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INTRODUCTION |
Virion infectivity factor
(Vif)1 protein of HIV-1 is
required for viral replication in vivo (1, 2). In cell
culture systems, HIV-1
vif viruses are incapable of
establishing infection in certain cells, such as H9 T-cells, peripheral
blood lymphocytes, and monocyte-derived macrophages (3-6). HIV-1
viruses with a defective vif gene are not able to complete
intracellular reverse transcription and endogenous reverse
transcription in cell-free virions when mild detergent is utilized to
make the viral envelope permeable (7-10). Most studies indicated that
the expression of viral components, including viral proteins and
nucleic acids, is not altered in the virions produced from
nonpermissive cells (3, 10, 11). However, the deletion of the
vif gene will result in alterations of virion morphology
(12-14). Various hypotheses have been proposed regarding the molecular
mechanisms of Vif protein. It has been reported that defect of
vif could affect the maturation of Gag precursor (15).
Furthermore, Vif could directly bind to the protease domain of
pol precursor and prevent the improper cleavage of Gag
precursors before viral assembly (16). It was also proposed that Vif
protein is required to counteract an unknown endogenous inhibitor(s) in
the virus-producing cells (17, 18). Recent studies further indicated
this endogenous inhibitor is CEM15, which is only expressed in the
nonpermissive cells. Introduction of CEM15 into the permissive cells
will generate a nonpermissive phenotype (19). However, the function of
CEM15 remains unknown. Because its sequence is similar with
APOBEC-1(apoB mRNA-editing catalytic subunit 1), a cytidine
deaminase that can change cytidine into uridine in the mRNA of
apolipoprotein B, CEM15 could affect the genomic RNA of HIV-1.
Interestingly, we and others show that Vif is an RNA-binding protein
and is an integral component of a messenger ribonucleotide
protein complex of viral RNA (20, 21). The Vif protein in this
ribonucleoprotein complex may protect viral RNA from various endogenous
inhibitors and could mediate viral RNA engagement with HIV-1 Gag
precursors. As such, Vif could play a key role in the proper
trafficking of the viral genetic substance (genomic RNA) in the
lentivirus-producing cells.
Because Vif is essential for HIV-1 replication, it is an important
target for anti-HIV therapeutics. However, because its molecular
mechanism in viral life cycle remains to be further determined, it is
quite difficult to generate a small molecule inhibitor(s) to block Vif
function at the present time. Recently, we have found that Vif proteins
are able to form multimer (22). It is well known that multimerization
is critical to the biological activity of many prokaryotic and
eukaryotic proteins and is a common mechanism for the functional
activation/inactivation of proteins. Therefore, multimerization has
been an ideal target for the development of inhibitors of various
proteins (23-25).
In this report, we demonstrate that Vif multimerization could be a
promising intervention target for anti-HIV-1 agent development. We have
found that a set of proline-enriched peptides is able to bind to Vif
protein, inhibit the Vif-Vif interaction, and inhibit viral replication
in cell culture. Our data demonstrates that, although the function and
structure of Vif remains uncertain, we have still successfully
developed the potent Vif antagonists, based upon the biochemical
characteristics of Vif protein.
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MATERIALS AND METHODS |
Plasmid Constructions, Expression of GST Fusion Proteins, and
Synthesis of 35S-Labeled Proteins by in Vitro
Translation--
The construction of pGEX-Vif, pCITE-Vif,
pCITE-Vif-(
151-192), and pCITE-Vif-(
151-164) were
described previously (20, 22). Vif(
PPLP) genes were generated by
PCR-mediated mutagenesis and then inserted into pGEX vector. The
vif(
PPLP) gene was also inserted into pCITE-4a vector
(Novagen, Madison, WI) for in vitro translation. 35S-Labeled Vif or its mutant proteins were synthesized by
in vitro transcription and translation utilizing SPT3 kits
(Novagen) in the presence of [35S]methionine (1,000 Ci/mmol; Amersham Biosciences), as described previously (20). The GST,
GST-Vif, and other GST fusion Vif mutant proteins were produced
according to the previously described methods (20, 22). The tyrosine
kinase Hck genes were generated by PCR amplification and then inserted
into the pGEX vector. GST-Hck fusion protein was expressed and purified
with the same procedure as for GST-Vif.
