A New Generation of Peptide-based Inhibitors Targeting HIV-1 Reverse Transcriptase Conformational Flexibility*

The biologically active form of human immunodeficiency virus (HIV) type 1 reverse transcriptase (RT) is a heterodimer. The formation of RT is a two-step mechanism, including a rapid protein-protein interaction “the dimerization step,” followed by conformational changes “the maturation step,” yielding the biologically active form of the enzyme. We have previously proposed that the heterodimeric organization of RT constitutes an interesting target for the design of new inhibitors. Here, we propose a new class of RT inhibitors that targets protein-protein interactions and conformational changes involved in the maturation of heterodimeric reverse transcriptase. Based on a screen of peptides derived from the thumb domain of this enzyme, we have identified a short peptide PAW that inhibits the maturation step and blocks viral replication at subnanomolar concentrations. PAW only binds dimeric RT and stabilizes it in an inactive/non-processive conformation. From a mechanistic point of view, PAW prevents proper binding of primer/template by affecting the structural dynamics of the thumb/fingers of p66 subunit. Taken together, these results demonstrate that HIV-1 RT maturation constitutes an attractive target for AIDS chemotherapeutics.

Human immunodeficiency virus type I (HIV-1) 4 is the primary cause of AIDS, a slow progressive and degenerative disease of the human immune system. Despite recent therapeutic developments and the introduction of highly active antiretroviral therapy, the rapid emergence of drug-resistant viruses against all approved drugs together with inaccessible latent virus reservoirs and side effects of currently used compounds have limited the efficacy of existing anti-HIV-1 therapeutics (1).Therefore, there is still an urgent need for new and safer drugs, active against resistant viral strains or directed toward novel targets in the replicative cycle, which will be useful for multiple drug combination.
HIV-1 reverse transcriptase (RT) plays an essential multifunctional role in the replication of the virus, by catalyzing the synthesis of double-stranded DNA from the single strand retroviral RNA genome (2,3). The majority of the chemotherapeutic agents used in AIDS treatments target the polymerase activity of HIV-1 RT, such as nucleoside reverse transcriptase inhibitors (NRTI) or non-nucleoside inhibitors (NNRTI) (4). The biologically active form of RT is an asymmetric heterodimer that consists of two subunits, p66 and p51, derived from p66 by proteolytic cleavage of the C-terminal RNase H domain (2,3,5).
The polymerase domain of both p66 and p51 subunit can be subdivided into four common subdomains: fingers, palm, thumb, and connection (6 -10). Determination of the threedimensional structures of RTs has revealed that, although the folding of individual subdomains is similar in p66 and p51, their spatial arrangement differs markedly (11). The p66 subunit contains both polymerase and RNase H active sites. The p66polymerase domain folds into an "open," extended structure, forming a large active site cleft with the three catalytic residues (Asp 110 , Asp 185 , and Asp 186 ) within the palm subdomain exposed in the nucleic acid binding site. The primer grip is responsible for the appropriate placement of the primer terminus at the polymerase active site and is involved in translocation of the primer-template (p/t) following nucleotide incorporation (12)(13)(14). In contrast, p51 predominantly plays a structural role in the RT heterodimer, by stabilizing the dimer interface thereby favoring loading of the p66 onto the p/t and maintaining the appropriate enzyme conformation during initiation of reverse transcription (15).
To propose new classes of HIV inhibitors, extensive efforts have been made in the design of molecules that target proteinprotein interfaces required for viral entry, replication, and maturation (16 -19). We (20,22) and others (5,24) have proposed that the heterodimeric organization of RT constitutes an interesting target for the design of new inhibitors. The formation of the active heterodimeric HIV-1 RT occurs in a two-step process. First a rapid association of the two subunits (dimerization step) via their connection sub-domains thereby yielding an inactive intermediate RT, followed by a slow conformational change of this intermediate (maturation step), generating the biologically active form of this enzyme. The maturation step involves contacts between the thumb of p51 and the RNase H of p66 as well as between the fingers of p51 with the palm of p66 (20 -23). NNRTIs have been reported to interfere with RT dimerization and to modulate the overall stability of the heterodimeric RT depending on their binding site on RT (24 -29). NNRTIs, including Efavirenz and Nevirapine, have been shown to promote HIV-1 RT maturation at the level of the Gag-Pol protein and to affect viral protease activation, resulting in the suppression of viral release from infected cells (30,31). Conversely, NNRTIs such as TSAO and BBNH derivatives act as destabilizers of RT subunit interaction (27).
We have demonstrated that preventing or controlling RT dimerization constitutes an alternative strategy to block HIV proliferation and has a major impact on the viral cycle (32). In protein-protein interactions the binding energy is not evenly distributed across the dimer interface but involves specific residues "hot spots" that stabilize protein complexes. We have shown that the use of small peptides targeting hot spot residues required for RT dimerization constitutes a new strategy to inhibit HIV-1 RT (16, 17) and have described a decapeptide "Pep-7"-mimicking p66/p51 interface that prevents RT dimerization by destabilizing RT subunit interactions and that blocks viral replication (23,32).
