Rationally Designed Peptides as Efficient Inhibitors of Nucleic Acid

Article Cite This: Biochemistry XXXX, XXX, XXX−XXX

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Rationally Designed Peptides as Efficient Inhibitors of Nucleic Acid Chaperone Activity of HIV‑1 Nucleocapsid Protein Volodymyr Shvadchak,†,# Sarwat Zgheib,†,# Beata Basta,†,# Nicolas Humbert,† Johannes Langedijk,‡ May C. Morris,§ Stefano Ciaco,† Ouerdia Maskri,∥ Jean-Luc Darlix,† Olivier Mauffret,∥ Philippe Fosse,́ ∥ Eleó nore Reá l,*,† and Yves Meĺ y*,† †

Laboratory of Bioimaging and Pathologies, UMR 7021 CNRS, Université de Strasbourg, 74 route du Rhin, 67401 Illkirch, France Pepscan Therapeutics BV, Lelystad, The Netherlands § Institut des biomolécules Max Mousseron, CNRS, UMR 5247, Université de Montpellier Faculté de Pharmacie, 15 av Charles Flahault 34093 Montpellier, France ∥ LBPA, ENS Paris Saclay, CNRS, Université Paris-Saclay, 94235, Cachan Cedex, France

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‡

S Supporting Information *

ABSTRACT: Due to its essential roles in the viral replication cycle and to its highly conserved sequence, the nucleocapsid protein (NCp7) of the human immunodeficiency virus type 1 is a target of choice for inhibiting replication of the virus. Most NCp7 inhibitors identified so far are small molecules. A small number of short peptides also act as NCp7 inhibitors by competing with its nucleic acid (NA) binding and chaperone activities but exhibit antiviral activity only at relatively high concentrations. In this work, in order to obtain more potent NCp7 competitors, we designed a library of longer peptides (10−17 amino acids) whose sequences include most of the NCp7 structural determinants responsible for its specific NA binding and destabilizing activities. Using an in vitro assay, the most active peptide (pE) was found to inhibit the NCp7 destabilizing activity, with a 50% inhibitory concentration in the nanomolar range, by competing with NCp7 for binding to its NA substrates. Formulated with a cell-penetrating peptide (CPP), pE was found to accumulate into HeLa cells, with low cytotoxicity. However, either formulated with a CPP or overexpressed in cells, pE did not show any antiviral activity. In vitro competition experiments revealed that its poor antiviral activity may be partly due to its sequestration by cellular RNAs. The selected peptide pE therefore appears to be a useful tool for investigating NCp7 properties and functions in vitro, but further work will be needed to design pE-derived peptides with antiviral activity. polyprotein precursor,5 is a small, basic nucleic acid (NA) binding protein with two CCHC type zinc-fingers connected by a short flexible basic linker. NCp7 binds to NAs via sequence specific and nonspecific interactions6,7 implicating the hydrophobic platform at the top of folded zinc fingers and the numerous basic residues distributed throughout the NCp7 sequence.8−11 NCp7 is also a NA chaperone that facilitates the remodeling of NA structures into the most thermodynamically stable conformations through its NA destabilization, aggregation, and rapid interaction kinetics.12−17 Mature NCp7 thus plays a critical role in ensuring the specificity and efficiency of reverse transcription.18,19 In addition, NCp7 is thought to stimulate the IN-mediated concerted integration of HIV-1 genome into the targeted cell genome.20 The nucleocapsid (NC) domain in the Gag polyprotein retains some chaperoning activity, participating in RNA dimer formation

The acquired immune deficiency syndrome (AIDS) pandemic has already caused the death of more than 35 million people, while nearly 37 million persons are living with human immunodeficiency virus type 1 (HIV-1) in 2016 according to the World Health Organization. The development of highly active antiretroviral therapy (HAART), which consists of the combination of drugs targeting essentially the three viral enzymes, i.e., reverse transcriptase (RT), protease (PR), and integrase (IN), has resulted in a drastic reduction of mortality and morbidity.1 However, due to the high sequence variability of HIV-1, virus strains resistant to these drugs have emerged2 with major consequences on long-term treatment strategies and therapeutic outcomes for people living with the virus. In this context and in the absence of a vaccine, there is an urgent need to develop new drugs targeting highly conserved viral proteins, thus eliciting less drug resistance. Among HIV-1 proteins, the nucleocapsid protein, NCp7, is of particular interest. NCp7 is a highly conserved3 structural component of the virion in which about 1500−2000 NCp7 molecules coat the genomic RNA.4 NCp7, derived from the Gag structural © XXXX American Chemical Society