Phage Display Peptide Screening--
Vif-binding peptides
displayed on M13 phages were selected using the Ph.D.-12 phage display
peptide library kit (New England Biolabs, Beverly, MA). Phage panning
procedures were performed according to the manufacturer's protocol
with some modifications. Briefly, GST-Vif fusion protein attached to
glutathione-conjugated agarose beads was used as a target for phage
panning. For each round of panning, 1011 phages were first
absorbed with GST followed by mixing with 3 ml of GST-Vif attached to
glutathione-agarose beads. After binding at room temperature for 1 h, the GST-Vif binding phages were then eluted by 5 mM
reduced glutathione. The eluted phages were amplified by mixing the
elution with 20 ml of Escherichia coli ER2738 culture (optical density at 0.6). After incubation at 37 °C with vigorous shaking for 4 h, the bacterial cells were pelleted, and the phages in the supernatant were precipitated by polyethylene glycol (20%)/NaCl (2.5 M). After resuspension in Tris-buffered saline and
reprecipitation by polyethylene glycol, the phages were suspended in
200 µl of Tris-buffered saline, 0.02%NaN3. The titration
of the eluted or amplified phages was determined by infecting the
E. coli ER2738 mixed in the conditioned medium-agar plates,
as described in the kit protocol. After three rounds of panning,
individual phage plaques from the GST or GST-Vif elution were selected
for amplification, respectively. Phage DNA was then purified and sequenced.
Determination of Binding Affinity by Enzyme-linked Immunosorbent
Assay (ELISA)--
An ELISA was performed to measure the relative
binding affinity of phages to GST, GST-Vif, or GST-Vif (
151-192).
The protocol supplied by the manufacturer was followed. Briefly, 150 µl of 100 µg/ml GST and GST-Vif in 0.1 M
NaHCO3, pH 8.6, was coated on 96-well microtiter plates,
respectively, and incubated at 4 °C overnight. The plates were
blocked with blocking buffer (0.1 M NaHCO3, pH
8.6, 5 mg/ml bovine serum albumin) for 2 h at room temperature.
The individual phage clones were 4-fold-serially diluted (from
1011 to 105), added to the wells coated with
GST, GST-Vif, or GST-Vif-(
151-192), and incubated for 2 h at
room temperature. After washing, horseradish peroxide-conjugated
anti-M13 antibody was added to bind the phages. After incubation at
room temperature for 1 h, the excess antibody was washed, the
substrate was added, and color development was allowed to proceed. The
phages captured by Vif were therefore semi-quantitated. Optical density
at 405 nm equal to or greater than 0.15 was considered as positive.
Peptide Synthesis--
HIV-1 consensus B Vif (15-mer) peptides
were provided by the National Institutes of Health AIDS Research and
Reference Reagent Program. All the other peptides were synthesized by
solid-phase techniques using a Symphony Multiplex synthesizer (Protein
Technologies, Inc., Tucson, AZ) and a 9050 Pepsynthesizer Plus
automated peptide synthesizer (Perseptive Biosystems, Cambridge, MA)
with N
-Fmoc
(N-(9-fluorenyl)methoxycarbonyl)/tert-butyl
chemistry. Biotin peptides were biotinylated by adding biotin (Sigma)
at the N terminus. The peptides were characterized by analytical HPLC
and matrix-assisted laser desorption/ionization time-of-flight mass
spectrometry. All the peptides were at least 95% pure, as determined
by HPLC.
In Vitro Vif-Vif/Vif-Hck Interactions and Their Inhibition by
Peptides--
A GST pull-down assay was used to study the in
vitro protein-protein interactions. The GST fusion proteins on
agarose beads are first generated as described previously except
without elution with 5 mM glutathione (20). The
35S-labeled, in vitro translated Vif proteins
were then mixed with GST fusion protein-conjugated agarose beads in
washing/binding buffer (150 mM NaCl, 10 mM
Tris-HCl, pH 8.0, and 0.1% Triton-X-100). Binding is allowed to
proceed at 23 °C for 20 min and then at 4 °C for 1 h. For
the inhibition of Vif-Vif/Vif-Hck binding by peptides,
35S-labeled Vif proteins were added to
GST-Vif/GST-Hck-conjugated agarose beads and incubated with peptides at
different concentrations in binding buffer at 4 °C for 1 h. The
beads were then washed with washing/binding buffer 3 times, and the
bead-bound 35S-labeled Vif proteins were fractionated by
SDS-PAGE followed by autoradiography and quantitated using a
PhosphorImager (Molecular Dynamics, Sunnyview, CA).