The thumb domain plays an important role in the catalysis and integrity of the dimeric form of RT, thereby constituting a potential target for the design of novel antiviral compounds (7,8,22). The p66-thumb domain is involved in p/t binding and polymerase activity of RT (7,8,13), and p51-thumb domain is required for the conformational changes associated with RT dimer maturation (22). We have designed a peptide, Pep-A, derived from a structural motif located between residues 284 and 300, corresponding to the end of helix ␣I, the loop connecting helices ␣I and ␣J and a part of helix ␣J. This peptide is a potent inhibitor of RT interfering with the conformational change associated with full activation of the enzyme. However, although it significantly blocks RT maturation in vitro, it lacks antiviral activity (22). In the present work, we have designed and evaluated a series of peptides derived from the thumb subdomain of RT using Pep-A as a template. We have identified a 17-residue peptide P AW , which constitutes a potent inhibitor of RT-polymerase activity of HIV-1 RT in vitro. We have demonstrated that P AW inhibits RT maturation and abolishes viral replication without any toxic side-effects. The characterization of the mechanism through which P AW inhibits RT, combining steady-state and pre-steady-state methods, together with sizeexclusion chromatography has revealed that P AW only binds dimeric RT and stabilizes it in an inactive/non processive dimeric conformation that prevents the proper binding of p/t. Taken together, these results demonstrate that conformational flexibility of HIV-1 RT during maturation constitutes an attractive target for AIDS chemotherapeutics.  was used for steady-state fluorescence titration and stoppedflow experiments, with 5Ј-TCCCTGTTCGGGCGCCACT-3Ј for the primer strand and 5Ј-TGTGGAAAATCTCATG-CAGTGGCGCCCGAACAGGGA-3Ј for the template strand. The sequence of the template strand corresponds to the sequence of the natural primer binding site (PBS) of HIV-1 (33). The primer was labeled at the 3Ј-end with 6-carboxyfluorescein on thymine base. Primer and template oligodeoxynucleotides were separately resuspended in water and diluted to 100 M in annealing buffer (25 mM Tris, pH 7.5, and 50 mM NaCl). Oligonucleotides were mixed together and heated at 95°C for 3 min, and then cooled to room temperature for 1 h.

Materials
Expression and Purification of HIV-1 RT Proteins-Histagged RTs were expressed and purified as previously described (23,34). Briefly, M15 bacteria (Qiagen) were separately transformed with all the constructs of p51 and p66 subunits. Cells were grown at 37°C up to ϳ0.3 A 595 , then cultures were cooled to 20°C and induced overnight with 0.5 mM isopropyl-1-thio-␤-D-galactopyranoside. Bacterial cultures expressing Histagged p66 subunit were mixed with cultures expressing the His-tagged p51 subunit to enable dimerization during sonication. For protein isolation and initial purification, the filtered supernatant was applied onto a Hi-Trap chelating column equilibrated with 50 mM sodium phosphate buffer, pH 7.8, containing 150 mM NaCl supplemented with 50 mM imidazole. The heterodimeric p66/p51 RT was eluted with an imidazole gradient and finally purified by size-exclusion chromatography on a HiLoad 16/60 Superdex 75 column equilibrated with a 50 mM Tris, pH 7.0, buffer containing 1 mM EDTA and 50 mM NaCl. Recombinant untagged HIV-1 BH 10 RT was expressed in Escherichia coli and purified as previously described (35). Highly homogeneous preparations from co-expression of the p66 and p51 subunits were stored in Ϫ80°C in buffer supplemented with 50% glycerol. Protein concentrations were determined at 280 nm using a molar extinction coefficient of 260 450 M Ϫ1 .cm Ϫ1 . Peptide Synthesis-Pep-A-derived peptides were purchased from GL Biochem, (Shanghai, China) and Genepep, SA (Prades le Lez, France). Pep-1 and P AW were synthesized using an (fluorenylmethoxy)carbonyl (Fmoc) continuous (Pionner, Applied Biosystems, Foster City, CA) starting from Fmoc-polyamide linker-poly(ethylene glycol)-polystyrene resin at a 0.05-mmol scale. Peptides were purified by semi-preparative reversedphase high performance liquid chromatography (HPLC) (C18 column Interchrom UP5 WOD/25 M Uptisphere 300 5 ODB, 250 mm ϫ 21.2 mm) and identified by electrospray mass spectrometry. P AW (1 mM) was coupled to FITC using maleimide-FITC (Molecular Probes. Inc., 5 mM) through overnight incubation at 4°C in PBS (Amersham Biosciences). Fluorescently labeled peptide was further purified by reversed-phase HPLC using a C18 reverse-phase HPLC column (Interchrom UP5 HDO/25 M Modulo-cart Uptisphere, 250 mm ϫ 10 mm) then identified by electrospray mass spectrometry.
RT-Polymerase Assay-RNA-dependent-DNA RT-polymerase activity was measured in a standard reaction assay using poly(rA)-(dT) 15 as p/t as previously described (5). Briefly, 10 l of RT at 20 nM was incubated at 37°C for 30 min with 20 l of reaction buffer (50 mM Tris, pH 8.0, 80 mM KCl, 6 mM MgCl 2 , 5 mM DTT, 0.15 M poly(rA-dT), 15 M dTTP, 0.3 Ci of [ 3 H]dTTP). For peptide evaluation, HIV-1 RT was incubated with increasing concentrations of peptide inhibitors for 23 h, and polymerase reaction was initiated by adding reaction buffer. Reactions were stopped by precipitation of nucleic acids with 5 ml of 20% trichloroacetic acid solution for 2 h on ice, then filtered using a multiwell-sample collector (Millipore), and washed with 5% trichloroacetic acid solution. Filters were dried at 55°C for 30 min, and radionucleotide incorporation was determined by liquid scintillation spectrometry. Data were fitted using a Dixon plot reporting the reciprocal of the velocity (1/v) as a function of inhibitor concentrations. K i values for the different peptides were estimated from the intercept on the concentration axis (36).
Steady-state Fluorescence Experiments-Fluorescence experiments were performed in buffer containing 50 mM Tris-HCl, pH 8.0, 50 mM KCl, 10 mM MgCl 2 and 1 mM DTT, at 25°C, using a SPEX-PTI spectrofluorometer in a 1-cm path-length quartz cuvette, with a band-pass of 2 nm for excitation and emission, respectively. Excitation was performed at 492 nm, and emission spectra were recorded from 500 to 600 nm. According to fluorescence experiments, a fixed concentration of FAM-labeled (19/36) p/t (50 nM) or of FITC-P AW (200 nM) was titrated with increasing protein concentrations from 5 nM to 1 M. Data were fitted as previously described (34,37), using a quadratic equation (GraFit, Erithacus Software).