Received: May 8, 2018 Revised: June 19, 2018

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DOI: 10.1021/acs.biochem.8b00527 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry

Figure 1. Sequence of NCp7 (A) and structure of its hydrophobic platform (B). In (A), the amino acids of the hydrophobic platform are in red and the basic residues are in blue. In (B), the lateral chains of the residues involved in NCp7 hydrophobic platform are highlighted. The structure of NCp7(12−55) is extracted from (pdb 2EXF).

and stabilization21 and annealing of the primer tRNALys3 to the primer binding site (PBS) in genomic RNA.22 Hence, the nucleocapsid either in its mature form or as a domain of Gag plays essential roles in viral replication. Its critical role is further illustrated by the fact that point mutations in the zinc fingers cause a complete loss of virus infectivity.23−26 Because of its small size, its high level of conservation, and its multiple roles in HIV-1 replication, NCp7 is a target of choice for the design of anti-HIV drugs. However, NCp7 has no enzymatic activity, which makes high throughput screening (HTS) tedious to design. This task is further complicated by the fact that several NCp7 functions rely on poorly specific NA/ protein interactions. Despite these difficulties, multiple strategies have been developed to inhibit NCp7 functions.27,28 The most popular one is based on zinc ejectors that unfold NCp7 zinc fingers, alter the binding of NCp7 to NA, and lead to a complete loss of virus infectivity. Most importantly, viruses resistant to zinc ejectors could not be generated, despite extended efforts,29 further validating the relevance of NCp7 as a target for antiviral therapy. Numerous chemical classes of zinc ejectors have been developed,30,31 but they suffer from limited specificity and rather high toxicity, so that their use is mainly envisioned as topical microbicides for the prevention of HIV transmission.32,33 In parallel, several classes of small molecules interacting with the hydrophobic plateau at the top of the folded fingers of NCp7, but with no zinc ejector potential, have been identified in virtual, medium- and high-throughput screens, using assays based on the NA binding and chaperone properties of NCp7.34−39 However, their higher specificity is associated with an antiviral activity in the micromolar range, suggesting that their structure should be further optimized to increase their affinity for NCp7 as well as their cell penetration potential. Small methylated oligoribonucleotides have also been designed and shown to be promising since they can impede HIV-1 replication in primary human cells at subnanomolar concentrations, when appropriately formulated with CPPs.40 Finally, short peptides of 6−12 amino acids (a.a.) containing both aromatic and basic residues41−44 were designed to compete with the NA binding and chaperoning activities of NCp7. However, these peptides only displayed antiviral activity at rather high concentrations, most likely as a consequence of their small size which limits their binding affinity to NA. In this context, our objective was to design longer peptides that could more efficiently compete with NCp7 for its NA targets. On the basis of the importance of the NCp7 hydrophobic platform in its NA binding properties8,9,11,45 and functions,24,46,47 we designed a series of 60 peptides

including the residues of this platform, separated by a.a. stretches of variable sizes and composition. Peptides were selected, based on a specific assay measuring their ability to prevent the NCp7-directed destabilization of NA secondary structure that is critically dependent on the properly folded fingers of NCp7.46 The five most efficient peptides selected were found to inhibit NCp7 destabilization properties with IC50 values in the nanomolar range, by competing with NCp7 binding to its NA targets. The most efficient peptide, formulated with a cell penetrating peptide (CPP), was observed to enter and accumulate efficiently into cells, with low cytotoxicity. However, this peptide showed no antiviral activity, likely as a consequence of its sequestration by cellular NAs and/or its probable inability to enter into the viral capsid. This peptide thus appears as an interesting tool for in vitro studies, but further efforts will be needed to obtain antiviral activity.