Peptide Internalization Experiment--
H9 cells were suspended
in serum-free RPMI 1640 supplemented with 4 mM
L-glutamine and incubated with the peptides for 30 min.
After washing 3 times with phosphate-buffered saline (PBS), the cells
were fixed with 4% formaldehyde in PBS for 10 min at room temperature.
Cells were then washed twice with PBS and treated with 0.1% Triton
X-100 in PBS for 10 min. After an additional 2 washes with PBS, cells
were incubated with blocking buffer (3% bovine serum albumin in PBS)
for 1 h at room temperature followed by incubation with
streptavidin-fluorescein isothiocyanate (Sigma) at 2 µg/ml in
blocking buffer for 5-10 min in the dark. Cells were then washed with
PBS, and cell suspensions were smeared on glass microscope slides for
fluorescence microscopy using an Olympus BX60 fluorescence microscope.
Viral Infectivity Assay--
H9 cells (1 × 106) were mixed with HIV-1NL4-3 viruses at a
multiplicity of infection of 0.01. After incubation at 37 °C for
5 h, the excess viruses were removed, and the cells were cultured
in the presence of RPMI 1640 medium plus 10% fetal bovine serum with
or without peptides at a concentration of 50 µM. Every 3-4 days, the supernatants were harvested and refreshed. The effects of these peptides upon viral infectivity were monitored by detecting the HIV-1 p24 antigen level in the cell culture supernatant via ELISA,
as described previously (20, 26, 27).
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RESULTS |
Identification of PXP Motif-containing Peptides Binding to Vif
Protein--
To search for the peptides that bind with HIV-1 Vif protein,
the phage peptide display method was employed. The procedures described
in the manual supplied by manufacturer were followed. After three
rounds of panning, the phage-displayed peptides that bind with GST-Vif
were identified by sequencing DNA in the knot region of the phages.
Through theses methods we have identified a set of 12-mer peptides
containing a PXP motif that bind to the Vif protein (Table
I).
To determine the binding affinity of various PXP
motif-containing peptides to Vif protein, a simple assay based upon an
ELISA was used to determine the relative affinity. The phages at
various concentrations captured by Vif were semi-quantitated. Fig.
1A demonstrates that among
PXP motif containing peptides, VMI 5, VMI 7, VMI 9, and VMI
16 bind to Vif at the highest affinities. The C terminus-deleted Vif
protein binds with PXP motif-containing peptides at low
affinity, indicating that PXP motif-containing peptides bind
to Vif protein through the C terminus of the Vif protein.

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Fig. 1.
A, relative affinity comparisons
between PXP motif-containing peptides. GST fusion protein
(100 µg/ml) of Vif, Vif-( 151-192), and GST only were coated onto
the 96-well plate. The phages clones isolated through the
GST-Vif-containing column were serially diluted and added. After
incubation to allow phage-Vif binding, excess phages were washed off.
Anti-M13 phage antibody conjugated with horseradish peroxide was added
to bind the phages that were captured by GST-Vif. After washing, the
substrate was added, and color development was allowed to proceed. The
phages captured by GST-Vif were, therefore, semi-quantitated. Optical
density at 405 nm equal to or greater than 0.15 was considered to be
positive. The phage sample number (VMI) is the same as shown in Table
I. B, in vitro binding affinity of peptides to
Vif. Various peptides (10 7, 5 × 10 7,
10 6, 10 5, 10 4,
10 3 M) were added to the mixture of
35S-labeled Vif and GST-Vif-conjugated agarose beads.
The 35S-labeled Vif binding to GST-Vif was dissociated from
beads by adding 2% SDS loading buffer and then analyzed by SDS-PAGE
followed by autoradiography and quantitation using PhosphorImager.
