hybond-C
super nitrocellulose membrane (Amersham Pharmacia Biotech) in 100 µl
of TBS (25 mM Tris, 100 mM NaCl, 0.1 mM DTT, 3 mM KCl, pH 7.4) for 3 h at room temperature. The membrane was treated with SuperBlock blocking buffer
(Pierce) for 3 h at room temperature to reduce nonspecific interactions and then incubated overnight at 4 °C either with NCp7,
(a-D)NCp7, (12-53)NCp7, or Vpr (2.1 µM) or without
proteins as a control in 4 ml of 5% dry milk in TBS buffer containing
0.1% Tween 20. After three washes in TBS-Tween and a 45-min incubation in superblocker, blots were revealed by incubation, first, with 1/15,000 anti-NCp7 mAb (HH3 for NCp7 and (a-D)NCp7, 2B10
for (12-53)NCp7) or 1/1000 anti-Vpr mAb for 3 h in 5% dry milk
TBS-Tween followed by treatment with peroxidase-conjugated anti-mouse
antibody for 1 h. These antibodies were not able to cross-react
with RT. Complexes were revealed by the ECL method (Amersham Pharmacia
Biotech) with peroxidase substrate incubation.
Alternatively, 2 µg of HIV-RT were loaded on a 15%
SDS-polyacrylamide gel electrophoresis (PAGE) and then transferred onto nitrocellulose membrane. The blot was incubated with 2.1 µM NCp7 or without NCp7 as control following the
procedure described above using HH3 mAb against NCp7.
NCp7/RT Binding Studies Using RT Immobilized on Sepharose
Beads--
HIV-1 RT was covalently linked to CNBr-activated Sepharose
4B beads (Amersham Pharmacia Biotech) using the standard procedure described by the supplier. Typically, 100 mg of CNBr-activated Sepharose beads were swollen and washed in 20 ml of HCl (1 mM), then equilibrated in coupling buffer (0.1 M NaHCO3, 500 mM NaCl, pH 8.3),
thus providing 350 µl of gel, which was then diluted in 350 µl of
coupling buffer. The batch was divided in two equal parts. 40 µg of
HIV-1 RT in 200 µl of coupling buffer were added to the first batch,
and only coupling buffer was added to the second one. After a night at
4 °C, excess ligand was washed away, and remaining unbound sites
were inactivated in 1 M 1,3-diaminopropane, pH 8, during
2 h at room temperature. The two bead batches were washed with 3 5-min cycles of alternating pH using 0.1 M acetate buffer
(500 mM NaCl, pH 4) and 0.1 M Tris-HCl buffer
(500 mM NaCl, pH 8). The efficiency of RT immobilization
was checked by detecting the quantity of unbound proteins remaining in
the supernatant using Western blot analysis and calculated to be
superior to 98% (data not shown), suggesting that 0.23 µg (2 × 10 Surface Plasmon Resonance Experimental Method--
Surface
plasmon resonance experiments were carried out on an Amersham Pharmacia
Biotech BIAcore 2000 apparatus. CM5 sensorchips, N-hydroxysuccinimide,
N-ethyl-N'-(dimethylaminopropyl)carbodiimide, and
surfactant p20 were supplied by Amersham Pharmacia Biotech. 1,3-Diaminopropane was from Aldrich. CM5 sensorchip was stabilized in
HBS running buffer (10 mM Hepes, 150 mM NaCl,
0.005% p20, pH 7.4) and activated using the
N-ethyl-N'-(1,3-diethylamide-propyl)carbodiimide/N-hydroxysuccinimide procedure described by Amersham Pharmacia Biotech. Then, about 6000 response units of RT were reproducibly bound following
a 20-µl pulse (5 µl/min) with a solution of RT protein (0.1 µg/µl) diluted in sodium acetate buffer, pH 4.5. To prevent
possible electrostatic interactions with (12-53)NCp7 and its
derivatives, remaining activated carboxyl groups were blocked by the
addition of 1,3-diaminopropane (1 mM, 30 µl). The bulk
refractive index due to injected proteins was corrected using a
nonderivatized channel as control. Binding experiments were performed
at 25 °C with a 20 µl/min flow rate in HBS running buffer (50 mM NaCl). 50 µl of (12-53)NCp7,
W16F37(12-53)NCp7,
L37(12-53)NCp7, and C23(12-53)NCp7 solutions
(1.6 µM) were injected three times on immobilized RT and
on the control channel. Flow cells were regenerated by a 5-µl pulse
of 0.05% SDS in HBS running buffer followed by 2.5 M NaCl solution.