HPLC Size-exclusion Chromatography-Chromatography was performed using one (Phenomenex S3000) or two HPLC columns in series (Phenomenex S3000 followed by Phenomenex S2000, both 7.5 mm ϫ 300 mm). Samples containing 3-10 M of RT or p51 were applied onto one or two HLPC columns and eluted with 200 mM potassium phosphate (pH 7.0) at a flow rate of 0.5 ml⅐min Ϫ1 (5).
Rapid Kinetic Experiments-Binding kinetics of p/t onto HIV-1 RT were performed with a FAM-labeled p/t in buffer containing 50 mM Tris-HCl, pH 8.0, 50 mM KCl, 10 mM MgCl 2 , and 1 mM DTT, using a stopped-flow apparatus (Hi-Tech Scientific, Salisbury, UK) at 25°C. A fixed concentration of FAMlabeled p/t (20 nM) was rapidly mixed with increasing concentrations of RT or RT⅐P AW complex formed at a 1/20 molar ratio (25-400 nM). 6-Carboxyfluorescein fluorescence was excited at 492 nm, and emission was detected through a filter with a cutoff at 530 nm. Data acquisition and analysis were performed using KinetAsyst 3 software (Hi-Tech Scientific), and traces were fitted according to a three exponential equation, as previously described (34). The rate constant for the first phase (k ϩ1 and k Ϫ1 ), corresponding to the formation of a RT⅐P AW ⅐p/t collision complex, was extrapolated from the slope and the intercept with the y axis of the plot of k obs1 versus RT concentrations. The k 2 (k ϩ2 ϩ k Ϫ2 ) and k 3 (k ϩ3 ϩ k Ϫ3 ) rate constants for the second and third phases corresponding to conformational changes of preformed RT⅐P AW ⅐p/t complex were directly obtained from the three exponential fitting.
Dissociation kinetics of HIV-1 RT were monitored by using bis-ANS as extrinsic probe Changes in bis-1 anilino-8 naphtalene sulfonate (bis-ANS) fluorescence provide a good signalto-probe variation in the exposure of the hydrophobic regions associated to RT dissociation in a time-dependent manner. 0.5 M RT was dissociated in the presence of 0.8 M bis-ANS, by adding 10% acetonitrile in the absence or in the presence of 10 M P AW . Kinetics of dissociation were monitored by following fluorescence resonance energy transfer between tryptophan residues of RT and bis-ANS. Excitation of RT-Trp residues was performed at 290 nm, and the increase of bis-ANS fluorescence emission at 490 nm was detected through a 420 nm cut-off filter. Data acquisition and analysis were performed using KinetAsyst 3 software (Hi-Tech Scientific), and traces were fitted according to a single-exponential equation.
Cell Culture, Transfection, and Indirect Immunofluorescence Microscopy-HeLa cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum at 37°C in a humidified atmosphere containing 5% CO 2 ). Cells were grown on glass coverslips to 75% confluency, then transfected with pcDNA3-p66RT plasmid using Lipofectamine 2000 reagent according to the manufacturer's instructions (Invitrogen). For colocalization experiments, cells were subsequently cultured for 32 h, before incubation with FITC-P AW or FITC-P AW /Pep-1 (complex obtained at a molar ratio 1/10) for 1 h. Coverslips were extensively rinsed with PBS, and cells were fixed in 4% paraformaldehyde for 10 min and permeabilized in 0.2% Triton. After saturation in PBS supplemented with bovine serum albumin 1% for 1 h, cells were incubated overnight with monoclonal 8C4 anti-HIV-1 RT antibody (AIDS Research Reference Reagent Program, National Institutes of Health, diluted 1:100 in PBS-bovine serum albumin 1%), followed by Alexa-555 anti-mouse (Molecular Probes). Immunofluorescence detection of HIV-1 RT and FITC-P AW was performed by epifluorescence microscopy using a PL APO 1.4 oil PH3 objective on a Leica DMRA 1999 microscope. Three-dimensional reconstitution of the 20 frames (interval, 0.3 m) realized from z stacking was performed using Imaris 6.0 software. ATCC H9 cells, stably transfected with pNL4.3 V-Rϩ plasmid and constitutively expressing Gag-Pol HIV-1 proteins (obtained from Dr. R. Marquet, Institut de Biologie Moléculaire et Cellulaire, France) were used for RT pulldown experiments. H9 cells were cultured in RPMI 1640 medium supplemented with 2 mM glutamine, 10% (w/v) fetal calf serum, 1% antibiotics (streptomycin 10,000 mg/ml, penicillin, 10,000 IU/ml) and G418 (1 mg/ml). P AW was incubated with 500 l of activated CNBr-activated Sepharose 4B beads at 4°C overnight. After centrifugation, supernatants were removed, and the beads were incubated with glycine, pH.8.0, for 2 h at 4°C with gentle stirring. The beads were then washed with 0.1 M sodium acetate buffer (pH 4.0), then 0.5 M bicarbonate buffer, and finally in PBS, three times each. The peptide bound to the beads were then saturated for 30 min in PBS/bovine serum albumin 0.1% and then incubated for 1 h at 4°C with equal amounts of H9 cells lysed for 30 min on ice in lysis buffer (Tris, 20 mM, pH 7.2, NaCl, 400 mM, EDTA, 1 mM, DTT, 1 mM, and Protease inhibitors, EDTA free) and sonicated 2 ϫ 5 s at 20%. Beads were washed with lysis buffer then twice with PBS, and the bound proteins were finally separated on 15% SDS-PAGE gel and analyzed by Western blotting using monoclonal 8C4 anti-HIV-1 RT antibody.