IC50 is the concentration of the peptides inhibiting 50%
35S-labeled Vif binding to GST-Vif in GST pull-down
assays.
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PXP Motif-containing Peptides Inhibit Vif-Vif Interaction in
Vitro--
We noted that Vif proteins of various HIV-1 strains all
contain proline-rich sequence at their C terminus
(161PPLP164 in NL4-3 strain), and we have
demonstrated that the proline-enriched domain
(151AALIKPKQIKPPLP164) is required for Vif
multimerization (22). Therefore, it is interesting to examine whether
PXP motif-containing peptides inhibit Vif-Vif interaction.
To this end, some of these peptides containing the PXP
domain, identified from phage display libraries, were chemically
synthesized and examined for their ability to inhibit Vif-Vif binding.
Fig. 1B indicates that peptides containing the PXP motif, such as SNQGGSPLPRSV (VMI 7) or LPLPAPSFHRTT (VMI
9), could significantly inhibit Vif-Vif interaction. The
IC50 for the inhibition of Vif multimerization is 7.43 µM for VMI 7 and 4.84 µM for VMI 9 (Fig.
1B). A Vif-derived 12-mer peptide,
155KPKQIKPPLPSV166 (Vif-(155-166)), which is
originated from the proline-enriched C terminus of Vif, also has the
similar inhibition activity upon Vif-Vif interaction (IC50 = 17.39 µM).
We have also screened a set of synthesized Vif peptides (15-mer), which
includes all the amino acids of HIV-1 Vif protein, for their ability to
block the Vif-Vif interaction in vitro. We demonstrated that
proline-enriched Vif peptides, such as
153LITPKKIKPPLPSVT167 and
157KKIKPPLPSVTKLTE171, which contain the
161PPLP164 domain, are able to inhibit the
Vif-Vif interaction significantly, further supporting that
PXP motif-containing peptides inhibit Vif multimerization
(Fig. 1B and Fig. 2).
Conversely, this result also suggests that the 151-165 region of Vif
is responsible for Vif-Vif binding. The peptides, derived from region
of 145-163, which is upstream of the
161PPLP164 domain, are also able to moderately
inhibit Vif-Vif interaction, suggesting that the amino acid residues at
this region could also participate in the Vif-Vif interaction.

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Fig. 2.
The inhibition of HIV-1 Vif (15-mer) peptides
upon Vif-Vif binding. HIV-1 consensus B Vif (15-mer) peptides (100 µM) were added to the mixture of 35S-labeled
Vif- and GST-Vif-conjugated agarose beads. The 35S-labeled
Vif binding to GST-Vif was dissociated from beads by adding 2% SDS
loading buffer and then analyzed by SDS-PAGE followed by
autoradiography and quantitation using a PhosphorImager
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The 161PPLP164 Domain Is Required for Vif
Multimerization--
Because the PXP motif is also shared
by Vif in the 161PPLP164 domain that is located
within the putative Vif multimerization domain and the PXP
motif-containing peptides are able to inhibit Vif-Vif interaction, it
is interesting to investigate whether the
161PPLP164 domain is required for Vif-Vif
interaction. To this end, site-directed mutagenesis was performed to
delete 161PPLP164,
151AALIKPKQIKPPLP164, and the Vif C terminus
(151-192). The mutants were expressed with an in vitro
translation system in the presence of [35S]methionine or
expressed as GST fusion proteins. The 35S-labeled Vif or
Vif mutants were then bound with GST-Vif or GST-Vif(
PPLP) that were
conjugated with glutathione-coated agarose beads. As described
previously, Vif mutant proteins deleted at the C terminus (151-192) or
151AALIKPKKIKPPLP164 have decreased binding to
Vif (Fig. 3) (22). Vif mutant protein just deleted at the 161PPLP1 64 domain also
showed a decrease in its binding to Vif. Interestingly, the
protein-protein interactions between the Vif mutants deleted at
161PPLP164 domain were significantly decreased
(Fig. 3). These data indicated that the
161PPLP164 domain is required for Vif-Vif
multimerization.

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Fig. 3.