Cross-linking of RT and NCp7 with
3,3'-Dithiobis[sulfosuccinimidyl Propionate] (DTSSP)--
RT and
NCp7 were cross-linked with a homobifunctional
N-hydroxysuccinimide ester-conjugated reagent DTSSP
(Pierce), leading to covalent bonds between amino groups of both
proteins. After incubation of 1 µg of RT and 4.6 µg of NCp7 (40 equivalents) for 1 h at room temperature in 10 µl of
phosphate-buffered saline (3 mM KCl, 1.5 mM
KH2PO4, 140 mM NaCl, 8 mM Na2HPO4, pH 7.5), DTSSP was
added (2 or 5 mM) for 30 min. The cross-linking reaction was stopped by incubating the mixture for 30 min in 50 mM
Tris, 6 mM glycine buffer. Proteins boiled in Laemmli
buffer without DTT were resolved by electrophoresis on a 12% SDS-PAGE,
transferred onto nitrocellulose membrane, and analyzed by two
successive Western blots using, first, rabbit polyclonal antibody
against RT (a generous gift from S. Litvak) and then, after
dehybridation in 0.1 M CH3COOH, a mouse mAb
against NCp7 (HH3) as described above.
Immunoprecipitations from HIV-1 (NL43 Strain) Crude
Extracts--
50 µl of crude extract obtained from 293T transfected
cells were rocked with 50 µl of ascite fluid containing mAb against NCp7 (HH3 or 2B10) overnight at 4 °C. Then, 25 µl of
protein G-Sepharose beads (Amersham Pharmacia Biotech) equilibrated in
80 mM Tris, 80 mM NaCl, pH 8.0, 1% Nonidet
P-40 were added, and the suspension was incubated for 4 h at
4 °C. Beads were collected by centrifugation (10,000 × g for 5 s) and washed at 4 °C twice with the
previous buffer and once with a low salt buffer (80 mM
Tris, pH 8.0, 0.1% Nonidet P-40, 0.05% sodium deoxycholate). Beads
were boiled in Laemmli buffer. Proteins were resolved by
electrophoresis on a 15% SDS-PAGE and analyzed by two successive
Western blots in order to reveal RT and NCp7 using the previously
mentioned procedure.
Evidence for a Molecular Interaction between HIV-1 RT and
Nucleocapsid Protein NCp7--
Interactions between NCp7 and
heterodimeric p66/p51RT or monomeric p66RT and p51RT forms alone were
investigated in vitro by far Western blot analysis using RT
samples immobilized on nitrocellulose membrane under nondenaturating
(Fig. 2A) or denaturating conditions (Fig. 2B).
The formation of a complex was evidenced using a mAb directed against
NCp7 or its derivatives, which is unable to cross-react with native RT.
As depicted in Fig. 2A, NCp7 (1st lane) and its central zinc-fingered domain, (12-53)NCp7 (3rd lane),
interact with the heterodimeric RT, whereas (a-D)NCp7 (2nd
lane), in which both zinc fingers have been replaced by two
Gly-Gly linkers (Fig. 1), was found
unable to bind RT. In contrast, even after a long exposure time, no
signal could be detected when Vpr, another basic HIV-1 protein (31),
was used instead of NCp7 (Fig.
2A, 4th lane). Likewise, p6, the second maturation product of p15, was found unable to
recognize RT (data not shown). Fig. 2B shows that NCp7 recognized both subunits of RT, independently. This interaction is not
critically dependent on salt concentration, because only a slight
difference was observed when binding experiments were performed using
buffers containing 100 up to 500 mM NaCl (Fig. 2C).