Antiviral Assay-The anti-HIV activities of the whole series of peptides were assayed according to previously described methods (38). Phytohemagglutinin-P (PHA-P)-activated peripheral blood mononuclear cells (PBMCs) were infected with the reference lymphotropic HIV-1-LAI strain (39). Virus was amplified in vitro on PHA-P-activated PBMCs. Viral stock was titrated using PHA-P-activated PBMCs, and 50% tissue culture infectious doses (TCID 50 ) were calculated using Kärber's formula (40). PBMCs were pretreated for 1 h with increasing concentrations of peptide (from 100 to 0.1 nM), then infected with 100 TCID 50 of the HIV-1-LAI strain. Peptides were maintained throughout the culture, and cell supernatants were collected at day 7 post-infection and stored at Ϫ20°C. Viral replication was measured by quantifying RT activity in cell culture supernatants. In parallel, cytotoxicity of the compounds was evaluated in uninfected PHA-P-activated PBMCs by colorimetric 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromite assay on day 7 (41). Experiments were performed in triplicate and repeated with another blood donor. Data analyses were performed using SoftMaxPro 4.6 microcomputer software: percentages of inhibition of RT activity or of cell viability were plotted versus concentration and fitted with quadratic curves; 50% effective doses (ED 50 ) and cytotoxic doses (CD 50 ) were calculated.

Design and Evaluation of Pep-A-derived Peptides-
We have previously demonstrated that the thumb domain of p51 subunit is involved in activation of heterodimeric RT and that a 17-residue peptide, Pep-A, corresponding to an extremely well conserved structural motif located between amino acids "284 and 300," can affect the maturation of HIV-1 RT (22). To optimize and identify major residues in Pep-A required for RT inhibition, a series of new peptides was derived from Pep-A sequence (RGTKALTEVIPLTEEAEC). First, the N-terminal arginine of Pep-A was removed to improve the solubility and facilitate the synthesis of Pep-A-derived peptides, and then additional peptides were then generated by performing an alanine scan on P1. Pep-A-derived peptides were evaluated using a standard polymerase RT assay, and K i values extrapolated using Dixon plot analysis (36) are reported in Table 1.
All peptides affected the polymerase activity of RT in a dosedependent manner, and four peptides, P1 (K i : 7.5 M), P6 (K i : 5.7 M), P10 (K i : 7.3 M), and P11 (K i : 7.0 M), possess an inhibition constant Ͻ10 M (Fig. 1). As a reference, we show that Pep-A inhibits RT-polymerase activity with an inhibition constant value of 35 M. Peptide analysis reveals that removing the Arg 1 residue in Pep-A increases the potency of the peptide (P1) 5-fold. In comparison to P1, mutation of residues Gly 1 , Ala 4 , Glu 7 , and Leu 11 into alanine significantly affects the potency of the peptide suggesting that the side chains of these residues are required for the interaction with RT. The nature of the side chain of Glu 7 seems to be a major requirement for the interaction with RT, because its substitution by alanine (P8), reduces the efficiency of the peptide 8-fold. In contrast, Lys 3 , Thr 6 , Val 8 , and Glu 14 residues have a minor impact because their mutation into alanine only reduces their potency by a factor of 2. Interestingly, the hydrophobic character of Ala 4 and Val 8 side chains plays a role in the binding of the peptide to RT, and reducing their length affects the potency of the corresponding peptides to inhibit RT 2.7-and 2-fold, respectively. Taking into account that Trp residues are generally involved in stabilization of protein-protein interfaces, the two residues Ala 4 and Val 8 were mutated into Trp, to favor the binding of the peptide to RT. As shown in Fig. 1, the corresponding peptide P AW significantly inhibits RT polymerase activity with an inhibition constant (K i ) of 0.7 M, revealing that mutation of these two residues into Trp improves peptide efficiency 50-fold over Pep-A and 10-fold in comparison to the best lead peptide from the Ala scan (P6) ( Fig. 1 and Table 1).
Antiviral Potency of Pep-A-derived Peptides-Antiviral activity of the five peptide leads (P1, P6, P10, P11, and P AW ) was

TABLE 1 Sequences and inhibition of polymerase activity of HIV-1 RT by Pep-A derived peptides
a RT polymerase activity was measured as described under "Experimental Procedures." The inhibition constants K i were calculated from Dixon plots, and reported data correspond to the mean of three separate experiments. evaluated on PHA-P-activated PBMCs infected with HIV-1-LAI. Results were reported as 50% efficient concentration (EC 50 ) and selectivity index corresponding to the ratio between EC 50 and the cytotoxic concentration (CC 50 ) inducing 50% death of uninfected PBMCs and relative to Pep-A and P8 (Table  2). To avoid any limitation due to the poor ability of peptides to cross cellular membranes, they were associated to the peptidebased nanoparticle delivery system Pep-1, at a 1/10 molar ratio.
Pep-1 has been successfully used for the delivery of peptides and proteins into numerous cell lines as well as in vivo (42,43). The inability of free peptides to block viral replication is directly associated to their poor cellular uptake as reported in Fig. 2A for fluorescently labeled peptide (FITC-P AW ). In contrast, when complexed at a molar 1/10 ratio with the Pep-1 delivery system, FITC-P AW rapidly (in Ͻ1 h) enters cells (Fig. 2B). FITC-P AW localization and RT⅐P AW interaction were characterized using three-dimensional reconstitution of frames from z stacks. Three-dimensional image analysis reveals that P AW does not enter the nucleus and partially localizes with RT at the periphery of the nucleus (Fig. 2, E and F).