Deletion of PPLP eliminates Vif-Vif
interaction. GST-Vif- or GST-Vif( PPLP)-conjugated agarose beads
were mixed with 35S-labeled Vif or its mutants in binding
buffer and incubated at 4 °C for 1 h. The
35S-labeled Vif or its mutants remaining on beads were
dissociated from beads by adding 2%SDS loading buffer and then
analyzed by SDS-PAGE followed by autoradiography and quantitation using
a Phosphor-Imager. A, GST-Vif/35S-Vif;
B, GST-Vif/35S-Vif-( 151-192); C,
GST-Vif/35S-Vif-( 151-164); D,
GST-Vif/35S-Vif( PPLP); E,
GST-Vif( PPLP)/35S-Vif; F,
GST-Vif( PPLP)/35S-Vif( PPLP).
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PXP Motif-containing Peptides Inhibit Vif-Hck Binding--
It has
been demonstrated that Hck kinase can also bind with Vif through the
161PPLP164 domain (28). It is possible that
PXP motif-containing peptides are able to block the
interaction between Vif and Hck and other protein kinases. To this end,
35S-labeled Vif was allowed to bind with GST-Hck in the
presence or absence of various peptides. As described by others, Vif is able to bind with Hck. In the presence of VMI 7, VMI 9, and Vif (155-166), the binding between Vif and Hck is significantly decreased (Fig. 4).

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Fig. 4.
The in vitro
inhibition by the peptides on Vif-Vif or Vif-Hck binding.
Various peptides (100 µM) were added to the mixtures of
35S-labeled Vif and GST-Vif- or GST-Hck-conjugated agarose
beads. The 35S-labeled Vif binding to GST-Vif or GST-Hck
was dissociated from beads by adding 2% SDS containing loading buffer
and then analyzed by SDS-PAGE followed by autoradiography and
quantitation using a PhosphorImager.
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PXP Motif-containing Peptides Inhibit HIV-1 Replication--
To
examine the inhibitory effects of PXP motif-containing
peptides upon viral infectivity in cell culture, the peptides must be
introduced into the virally infected cells by a reliable method. As
antennapedia homeodomain (Ant, RQIKIWFQNRRMKWKK) has been widely used
to effectively carry peptides into various living cells (29-34), Ant
fusion peptides, Ant-VMI 7, Ant-VMI 9, and Ant-Vif (155-166), were
synthesized, and their activity for in vivo inhibition of HIV-1 replication were investigated. These peptides did not show any
toxicity to H9 cells at the concentration of 50 µM (data
not shown). To examine whether these fusion peptides are able to enter the H9 cells, the cell permeability of biotin Ant-VMI 9 was determined. Fig. 5A indicates that the Ant
fusion peptide can efficiently enter the H9 cells and locate in the
cytoplasm. Because Vif mainly locates in the cytoplasm of
virus-infected cells, the fusion peptide should physically interact
with Vif protein.

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Fig. 5.
A, internalization of peptides. H9 cells
were incubated with biotinylated peptides VMI 9 and Ant-VMI 9 for 30 min. The excess peptides were then washed off. After fixing, the
internalized peptides were detected with streptavidin-fluorescein
isothiocyanate followed by visualization with fluorescence microscopy.
A, Ant-VMI 9, fluorescence; B, Ant-VMI 9, phase-contrast; C, VMI 9, fluorescence; D, VMI 9, phase contrast. B, Ant fusion peptides inhibit HIV-1
replication. H9 cells were infected by HIV-1NL4-3 virions
at 37 °C for 4 h. The infected H9 cells (1 × 106) were then cultured in duplicate in 2 ml of RPMI 1640 medium plus 10% fetal bovine serum without or with peptides (50 µM). Portions of the supernatants (0.5 ml) were collected
every 3-4 days. The HIV-1 p24 antigen levels were determined by ELISA.
This data represent three independent experiments.
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The fusion peptides were then added into the cell culture to examine
their capability to inhibit HIV-1 replication. H9 cells, a
nonpermissive cell line that requires Vif to counteract the endogenous
inhibitor, were infected with HIV-1 viruses in the presence or absence
of various fusion peptides. At the concentration of 50 µM, the fusion peptides, Ant-VMI 7, Ant-VMI 9, and
Ant-Vif (155-166) are able to effectively inhibit HIV-1 replication.