The affinity of NCp7 for RT was measured by means of an affinity test
based on the immobilization of p66/p51RT onto Sepharose beads through
the formation of covalent bonds between amino groups of RT and
CNBr-preactivated sites on the resin. The beads were incubated with
increasing concentrations of NCp7 from 0.1 up to 4.2 µM
or without NCp7 as control. After washes, the bound NCp7 molecules were
released from the beads by denaturation and analyzed by successive 20%
SDS-PAGE, nitrocellulose membrane transfer and Western blot analysis
with NCp7 mAb (32). Fig. 3A,
which represents the total binding of NCp7 on immobilized RT, confirmed
the formation of a dose-dependent RT/NCp7 complex,
previously characterized by far Western analysis. The nonspecific
binding of NCp7 (Fig. 2C, 1st lane) was
significantly reduced by adding 1,3-diaminopropane to the RT
substituted resin, which blocked the remaining CNBr-activated groups
and provided positive charges at the surface of the beads, resulting in
electrostatic repulsion of the positively charged NCp7. The specific
saturation curve and the binding parameters were determined from
Scatchard representation. The calculated apparent affinity
KD was 0.60 µM (± 0.07) for a 1:1
stoichiometry (n = 0.86 ± 0.04), corresponding to
about 1 NCp7 molecule for 1 molecule of the heterodimer p66/p51RT (Fig.
3, B and C).
Structure-Activity Relationships--
Since it appears from the
experiments with (a-D)NCp7 (Fig. 2A) that the N- and
C-terminal parts of NCp7 are not critical for RT recognition, the
contribution of the zinc finger domains was investigated by measuring
the direct binding of (12-53)NCp7 and its various mutants,
W16F37(12-53)NCp7,
L37(12-53)NCp7, and C23(12-53)NCp7, to RT
using surface plasmon resonance experiments. One concentration (1.6 µM) of each peptide was injected to 6000 response units
of RT immobilized on the sensorchip in HBS buffer (10 mM
Hepes, 50 mM NaCl, 0.005% p20, pH 7.4). As shown on Fig. 4A, (12-53)NCp7 was able to
recognize RT. The inversion of aromatic residues in
W16F37(12-53)NCp7 induced a 50% decrease of
RT binding as compared with wild-type (12-53)NCp7. Moreover, the
replacement of Trp37 by Leu or His23 by Cys
dramatically disturbed the RT recognition, because in both cases, only
20% of wild-type binding was retained. These results were confirmed by
competition experiments using immobilized RT on Sepharose beads. For
this purpose, the beads were preincubated with two different
concentrations of (12-53)NCp7 or various mutants. Wild-type NCp7 was
then added at 0.4 µM, and the collected samples were
analyzed by Western blot as above in order to quantify the amount of
NCp7 bound to RT. The monoclonal antibody used (HH3) interacts selectively with NCp7 and is unable to cross-react with the
competing NCp7 derivatives. The addition of 10 equivalents of
(12-53)NCp7 induced a complete disappearance of the NCp7 band (Fig.
4B, lanes 3-4), supporting the importance of the
finger domain in RT recognition. Moreover, the structure of the zinc finger domain appears important for the interaction with RT because W16F37(12-53)NCp7 appeared to be able to
decrease NCp7 binding only at 50 equivalents (Fig. 4B,
lanes 9-10), whereas L37(12-53)NCp7
(lanes 5-6) and C23(12-53)NCp7 (lanes
7-8) were unable to impede RT/NC recognition.
Cross-linking of RT and NCp7 with DTSSP--
The interaction
between RT and NCp7 was confirmed by cross-linking experiments using 2 and 5 mM DTSSP, which cross-links compounds in close
spatial proximity by formation of covalent amide bonds with amino
groups of interacting proteins (Fig. 5,
A and B). Therefore, NCp7 and RT were
preincubated to allow the complex to be preformed. Then DTSSP was
added. After quenching the reaction with Tris-glycine buffer, samples
were analyzed by two successive Western blots using antibodies directed
either against RT (Fig. 5A) or against NCp7 (Fig.
5B). As expected, this resulted in the formation of various
species with apparent molecular masses of 59, 74, 117, and 134 kDa that
reacted with antibodies directed against RT (Fig. 5A). The
band at 117 kDa corresponds to the coupling of the p51 and p66
subunits. When anti-NCp7 mAb was used (Fig. 5B), NCp7 could
be detected on the previously mentioned adducts migrating at the 59-, 74-, and 134-kDa positions. The first and second compounds correspond
to the covalent binding of NCp7 to the p51 and p66 subunits of RT,
respectively, and the third one to the binding of two molecules of NCp7
to p66/p51 RT. Interestingly, the p66/NCp7 cross-linked compound is the
most abundant adduct. Weak bands at 16, 24, and 32 kDa, corresponding
to dimers, trimers, and tetramers of NCp7, were observed.