When associated with Pep-1, peptides P1, P6, P10, and P11 block viral proliferation with IC 50 values in the low micromolar range, which correlates with their ability to inhibit HIV-1 RT in vitro ( Table 2). In contrast, in agreement with previous findings no antiviral activity was observed with Pep-A when associated to Pep-1 (22). When complexed with Pep-1, P AW exhibits a marked antiviral activity with an EC 50 of 1.8 nM and a therapeutic/selectivity index of ϳ550. The 44-and 161-fold greater potency of P AW over peptides harboring mutations at Glu 7 (P8) or lacking Trp residues (P1) confirms the requirement of these residues for targeting RT both in vitro and in cellulo. P AW constitutes a powerful inhibitor of polymerase activity and possesses a very potent antiviral activity without any toxic effect.
We therefore, further investigated its mechanism of action on RT. P AW Peptide Interacts with HIV-1 RT in a Cellular Context-To confirm that P AW targets HIV-1 RT in a cellular context, we further investigated its ability to form stable complexes with HIV-1 RT expressed in cells by using pulldown experiments. The peptides P AW and P8, covalently associated with CNBr-Sepharose beads, were incubated in the presence of cell lysates of H9 cells expressing Gag-Pol gene products of HIV-1. Analysis of the presence of RT by Western blotting revealed than only P AW was able to form a stable complex with RT in a cellular context and to retain RT on beads (Fig. 2G). In contrast, no RT was associated to free or P8 beads.
Binding of P AW Peptide to the Dimeric Form of HIV-1 RT-To further understand the mechanism through which P AW inhibits RT, we investigated its potency to interact with the dimeric form of HIV-1 RT in the absence or presence of DNA/ DNA p/t. The binding of P AW to RT was monitored using a fluorescently labeled peptide (FITC-P AW ). We first evaluated the impact of P AW labeling on the C-terminal cysteine FIGURE 2. HIV-1 RT interacts with P AW in cultured cells. A-F, Cellular localization of P AW and its interaction with HIV-1 RT in cellulo was monitored using HeLa cells expressing RT transfected with FITC-P AW ⅐Pep-1 complex formed at a 1/10 molar ratio. HIV-1 RT (Alexa 555 secondary antibody) and FITC-P AW were visualized, respectively, through a Cy3 and a GFP filter. HeLa cells transfected with free FITC-P AW (A) or complexed with Pep-1 (B). Cultured HeLa cells were transfected (D) with pcDNA3-p66RT. HeLa cells co-transfected with both pcDNA-p66RT and FITC-P AW ⅐Pep-1 at a 1/10 molar ratio. The RT⅐P AW co-localization was analyzed by the threedimensional image reconstitution with Imaris 6.0 software of 20 frames from z stacks (E and F). RT, P AW , and nuclear staining with Hoechst are reported in red, green, and blue, respectively. Global view (D), three-dimensional image analysis of a selected cell (red arrow) reveals that P AW and RT localize in the cytoplasm at the periphery of the nucleus. F, zoom of the box reported in panel E. G, interaction between P AW and HIV-1 RT detected in a CNBr pulldown assay. Experiments were performed as described under "Experimental Procedures." 30 g (total protein) per lane were separated on 15% SDS-PAGE and subjected to Western blotting using rabbit anti-RT antibody. Lanes correspond to control free beads, P8 and P AW beads, and total proteins loaded on the gel, respectively. with an FITC probe on its ability to inhibit RT polymerase activity. As reported in Table 1, FITC-P AW blocks RT polymerase activity with a K i of 2.7 Ϯ 0.7 M, 3.8-fold greater than for P AW , suggesting that labeling has only a minor effect on P AW inhibitory property. As reported in Fig. 3A, upon binding to the dimeric form of RT, the fluorescence of FITC-P AW was quenched by 39% and analysis of the titration curves revealed that P AW tightly binds heterodimeric RT with a dissociation constant (K d ) of 33 Ϯ 10 nM. When RT is first incubated with DNA/DNA p/t (18/36-mer), the quenching of FITC-P AW fluorescence associated with its binding was of 57%, and the affinity of FITC-P AW for RT increased 5-fold (K d : 7.1 Ϯ 2.8 nM), suggesting that the presence of p/t on RT facilitates the binding of P AW . The association of unlabeled P AW to RT was also evaluated by monitoring changes in fluorescently labeled p/t bound to RT (Fig. 3B). Binding of P AW results in a 39% quenching of fluorescence and a K d value of 40 Ϯ 18 nM was estimated from the titration binding curve. The 5.6-fold lower K d of labeled FITC-P AW over unlabeled peptide suggests that the dye contacts RT and stabilizes the peptide within its binding site.
Because both Trp 24 and Phe 61 located on the fingers domain of p66 subunit have been reported to be involved in the control of p/t binding and in the dynamics of the thumb-fingers subdomain interactions (34,44,45), we then evaluated the binding of P AW on RT harboring single Phe 61Gly and double Phe 61Gly and Trp 24Gly mutations on the p66 subunit. In comparison to wildtype RT, the affinity of P AW was reduced 6-fold (K d : 207 Ϯ 62 nM) for p66 F61G /p51 wt and 4.5-fold (K d : 149 Ϯ 38 nM) for p66 DM /p51 wt (Fig. 3A).