As a control, the Ant peptide itself does not have any anti-HIV-1 activity (Fig. 5B).
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DISCUSSION |
We have demonstrated that the
151AALIKPKQIKPPLP164 domain of HIV-1 Vif is
critical for Vif multimerization, which is required for Vif function
(22). In this report, we have further demonstrated that the
161PPLP164 domain plays a key role in Vif-Vif
interaction. Our current results suggest that Vif-Vif binding occurs,
at least in part, through the direct interaction between
161PPLP164 domains in each Vif molecule.
Because the function of Vif remains unknown, it is difficult to
investigate the molecular mechanism regarding how Vif multimerization
is required for Vif function. However, recent studies indicate that Vif
is required to counteract the endogenous inhibitor CEM15, which is a
putative cytidine deaminase (19, 35). Because Vif binds to HIV-1 RNA,
it is reasonable to assume that Vif-RNA binding could protect the
HIV-RNA from RNA editing (20, 21). If so, Vif-RNA binding could be the major mechanism for Vif function. It is therefore quite important to
study the correlation between Vif-RNA binding and Vif-Vif interaction. Because Vif binds to RNA through its N terminus, whereas Vif-Vif interaction takes place at the C terminus, Vif-Vif interaction could be
correlated with Vif-RNA binding. Conversely, Vif is able to bind with
Gag protein through the positive-charged amino acids in the 151-164
region at the C terminus, and Vif binds to Hck also through the
161PPLP164 domain. Therefore, the Vif-Vif
interaction could be reversibly correlated with Vif-Gag binding or
Vif-Hck binding (13, 28). These hypotheses remain to be fully tested.
Through screening phage display peptide libraries, a set of
proline-enriched peptides binding to Vif was identified and is able to
block the Vif-Vif interaction. The proline-enriched sequence is a
hydrophobic region and usually binds to the hydrophobic interface of
SH3/WW domains in protein-protein interactions (36). Vif-Vif interaction could occur between the two
161PPLP164 domains or the
161PPLP164 domains and other regions in the Vif
protein. It seems that the PXP motif-containing peptides
mimic the hydrophobic structure of the
161PPLP164 domain and bind to the hydrophobic
interface of Vif, which is quite critical for Vif multimerization.
Among these proline-enriched peptides, the peptides containing the
PXPXP motif have the higher binding affinity to
Vif protein. We have also tested the synthesized peptides derived from
the Vif protein upon Vif-Vif interaction. Our data demonstrated that
the peptides containing the 161PPLP164 domain
are able to inhibit Vif-Vif interaction, indicating that the
161PPLP164 domain plays a key role in Vif-Vif interaction.
In this report, we demonstrated that proline-enriched PXP
motif-containing peptides not only inhibit Vif-Vif interaction but also
the binding between Vif and Hck. It is notable that the PXP motif-containing peptides have been shown to inhibit the activation of
various SH3 domain-contained protein kinases (36, 37). Because the
peptides identified in this report do not have any toxicity to the
cultured cells at concentrations used to inhibit HIV-1 replication,
they should have certain specificity in blocking Vif-Vif or Vif-Hck
interactions rather than inhibiting the activation of other protein
kinases used in maintaining the normal functions of the cells.
Because Vif is required for HIV-1 replication and Vif multimerization
is important for the function of Vif, the inhibitor(s) that blocks the
formation of Vif multimer should inhibit HIV-1 replication. A reliable
method was used to allow the peptides that inhibit Vif-Vif interaction
to effectively enter HIV-1-infected cells. Indeed, the peptides that
effectively inhibit Vif-Vif interaction potently inhibit HIV-1
replication in cell culture (Fig. 5B).
In this report, we have shown that the
161PPLP164 domain of Vif is a valuable target
for developing Vif inhibitors. Because the PXP
motif-containing peptides potently inhibit Vif-Vif interaction and
inhibit HIV-1 replication in nonpermissive cells, it is interesting to
further investigate the structural mechanisms of these peptide inhibitors and develop more potent nonpeptide Vif inhibitors, such as
peptidomimetic or small organic molecular inhibitors. Because of the
essential role of Vif in HIV-1 replication, we believe that the
development of these Vif inhibitors may represent a new strategy for
anti-AIDS therapy (38, 39).