Co-immunoprecipitation of RT/NCp7 Complex--
The existence of a
RT/NCp7 complex was confirmed by co-immunoprecipitation using a crude
extract from 293T cells transfected by pNL43 HIV-1 plasmid. The viral
preparation was treated with HH3 or 2B10 mouse monoclonal
antibody. Antigen-antibody complexes were isolated by affinity
absorption to protein G-Sepharose beads and subsequent elution with an
SDS-containing buffer. After gel electrophoresis and transfer onto
nitrocellulose membrane, the immunoprecipitated proteins were probed
with a rabbit polyclonal antibody against RT (Fig.
6A) then, after dehybridation,
with a mouse mAb against NCp7 (Fig. 6B). Significative
amounts of NCp7 were retrieved in both cases (Fig. 6B,
lanes 1-2), whereas no nonspecific binding on the beads was
observed (Fig. 6B, lane 3). Moreover, when using
HH3 mAb directed against the C-terminal part of NCp7, both
subunits, but principally p66, co-immunoprecipitated (Fig.
6A, lane 1), whereas in the case of 2B10
recognizing the first zinc finger of NCp7, essentially p51 was found
(Fig. 6A, lane 2). The light and heavy chains of
immunoprecipitating mAbs were also stained (Fig. 6B),
because these antibodies and the probing secondary anti-NCp7 antibody
were both elicited in mice.
The role of the nucleocapsid protein NCp7 in HIV-1 viral
replication is not as yet well defined. In addition to its involvement in dimerization and packaging of genomic RNA and in virus morphogenesis (2), several studies suggested that NCp7 may function as a key element
of the reverse transcriptionally active ribonucleoprotein complex.
Thus, in vitro, NCp7 was shown (i) to promote annealing of
the tRNA3Lys to the primer binding site
of HIV-1 genome (16, 17), (ii) to accelerate minus-strand DNA transfers
during proviral DNA synthesis (18-21), and (iii) to enhance
processivity and RNase H activity of the reverse transcriptase
(23-26). Most of these functions are related to the chaperone activity
of NCp7 (34), which shows a high affinity for nucleic acids, in
particular for single-stranded RNA. Nevertheless, a direct interaction
between NCp7 and RT increasing reverse transcription efficiency was
hypothesized (18, 20, 22-24, 26, 27, 35) to account for the lack of
infectivity resulting from mutations in the zinc finger domains
(13).
In this work, we clearly demonstrate that NCp7 is able to bind in
vitro in a dose-dependent manner the heterodimeric
form of RT as well as both subunits independently. This interaction seems to be essentially hydrophobic since increasing NaCl
concentrations have little effect on NC/RT complex formation. Under our
experimental conditions, this interaction appears to be specific since
Vpr, another small and positively charged retroviral protein, and p6 were unable to recognize RT. The binding constant KD was found to be 6.0 ± 0.7 × 10 Direct binding experiments with various NCp7 fragments demonstrated
that the flexible N- and C-terminal parts of NCp7 are not involved in
NCp7/RT complexation, since the finger domain-deficient (a-D)NCp7 did
not recognize RT. In contrast, the central region, encompassing both
zinc fingers and the basic linker RAPRKKG, seems to be crucial. The
importance of the conserved structure of zinc finger domains (4, 37,
38) in NCp7/RT binding was confirmed by surface plasmon resonance and
competition experiments. Thus, although the structure of
W16F37(12-53)NCp73
was found close to that of (12-53)NCp7, this mutant was twice less
active than (12-53)NCp7 to bind RT. The replacement of
Trp37 by Leu in L37(12-53)NCp7, a modification
that does not perturb the zinc finger folding3 but impairs
NCp7-induced annealing of tRNA3Lys to
the primer binding site (17), strongly reduced the inhibitory effect of
this mutant, suggesting that Trp37 is important for NCp7/RT
interaction (Fig. 4A, lanes 5 and 6). The mutation of His23 to Cys23(12-53)NCp7
induces a rearrangement of the residues around the zinc ion in the
first finger and increases the distance between the two zinc fingers
(6). This mutant was also unable to impede RT/NCp7 recognition (Fig.
4A, lanes 7 and 8). From these data, it can be to concluded that at least in vitro, the
interaction between NCp7 and RT requires the central zinc finger domain
of NCp7. It is therefore tempting to explain the complete loss of infectivity observed with mutants characterized by conformational modifications of NCp7 by a defect in retrotranscription rather than by
modifications in virus morphology or reduction in packaged genomic RNA
(6, 12, 13).