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RESULTS AND DISCUSSION Strategy for Peptide Design. As a NA chaperone, the HIV-1 NCp7 promotes the two obligatory strand transfers required for conversion of genomic RNA by the RT into double-stranded DNA (dsDNA) flanked by the long terminal repeat (LTR). The first strand transfer reaction involves regions containing imperfect stem-loop (SL) structures, i.e., the transactivation response element (TAR) RNA sequence and its complementary DNA sequence (cTAR) at the 3′ end of the newly made strong-stop DNA. In vitro, NCp7 activates the transient melting of the cTAR DNA structure14 and stimulates its annealing with TAR RNA as well as with its DNA equivalent, dTAR.48 The NA binding and chaperone properties of NCp7 are supported by the hydrophobic plateau (V13, F16, T24, A25, W37, Q45, and M46) at the top of the folded fingers and the basic residues distributed throughout the NCp7 sequence (Figure 1).8,11,12,45−47,49−52 Herein, we designed a series of 60 peptides (10 to 17 a.a. long, Supplementary Table S1) that contain the a.a. of NCp7 hydrophobic plateau in order to compete with NCp7 binding to its NA target and thus inhibit NCp7 chaperone activity. The structure of the peptides was based on the assembly of four blocks of variable sizes (Ba, Bb, Bc, and Bd) organized around the seven residues of the NCp7 hydrophobic plateau (Figure 2) and completed with basic residues in order to increase the peptide affinity for NA. Variation in the peptide sequences presented in Figure 2 also included acetylation of the N-terminal K residue in block Ba, amidation of the C-terminus in block Bd, and replacement of L-Trp (W) by D-Trp (w) in block Bc (Supplementary Table B


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Figure 2. Schematic organization of the 60 peptides synthesized for screening. The peptides are divided into four blocks centered on the amino acids involved in the hydrophobic plateau of NCp7 (in red). For each block, the sequences found in the peptides of the library are listed. Figure 3. Schematic representation of the screening assay (A) and structure−activity relationship of selected peptides (B). In (A), the emission spectra of Rh6G-5′-cTAR-3′-Dabcyl in the absence and presence of NCp7(11−55) are in red and green, respectively. Peptides competing with NCp7(11−55) for the binding to cTAR and inhibiting NCp7(11−55) destabilizing activity restore the low emission of the doubly labeled cTAR, observed in the absence of NCp7(11−55) (black dash-dotted line). In (B), the sequence of the best peptide pE is given at the top row. One of the least active peptide pZ was used as a negative control in gel experiments.

S1) which are used to protect the peptides from cellular peptidases. Peptide Screening Assay. Since destabilization of NA secondary structure is a key component of NCp7 chaperone activity14,53,54 that critically relies on the NCp7 hydrophobic platform,46,47 we investigated the ability of the designed peptides to inhibit the NCp7-induced destabilization of a cTAR sequence labeled at its 5′- and 3′-ends by Rh6G and Dabcyl, respectively. In this assay, we used the truncated NCp7(11−55) peptide that retains the zinc finger domain instrumental for NA specific binding and destabilizing properties,12 but lacks the basic N-terminal domain responsible for NA aggregating properties.55 In the absence of NCp7(11−55), the proximity of cTAR ends induces a strong quenching of Rh6G fluorescence by the Dabcyl group.56 Addition of NCp7(11−55) results in a strong increase of Rh6G fluorescence as a consequence of its ability to melt the lower half of the cTAR stem and thus increase the distance between the two dyes (Figure 3A). The Rh6G fluorescence increases with the NCp7(11−55)/cTAR ratio14 and reaches a plateau when NCp7(11−55) fully coats the cTAR sequence (at a ratio of 8 NCp7(11−55) molecules per cTAR). To ensure optimal sensitivity of the assay and reproduce the coating observed in viral particles,12 the peptides were tested on Rh6G-cTAR-Dabcyl molecules fully coated by NCp7(11−55). In these conditions, a peptide that can inhibit the NCp7(11−55) destabilizing activity, by competing with NCp7(11−55) for the binding to the doubly labeled cTAR, will be detected through a partial or total reversal of the NCp7(11−55)-induced increase of Rh6G fluorescence. The 60 peptides were screened at 1 or 10 μM concentrations corresponding to peptide/NCp7(11−55) ratios of 1/1 and 10/1 and peptide/DNA ratios of 10/1 and 100/1, respectively. By measuring their effect after 10 or 60 min of incubation, a relative peptide inhibitory activity (R) was calculated, and the peptides were ranked according to a rating corresponding to the sum of the R values (from 0 to 4) in the four measurements (Supplementary Table S1). The 60 peptides can be classified in three classes, with class A corresponding to highly active peptides with a rating greater than 2.8 (9 peptides), class B to moderately active peptides