Effect of P AW Peptide on Primer/Template Binding to HIV-1 RT-The impact of P AW peptide on the ability of HIV-1 RT to bind p/t was then investigated at both steady-state and presteady-state levels using a 19/36-mer p/t labeled at the 3Ј-end of the primer with FAM-derivative as previously described (34,37). As reported in Fig. 4A, the presence of a saturating concentration of P AW (10 M) decreases the affinity of fluorescently labeled p/t for RT 4.5-fold with a K d value of 99 Ϯ 40 nM in comparison to 22 Ϯ 5 nM obtained in the absence of P AW . The binding of unlabeled p/t induces a 42% change in the fluorescence of P AW -FITC pre-bound to RT and leads to a similar K d value of 66.5 Ϯ 19 nM (Fig. 4A). These results suggest that p/t interacts close to P AW binding site on RT, inducing a change in the orientation of FITC linked to P AW , but does not share the same binding site.
RT⅐p/t pre-steady-state binding kinetics follow a three-step mechanism in the presence or in the absence of P AW , including a rapid diffusion controlled second order step leading to the formation of the RT⅐p/t collision complex, followed by two slow, concentration-independent, conformational changes (34). The plot of the pseudo-first order rate constant for the initial association of the p/t with RT against RT concentration is linear. In the absence of P AW , k ϩ1 and k Ϫ1 rate constant values of 4.23 ϫ 10 8 M Ϫ1 ⅐s Ϫ1 and 29.9 s Ϫ1 were calculated from the slope and the intercept with the y axis of the graph (Fig. 4, B and  C). Analysis of the second and third slow phases yielded rate constants of k 2 ϭ 5.8 s Ϫ1 and k 3 ϭ 0.76 s Ϫ1 for RT. The presence of P AW did not alter the overall K d1 for the initial formation of the RT⅐p/t complex as both the "on" (k ϩ1 ϭ 1.05 ϫ 10 8 M Ϫ1 ⅐s Ϫ1 ) and the "off" (k Ϫ1 ϭ 7.9 s Ϫ1 ) rates of the first step are decreased by ϳ4-fold. In contrast, the presence of P AW on RT significantly reduced the rate constants of the slow conformational steps (k 2 ϭ 1.99 s Ϫ1 and k 3 ϭ 0.22 s Ϫ1 ), affecting the proper binding of the p/t (Fig. 4, B and C).
Effect of P AW on the Stability and Dimerization of HIV-1 RT-The impact of P AW on the stability and formation of heterodimeric RT was investigated in detail by size-exclusion chromatography as previously described (17). As reported in Fig. 5A, heterodimeric RT incubated or not in the presence of an excess of P AW (100 M) for 1 h and 30 min at room temperature, is fully dimeric and eluted as a single peak at 16.7 min. The interaction of P AW with RT was monitored by size-exclusion chromatography using HIV-1 RT preincubated with FITC-P AW . Chromatography analysis reveals that FITC-P AW co-elutes with heterodimeric RT in a single peak at 16.7 min (Fig. 5A), demonstrating that P AW binds heterodimeric RT and does not induce RT dissociation. We A fixed 200 nM concentration of FITC-P AW was titrated with increasing concentrations of HIV-1 RTs or RT⅐p/t. The binding of P AW to RT was monitored by following the quenching of extrinsic P AW fluorescence at 512 nm, upon excitation at 492 nm. B, titration of P AW binding to RT⅐FAM⅐p/t. A fixed 20 nM concentration of RT⅐p/t was titrated with increasing concentrations of P AW . The binding of P AW to RT was monitored by following the quenching of extrinsic fluorescence of FAM-labeled p/t at 512 nm, upon excitation at 492 nm. K d values were calculated using a quadratic equation and correspond to the mean of at least three separate experiments.
then evaluated the ability of P AW to interact with p66 or p51 monomeric forms. Experiments performed with a partially dissociated RT⅐P AW (50%) complex by 10% acetonitrile, showed that P AW remains associated only with the dimeric fraction of RT and does not bind monomeric p66 or p51 subunits, which are eluted at 17.5 min and 18.2 min, respectively (Fig. 5B).
We then investigated the ability of P AW to prevent HIV-1 RT dimerization. Dissociation of RT was achieved at room temperature with 17% acetonitrile, and then association of the subunits was induced by a 10-fold dilution of the sample in an acetonitrile-free buffer in the absence or presence of 100 M of P AW . At this concentration (1.7%) of acetonitrile no dissociation of RT could be detected. As shown in Fig.  6A, heterodimeric RT was fully reassociated 5 h after dilution in an acetonitrile-free buffer, both in the absence or in the presence of P AW (100 M), indicating that P AW does not block RT dimerization (Fig. 6A). The impact of P AW was further investigated on the kinetics of RT dimerization. The level of dimeric RT was evaluated 30 min and 2 h, respectively, after dilution in free acetonitrile buffer by size-exclusion chromatography (Fig. 6, B and C). In the presence of P AW 21 and 59% of dimeric RT was quantified after 30 min and 2 h, respectively (Fig. 6B). In comparison only 16% (30 min) and 29% (2 h) of dimeric RT were detected in the absence of peptide (Fig. 6C), suggesting that the presence of P AW favors the kinetics of RT dimerization. P AW Peptide Favors Dimerization of the Small p51 Subunit-p51 subunits are mainly monomeric, and dissociation constants for p51/p51 homodimer have been reported to be either in the micromolar (25) or millimolar (5) range depending on the technology used to quantify the interactions. We have investigated the ability of P AW to favor p51/p51 dimerization by size exclusion chromatography, using two HPLC columns in series. Experiments were performed at a p51 concentration of 3.5 M at which it is entirely monomeric and elutes as a single peak at 32.7 min (Fig. 7). Monomeric p51 (3.5 M) was incubated in the presence of FITC-labeled P AW (20 M) for 1 h at room temperature then analyzed by size exclusion chromatography. As reported in Fig. 7, in the presence of fluorescently labeled P AW 4.6% of p51 are dimeric and associated to P AW , suggesting that FIGURE 4. Impact P AW peptide on the binding of primer/template to HIV-1 RT. A, titration of fluorescently labeled p/t binding to RT (E) or RT⅐P AW (F) and of FITC-P AW /RT binding to p/t (OE). A fixed 50 nM concentration of fluorescently labeled p/t was titrated with increasing concentrations of RT or RT⅐P AW . The binding of p/t to RT was monitored by following the quenching of p/t extrinsic fluorescence at 512 nm, upon excitation at 492 nm. A fixed 100 nM concentration of FITC-P AW ⅐RT complex was titrated by increasing concentrations of p/t (18/36). The binding of FITC-P AW ⅐RT to p/t was monitored by following the quenching of P AW extrinsic fluorescence at 512 nm, upon excitation at 492 nm. K d values were calculated using a quadratic equation as previously described (20) and correspond to the mean of at least three separate experiments. Kinetics of binding of fluorescently labeled p/t to RT (B) and RT⅐P AW (C). Typical stopped-flow time courses are reported, where a fixed 20 nM concentration of FAM-labeled p/t was rapidly mixed with 100 nM of RT (B) or RT⅐P AW (C). Data collection acquisition and analysis were performed using KinetAsyst 3 software, and kinetics was fitted using a threeexponential equation. D, secondary plot of the dependence of the fitted pseudo-first order rate constants for the first phase on RT (E) or RT⅐P AW (F) concentration. P AW promotes p51/p51 homodimer and only p51/p51 homodimer.