Another striking result is that NCp7 is able to recognize both subunits
of RT, although in chemical cross-linking experiments, the covalent
binding of NCp7 occurred preferentially with p66. On the other hand,
immunoprecipitation experiments performed on the supernatant of cells
transfected by HIV-1 pNL43 plasmid using two different antibodies
against NCp7 resulted in precipitates containing NCp7 and either p66 or
p51 subunit. When HH3 recognizing the C-terminal part
(52-67) of NCp7 was used, NCp7 seems to interact only with p66. On the
other hand, when the 2B10 mAb covering an epitope located in the first
zinc finger (19-27) of NCp7 was used, the nucleocapsid protein was
found to bind only p51. This suggests that NCp7 could interact at the
interface between the two subunits with the N-terminal zinc finger in
proximity of p66 and the C-terminal zinc finger directed toward p51
(Fig. 7).
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
7 M. The structure-activity study has
emphasized the critical role of the zinc finger domain in the
complexation, giving insights into the loss of virus infectivity of
mutants with structurally altered NCp7. Co-immunoprecipitation of NCp7
and RT in a viral environment and cross-linking experiments suggest
that NCp7 could enhance RT processivity through stabilization by its
zinc finger domain of the p51-induced active form of p66 RNase H domain
(28). Moreover, the N-terminal part of NCp7 could compensate for the absence in RNase H domain of HIV-1 p66 RT of the sequence present in
the Escherichia coli RT (29). The critical role of the
complex during viral DNA synthesis suggests that inhibition of this
protein-protein interaction could be an interesting way for designing
new antiviral agents.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
12 mol) of RT has been captured by µl of beads. Then,
20 µl of these beads corresponding to about 4.6 µg of protein
(3.9 × 10
11 mol) were equilibrated in TBS, 0.1%
Tween 20, 0.1% Nonidet P-40 and incubated with increasing
concentrations of NCp7 from 0.1 up to 4.2 µM in 100 µl
of the same buffer for 5 h at room temperature. After
centrifugation (6500 rpm), the beads were washed twice with the
previous cold buffer at 4 °C. NCp7 bound to immobilized RT or
nonspecifically to the beads was recovered by heating at 80 °C for 5 min in 30 µl of Laemmli buffer (50 mM Tris, 10%
glycerol, 2% SDS, 0.05% bromphenol blue, 200 mM DTT).
Collected samples were loaded on a 20% SDS-PAGE, transferred onto
nitrocellulose, and analyzed by Western blot using HH3 mAb
in order to reveal NCp7. The effect of NaCl concentration on NCp7/RT
complex formation was measured using the same procedure, except that
the beads were incubated with a constant concentration of NCp7 (0.9 µM) in TBS-Tween-Nonidet P-40 buffer containing 100 to
500 mM NaCl. When (12-53)NCp7 or its derivatives were used
as competitors, two concentrations of these peptides (4 and 20 µM) were preincubated for 90 min with immobilized RT
before the addition of 0.4 µM NCp7. In this case, the
monoclonal antibody used (HH3) was selective for NCp7 and was unable to cross-react with (12-53)NCp7 or its derivatives. The
affinity and stoichiometry of the NCp7/RT complex were determined by
quantification of NCp7 bound to immobilized RT or nonspecifically bound
to the beads using a standard curve of pure NCp7 and a Bio-profile imager (Vilber-Lourmat). Nonspecific binding was subtracted from the
total binding, thus enabling us to calculate the concentrations of
bound and free NCp7. The binding parameters were calculated using the
Scatchard equation from Enzfit software. Results are means of three
independent experiments performed in duplicate.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Primary sequences of NCp7 and (a-D)NCp7.
(a-D)NCp7 was obtained by replacing both zinc fingers by two Gly-Gly
linkers located between Lys14 and Arg24 and
between Gly35 and Thr50, respectively (30). The
sites of His23 Cys, Trp37
Leu and
Phe16, Trp37
Trp16,
Phe37 mutations are indicated.
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Fig. 2.
