Potent Suppression of Viral Infectivity by the Peptides That Inhibit Multimerization of Human Immunodeficiency Virus Type 1 (HIV-1) Vif Proteins*

Virion infectivity factor (Vif) is essential for the replication of human immunodeficiency virus type 1 (HIV-1) in vivo , but its function remains uncertain. Re-cently, we have shown that Vif proteins are able to form multimers, including dimers, trimers, or tetramers. Because the multimerization of Vif proteins is required for Vif function in the viral life cycle, we propose that it could be a novel target for anti-HIV-1 therapeutics. Through a phage peptide display method, we have identified a set of 12-mer peptides containing a P X P motif that binds to HIV-1 Vif protein. These proline-enriched peptides potently inhibited the Vif-Vif interaction in vitro . We have also screened a set of synthesized Vif peptides (15-mer), which covers all the amino acids of the HIV-1 Vif protein sequence, for their ability to inhibit the Vif-Vif interaction in vitro . We demonstrated that Vif-derived proline-enriched peptides that contain the 161 PPLP 164 domain are able to inhibit the Vif-Vif interaction. Conversely, the deletion of the 161 PPLP 164 domain of Vif protein will significantly impair the capability of Vif proteins to interact with each other, indicating that the 161 PPLP 164

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)(4)(5)(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)(8)(9)(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)(13)(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)(24)(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 inter-action, 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. 35 S-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. 35 S-Labeled Vif or its mutant proteins were synthesized by in vitro transcription and translation utilizing SPT3 kits (Novagen) in the presence of [ 35 S]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.

Plasmid Constructions, Expression of GST Fusion Proteins, and Synthesis of
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, 10 11 phages were first absorbed with GST followed by mixing with 3 ml of GST-Vif attached to glutathioneagarose 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 l of Tris-buffered saline, 0.02%NaN 3 . 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 l of 100 g/ml GST and GST-Vif in 0.1 M NaHCO 3 , 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 NaHCO 3 , 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 10 11 to 10 5 ), 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 proteinprotein interactions. The GST fusion proteins on agarose beads are first generated as described previously except without elution with 5 mM glutathione (20). The 35 S-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, 35 S-labeled Vif proteins were added to GST-Vif/GST-Hckconjugated 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 35 Slabeled 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 g/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 ϫ 10 6 ) were mixed with HIV-1 NL4 -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 M. 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).

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 phagedisplayed 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  (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 IC 50 for the inhibition of Vif multimerization is 7.43 M for VMI 7 and 4.84 M for VMI 9 (Fig. 1B). A Vif-derived 12-mer peptide, 155 KPKQIKPPLPSV 166 (Vif-(155-166)), which is originated from the proline-enriched C terminus of Vif, also has the similar inhibition activity upon Vif-Vif interaction (IC 50 ϭ 17.39 M).
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 153 LITPKKIKPPLPSVT 167 and 157 KKIKPPLPSVTKLTE 171 , which contain the 161 PPLP 164 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 161 PPLP 164 domain, are also able to moderately inhibit Vif-Vif interaction, suggesting that the amino acid residues at this FIG. 1. A, relative affinity comparisons between PXP motif-containing peptides. GST fusion protein (100 g/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 Ϫ7 , 5 ϫ 10 Ϫ7 , 10 Ϫ6 , 10 Ϫ5 , 10 Ϫ4 , 10 Ϫ3 M) were added to the mixture of 35 Slabeled Vif and GST-Vif-conjugated agarose beads. The 35 S-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 Phos-phorImager. IC 50 is the concentration of the peptides inhibiting 50% 35 S-labeled Vif binding to GST-Vif in GST pull-down assays.
region could also participate in the Vif-Vif interaction.
The 161 PPLP 164 Domain Is Required for Vif Multimerization-Because the PXP motif is also shared by Vif in the 161 PPLP 164 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 161 PPLP 164 domain is required for Vif-Vif interaction. To this end, site-directed mutagenesis was performed to delete 161 PPLP 164 , 151 AALIKPKQIKPPLP 164 , and the Vif C terminus (151-192). The mutants were expressed with an in vitro translation system in the presence of [ 35 S]methionine or expressed as GST fusion proteins. The 35 S-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 151 AALIKPKKIKPPLP 164 have decreased binding to Vif (Fig. 3) (22). Vif mutant protein just deleted at the 161 PPLP 1 64 domain also showed a decrease in its binding to Vif. Interestingly, the protein-protein interac-tions between the Vif mutants deleted at 161 PPLP 164 domain were significantly decreased (Fig. 3). These data indicated that the 161 PPLP 164 domain is required for Vif-Vif multimerization.
PXP Motif-containing Peptides Inhibit Vif-Hck Binding-It has been demonstrated that Hck kinase can also bind with Vif through the 161 PPLP 164 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, 35 S-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).
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, RQIKIWFQNR-RMKWKK) has been widely used to effectively carry peptides  35 S-labeled Vif-and GST-Vif-conjugated agarose beads. The 35 S-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 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 M (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.
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 M, 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). DISCUSSION We have demonstrated that the 151 AALIKPKQIKPPLP 164 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 161 PPLP 164 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 161 PPLP 164 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 positivecharged amino acids in the 151-164 region at the C terminus, and Vif binds to Hck also through the 161 PPLP 164 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 161 35 Slabeled 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. 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 161 PPLP 164 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). 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, phasecontrast; C, VMI 9, fluorescence; D, VMI 9, phase contrast. B, Ant fusion peptides inhibit HIV-1 replication. H9 cells were infected by HIV-1 NL4 -3 virions at 37°C for 4 h. The infected H9 cells (1 ϫ 10 6 ) were then cultured in duplicate in 2 ml of RPMI 1640 medium plus 10% fetal bovine serum without or with peptides (50 M). 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.