P AW Peptide Prevents HIV-1 RT Dissociation-Finally, the impact of P AW on HIV-1 RT stability and dissociation were investigated at the steady state level by size exclusion chroma-tography and at the pre-steady-state level by stopped-flow rapid kinetics. HIV-1 RT was preincubated in the presence of 100 M P AW , for 2 h, prior dissociation with 17% or 10% of acetonitrile, and the level of dimeric form was then assessed by size exclusion chromatography and the rate of dissociation by presteady-state kinetics. As reported in Fig. 8A, the presence of P AW protected RT from the acetonitrile dissociation, because 17% remained dimeric whereas "free" RT was completely dissociated with 17% acetonitrile.
The protection by P AW of acetonitrile-associated RT dissociation was further investigated by monitoring pre-steady-state dissociation kinetics of HIV-1 RT, using bis-ANS as an extrinsic probe (46). Binding of bis-ANS to dissociated RT resulted in a large increase in the fluorescence of the probe due to noncovalent interactions of bis-ANS to exposed hydrophobic surfaces on RT subunits, therefore providing a good signal for following RT dissociation in a time-dependent manner. Experiments were performed by adding bis-ANS to HIV-1 RT prior dissociation of the enzyme by 10% acetonitrile and monitoring FRET between exposed Trp of RT and Bis-ANS. As reported in Fig. 8B, the kinetics of increased ANS fluorescence upon dissociation of RT in the absence of P AW followed a single-exponential reaction, with a dissociation rate constant k dis of 5.30 Ϯ 0.01 s Ϫ1 , which was reduced 3.8-fold (k dis ϭ 1.42 Ϯ 0.007 s Ϫ1 ), when RT was incubated with P AW .
Optimization of P AW and Selection of a Minimal Inhibitory Peptide Motif-Taken together our results demonstrate that P AW constitutes a potent conformational inhibitor of RT and exhibits a potent antiviral activity. To define the minimal peptidic sequence for RT inhibition, new peptides derived from P AW were designed and evaluated ( Table 3). As the interaction between P AW and RT seems to involve both the N-terminal part and Trp residues of the peptide, the peptidic sequence was shortened at the N-and/or C-terminal extremities, and the positional effect of the Trp was evaluated. All peptides were tested in standard RT assays (Table 3) and were evaluated on PBMCs infected by HIV-1-LAI (Table 2). Reducing P AW sequence by two residues at the N terminus reduced efficiency 2.5-fold (P26: K i ϭ 1.8 Ϯ 0.7 M). In contrast, the five last residues at the C terminus of P AW can be removed without affecting its potency to inhibit RT polymerase activity or to block viral replication (P24: K i ϭ 0.7 Ϯ 0.05 M and EC 50 ϭ 2.3 nM). That P18 does not inhibit RT polymerase activity confirms that the Trp residues form the major interface with RT. Moving  respectively. Interestingly, removing the last two residues of P AW increases its efficiency 14-fold (P27: K i ϭ 50 Ϯ 0.01 nM) and is also associated with an increase in its antiviral activity with an IC 50 Ͻ 0.32 nM and a therapeutic/selectivity index Ͼ 3100.

DISCUSSION
Targeting the conformational flexibility of heterodimeric RT has provided new concepts for the design of drugs active on viruses resistant to currently used RT inhibitors (5,17,26,28,47). RT activation involves a two-step dimerization process initiated by a rapid monomer/monomer association generating an inactive intermediate heterodimer, followed by a slow isomerization yielding the biologically active enzyme (5,20). Considering that HIV-1 RT is extremely stable (25,46,48), selection of compounds that are able to dissociate the complex remains challenging. In contrast, because maturation of RT requires less energy than dimerization, targeting conformational changes involved is a very attractive approach for the design of novel antiviral compounds. Maturation of the inactive intermediate heterodimer corresponds to conformational changes involving interactions between the thumb of p51 and the RNase H of p66 and between the fingers of p51 and the palm of p66 (20). We previously demonstrated that the thumb domain of p51 plays a major role in RT maturation and that a synthetic peptide (Pep-A) derived from this domain selectively inhibited activation of HIV-1 RT (22). In the present work, we report the design of a new generation of peptide inhibitors derived from the thumb domain and have identified a lead peptide P AW that efficiently blocks both maturation of RT and viral replication. P AW Peptide Preferentially Binds Dimeric RT in the "Open" Conformation-From size exclusion chromatography, we clearly demonstrated that P AW only binds dimeric forms of RT (p66/p51 and p51/p51). Moreover, as already reported for Pep-A (22), P AW does not induce heterodimer dissociation nor prevent the monomer/monomer association, and instead significantly increases stability of the heterodimer and favors dimerization. According to the twostep process mechanism, we propose that P AW blocks RT maturation by stabilizing the inactive intermediate of RT in a nonprocessive conformation.