Interaction of NCp7 and RT using far Western
blot analysis. A, 2 µg of p66/p51 RT were immobilized
on nitrocellulose membrane and incubated overnight at 4 °C in TBS
buffer with NCp7, (a-D)NCp7, (12-53)NCp7, or Vpr (2.1 µM). Detection was performed using mAbs directed either
against NCp7 (HH3 or 2B10) or against Vpr, followed by the
addition of peroxydase-conjugated anti-mouse antibody. B, 2 µg of p66/p51 RT were loaded on 15% SDS-PAGE, transferred onto
nitrocellulose membrane, and subjected to far Western blot analysis
with 2.1 µM NCp7 as probe. C, p66/p51 RT was
immobilized on CNBr-preactivated Sepharose beads and incubated with 0.9 µM NCp7 in TBS buffer containing increasing
concentrations of NaCl from 100 to 500 mM. After washes,
bound NCp7 was released through denaturation of beads in Laemmli
buffer. Samples were loaded on 20% SDS-PAGE and subjected to Western
blot analysis using antibodies directed against NCp7 (HH3).
The 1st lane represents the nonspecific binding of NCp7 on
the beads at 100 mM NaCl.
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Fig. 3.
Quantitative analysis of NCp7/RT
binding. A, total binding of NCp7 on immobilized RT.
p66/p51 RT was immobilized on CNBr-preactivated Sepharose beads and
incubated at room temperature for 5 h with increasing
concentrations of NCp7 from 0.1 to 4.2 µM in TBS, 0.1%
Nonidet P-40, 0.1% Tween 20. Unbound NCp7 molecules were removed by
centrifugation followed by two successive washes at 4 °C. Bound NCp7
molecules were recovered by heating in Laemmli buffer and resolved by
Western blot analysis using mAb directed against NCp7
(HH3). B, specific saturation curve. Nonspecific
binding of NCp7 on the beads was subtracted from the total binding for
each NCp7 concentration, providing the specific binding of NCp7 on RT,
which was quantified using a standard curve of pure NCp7. Results are
means (±S.D.) of three independent experiments performed in duplicate.
C, Scatchard representation of NCp7/RT binding. The
calculated binding parameters are: KD = 6.0 ± 0.7 × 10 7 M, n = 0.86 ± 0.04.
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Fig. 4.
RT/NCp7 recognition: zinc finger
structure-activity relationships. A, direct binding of
(12-53)NCp7 and its mutants to RT using surface plasmon resonance
technology. The same concentration (1.6 µM) of
(12-53)NCp7, W16F37(12-53)NCp7,
L37(12-53)NCp7, or C23(12-53)NCp7 was
injected to 6000 response units of RT immobilized on the sensorchip in
HBS buffer at 25 °C. Each sensorgram represents one typical
experiment. B, immobilized RT was preincubated or not with
(12-53)NCp7 and its derivatives (10 and 50 equivalents) for 1.5 h
before the addition of 0.4 µM wild-type NCp7 in
TBS-Tween. Unbound proteins were removed by centrifugation followed by
two successive washes with TBS-Tween at 4 °C. Bound NCp7 was
recovered after denaturation of complexes in Laemmli buffer. Detection
of NCp7 was carried out by Western blot analysis using antibody
selective for wild-type NCp7 (HH3). Lane C+
corresponds to specific binding of NCp7 to immobilized p66/p51RT, and
lane C corresponds to the same experiment with beads
without RT.
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Fig. 5.
Chemical cross-linking of NCp7 and RT by
DTSSP. NCp7 and RT were preincubated in phosphate buffer 1 h
at room temperature, then the cross-linking agent DTSSP was added at a
final concentration of 2 or 5 mM. After 30 min, the
reaction was stopped by the addition of Tris-glycine buffer. Samples
were then analyzed by two successive Western blots using a polyclonal
antibody specific of RT (A) followed by dehybridation and
subsequent revelation using a monoclonal antibody directed toward NCp7
(B).
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Fig. 6.
Co-immunoprecipitation of NCp7/RT complex
from HIV-1 viral extract. RT and NCp7 were co-immunoprecipitated
from supernatant of 293T cells transfected by pNL43 HIV-1 plasmid with
monoclonal antibody recognizing NCp7: lanes 1,
HH3; lanes 2, 2B10. Samples were developed by
Western blot analysis. Detection was performed with either polyclonal
antibody directed against RT (A) or monoclonal antibody
directed against NCp7 (B). Lanes 3 represent the
nonspecific binding of RT and NCp7 on the protein G-Sepharose
beads.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
7 M
for a 1:1 NCp7-p66/p51RT complex. This affinity is in agreement with
both the mM concentration of NCp7 in the virion and the
1/40 RT/NCp7 ratio (36). Because NCp7 interacts in vitro
with both subunits of RT, the binding site of NCp7 on the p66/p51
heterodimer could be located at the region connecting the two subunits.