Determination of crystal structures of the HIV-1 RT associated or not with a p/t has revealed that the binding of p/t to RT triggers major conformational changes in the overall structure of the enzyme, including the increase in the compactness together with conversion of RT from a "closed" to an open conformation (7)(8)(9). The structure RT adopts two conformational states: a closed conformation stabilized by interactions between fingers and thumb domains of p66 and an open conformation associated with a change in the orientation of the thumb domain and a shift of the fingers domain, which are induced by p/t binding (8,9). We demonstrate that P AW tightly binds RT preferentially in the open conformation, because its affinity is increased 5-fold in the presence of p/t. The dynamics of the thumb and fingers domains of p66 and the conformational changes of RT associated with p/t binding exposes the binding site of P AW , was strengthened by the fact that mutation of Phe 61 into glycine on the fingers domain of p66 subunit altered binding of P AW to RT (6-fold). Phe 61 is located in the fingers domain and together with Trp 24 and Arg 78 is involved in the stabilization of the closed conformation of RT, by contacting the loop between helices ␣I and ␣J of the thumb domain. Given that mutation of Phe 61 favors the open conformation of RT (34), but dramatically reduces the affinity of P AW for RT, suggests that this residue is directly involved in the binding of P AW . In addition, the P AW binding site is located close to this residue on the thumb or the fingers domain of p66.   (20). We propose that P AW interacts directly with the intermediate inactive form, which stabilizes in a non-processive conformation. From a mechanistic point of view, P AW acts as non-competitive inhibitor affecting conformational changes required for proper folding of the p/t binding site. P AW does not displace the RT⅐p/t complex but reduces both "off" and "on" rates of the collisional binding of p/t to RT; it did not affect the overall dissociation constants of the collisional step, with k 1 values of 70.4 nM and 70.6 nM obtained in the presence or the absence of P AW , respectively. In contrast, P AW dramatically affects the proper binding and conformational changes that place correctly the p/t for catalysis. The corresponding rate constants are reduced by 3-fold (k 2 ) and 3.4-fold (k 3 ), respectively. Taken together that P AW binds heterodimeric and homodimeric RTs (p66/p51 and p51/p51), preferentially in the open conformation, and that it specifically inhibits polymerase activity and not RNase H, demonstrates that P AW interacts with the inactive intermediate form of RT and prevents conformational changes required for the proper folding of the p/t binding site.
Similarly, NNRTIs are non-competitive inhibitors that block RT in a non-processive conformation (24 -28). NNRTIs bind near the RT-polymerase catalytic site and affect the dynamics of thumb-finger domain interaction on p66 subunit and maintain RT in an open conformation (6,8,10). Although the molecular mechanism by which NNRTIs inhibit RT is not entirely clear, evidence reports that binding of NNRTIs restricts the mobility of the thumb domain, slowing or preventing p/t translocation and thereby inhibiting elongation of nascent DNA. NNRTIs such as Efavirenz favor RT dimerization in vitro and in cultured cells (24), but in contrast to P AW , they improve the binding of p/t (35,49), which excludes that P AW and NNRTI binding sites overlap and favors a P AW binding site close to the interface between the thumb and fingers domain of p66. P AW Is a Potent Antiviral Compound-When associated with Pep-1-based nanoparticles, P AW is a potent non-toxic inhibitor of viral replication (EC 50 : 1.8 nM). This peptide constitutes a major improvement in comparison to Pep-A, which is 50-fold less potent in vitro on RT activity and does not exhibit any antiviral activity in the same delivery conditions. We demonstrate that, when delivered into cells using Pep-1, P AW interacts with RT in both cells expressing only p66 and in the context of the full Pol-polyprotein. Taken together, this result, combined with the fact that P AW EC 50 values on HIV-LAI are similar to its dissociation constant for RT, confirms that P AW inhibition occurs via conformational changes on RT and not through direct inhibition of polymerase activity. The analysis of P AW sequence has revealed that essential residues involved in RT binding and antiviral activity are located in the N terminus of the peptide. In particular, Glu 7 , Leu 11 , Trp 4 , and Trp 8 are required for binding of P AW to RT. The hydrophobic character of the side chain of Trp residues at positions 4 and 8 plays a major role in stabilizing the P AW ⅐RT complex. We show that P AW can be reduced to 12 residues (P24) without affecting either its in vitro or in cellulo potency. Interestingly, removing the last two residues of P AW (P27) increases in vitro and antiviral efficiency 12-fold and 10-fold, respectively. These results suggest that, although the Trp residues are key residues for the potency of the peptide, residues 12-14 in the sequence of P AW are also required to stabilize P AW in its binding site.

CONCLUSIONS
In the present work, we have demonstrated that the dynamics of the finger/thumb domains of p66 play an essential role in the stabilization and maturation of heterodimeric HIV-1 RT. As such, we have established a proof of concept that targeting conformational changes required for RT flexibility can lead to highly potent antiviral molecules. We have identified a new RT inhibitor, P AW , that alters finger/thumb dynamics and maintains RT in a non-processive conformation, by altering the proper binding of p/t to RT. Development of this type of inhibitor together with a better knowledge of its mechanism at the viral level will provide new perspectives for designing specific inhibitors of the "niche" of highly resistant strains.