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Fig. 7.
Proposed mechanism of recognition between
NCp7 and RT. NCp7, captured from the structure of NCp7 complexed
with SL3, has been located at the connection site between p66 and p51
subunits of RT (adapted from Katz and Skalka (15)) with the N-terminal
zinc finger facing p66 and the C-terminal one directed toward p51. This
accounts for the results presented here. The two zinc fingers could be
considered as a hinge element, stabilizing the p51-induced active form
of p66 RNase H domain. The N-terminal helical part of NCp7 has been
disposed in the position that it takes in the structure of the complex
between NCp7 and the double-stranded stem part of SL3 (43).
NCp7 has been shown to enhance the global processivity of reverse
transcription by accelerating RT-catalyzed DNA strand transfer reactions through modulation of RNase H activity (20, 23). The crystal
structure of the RNase H domain of HIV-1 p66-RT shows that the protein
folding is similar to that of E. coli RNase H despite a low
(24%) amino acid homology between both proteins (29). But in contrast
to E. coli RNase H, the HIV-1 RNase activity is critically
dependent of the presence of the other domains of RT. In the viral
enzyme, this could be related to the C-terminal region of HIV-1 RNase H
encompassing the helix E, which was found flexible and unstructured
by NMR (39) and crystallographic (29) studies. Accordingly, the
addition of purified p51 to the isolated domain led to the
reconstitution of HIV-1 RNase H activity in vitro (28).
Therefore, the biological relevance of the present results could be
that the zinc finger domains of NCp7, by interacting with RT at the
interface between the two subunits, reinforce the required
structuration of the catalytic HIV-1 RNase H domain ensured by p51
subunit, thus improving its activity. This hypothesis is consistent
with the model proposed by Cameron et al. (14), since their
experiments showed that NCp7 was able to restore strand transfer
efficiency and RNase H activity of a defective RT mutant containing a
deletion in the C-terminal part of p51 found in spatial proximity of
the RNase H domain.
On the other hand, a highly basic helix/loop region present in E. coli enzyme is missing in the HIV-1 domain. Interestingly, the
introduction of this sequence in wild-type HIV-1 RNase H induced enzyme
activity (40). In E. coli, this basic helix C, QWIHNWKKR, has been proposed to contribute to the proper binding and positioning of the double-stranded RNA/DNA hybrid substrate, thus improving the
catalytic process (41, 42). Moreover, the basic N-terminal sequence of
NCp7 is flexible in solution but was recently found to interact with
the double-stranded RNA stem of the SL3 domain of genomic HIV-1 RNA
under a helical form (43). Therefore, in agreement with the present
results, it is tempting to propose that NCp7 could improve HIV-1 RNase
H activity through RNA/DNA recognition by means of its N-terminal
domain and stabilization of p66/p51 heterodimer by the zinc finger
domain. Finally, this study provides an interesting new approach for
the rational design of antiviral agents.
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ACKNOWLEDGEMENTS |
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We thank H. Buc (Institut Pasteur, France) and S. Litvak (CNRS EP 630, France) for the generous gifts of RT and antibodies against RT, respectively. We acknowledge P. Petitjean for peptide synthesis and C. Dupuis for expert manuscript drafting.
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FOOTNOTES |
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* This work was supported by the French programs against AIDS (ANRS and SIDACTION) and has been presented in part during the International Retroviral Nucleocapsid Symposium, June 21-24, 1998 at NCI, Frederick Research and Development Center, Frederick, MD.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Institut Pasteur, RTG 25, rue du Docteur Roux,
75015 Paris, France.
§ To whom correspondence should be addressed: Département de Pharmacochimie Moléculaire et Structurale, U 266 INSERM-UMR 8600 CNRS, UFR des Sciences Pharmaceutiques et Biologiques 4, avenue de l'Observatoire, 75270 Paris Cedex 06, France. Tel.: (33)-1-53.73.96.88./89; Fax: (33)-1-43.26.69.18; E-mail: roques{at}pharmacie.univ-paris5.Fr.
2 H. de Rocquigny, A. Caneparo, C. Z. Dong, P. Petitjean, T. Delaunay, and B. P. Roques, submitted for publication.
3 N. Morellet, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: HIV-1, human immunodeficiency virus type 1; RT, reverse transcriptase; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; DTT, dithiothreitol; DTSSP, 3,3'-dithiobis[sulfosuccinimidyl propionate].
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