exhibiting a rating between 1.2 and 2.8 (17 peptides), and class C including all low active peptides with a rating lower than 1.2 (34 peptides). Sequence comparison of the most active peptides reveals some structure−activity relationships (Figure 3B and Supplementary Table S1). Deletion in block Ba of the N-terminal K residue is detrimental for the peptide activity, while replacement in this block of F by W or acetylation of the Nterminal K shows marginal effects. In Bb, motifs of 4−6 a.a. containing two positively charged R residues close to each other are beneficial for the activity with the best activity obtained with 4 a.a. including two adjacent R residues. Particularly, addition of A residues or deletion of R residues inside or at the end of the block Bb is detrimental for the activity. In Bc, the G residue appears important for peptide activity, while replacement of L-Trp by D-Trp results in a variable effect with an activity increase for some peptides (three of them) and a decrease for others (four of them). Finally, the GR motif and the C-terminal K in Bd appear to be beneficial but not critical for activity. Taken together, our data show that, in addition to the residues of the NCp7 hydrophobic platform, a number of positively charged residues and a controlled distance between key a.a. are necessary for the peptide to be active. The best peptide of class A (Figure 3B) was named pE and selected for further investigation. Characterization of the NA Binding Properties of pE. In order to understand the mode of action of the inhibitory peptide pE, we analyzed its ability to bind to cTAR through a fluorescence anisotropy titration. Addition of pE to fluoresceinlabeled cTAR (Fl-5′-cTAR) resulted in a fluorescence anisotropy increase, due to the binding of pE to cTAR (Figure C


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Biochemistry 4). An apparent dissociation constant Kd = 0.15 (±0.01) μM was obtained, assuming a number of peptide binding sites of 8

cTAR migrates in the form of a complex with pE, we observed an accumulation of cTAR in the form of aggregates that did not enter the gel at higher concentrations of pE. In contrast, the class C peptide pZ (Figure 3B) was unable to bind to cTAR (Figure 5A,B). To further characterize the binding of pE to cTAR, we labeled cTAR at five selected positions (Figure 6) by 2aminopurine (2-AP), a fluorescent analogue of adenine whose fluorescence is sensitive to NCp7 binding.57 In the absence of pE, the 2-AP-labeled oligonucleotides exhibit a rather low quantum yield in comparison to that of free 2-AP,58 as a consequence of the dynamic quenching of 2-AP fluorescence by the neighboring bases, and notably the flanking G residues.59 In contrast to NCp7(11−55), binding of pE led to marginal changes in the quantum yield of 2-AP residues at all positions (Figure 6), including those close to G10 and G50 residues which play a key role in the NCp7-induced destabilization of cTAR stem.57 These data strongly suggest that pE is unable to restrict the dynamic interaction of 2-AP residues with their neighboring bases and to preferentially destabilize the lower part of cTAR stem. In fact, the results obtained with pE are similar to those obtained with the SSHS NCp7 mutant, wherein the Cys residues in the ZFs are substituted with Ser residues preventing the binding of zinc required for proper folding of the zinc fingers and the NA destabilizing activity of NCp7.57 This suggests that pE and the SSHS NCp7 mutant share similar binding modes. Characterization of the Inhibition by pE of the NCp7Promoted cTAR Destabilization. We first checked whether pE could destabilize cTAR by adding increasing concentrations of pE to a fixed amount of Rh6G-5′-cTAR-3′-Dabcyl. While NCp7(11−55) used at a ratio of 11 NCp7(11−55) per cTAR causes a 6−7-fold increase in the Rh6G fluorescence, pE induces an increase of less than 20% when used at a peptide/ cTAR ratio of 65 (data not shown). This indicates that pE does not significantly destabilize cTAR and thus that the hydrophobic residues of pE and NCp7(11−55) do not interact similarly with cTAR. This conclusion is in line with the data of Figure 6 and the key role of the proper folding of NCp7 zinc fingers in the destabilization activity.14,46 To quantitatively characterize the capacity of pE to inhibit the NCp7(11−55)-induced cTAR destabilization, we determined its 50% inhibitory concentration (IC50), by adding increasing amounts of pE to Rh6G-5′-cTAR-3′-Dabcyl fully coated with NCp7(11−55). Inhibition was assessed by monitoring the decrease of Rh6G fluorescence as a result of the refolding of cTAR which brings Rh6G and Dabcyl close back together. Under the conditions used in the screening assay, pE inhibited NCp7(11−55)-induced cTAR destabilization with an IC50 value of 500 nM. By checking the dependence of the IC50 values on NCp7(11−55) and cTAR concentrations, we found a close match of the IC50 values with the NCp7(11−55) but not the cTAR concentrations (Table 1), confirming that the inhibitory activity of pE resulted from a competition with NCp7(11−55). IC50 values as low as 64 nM were obtained and were thus markedly lower than those reported for previous peptide inhibitors.60 Cell Entry and Antiviral Activity of pE. In order to exert an antiviral activity, the peptide has first to cross the cell plasma membrane and then to enter into the cytoplasm in a biologically active form that may target the incoming virions. Charged peptides are known to poorly cross the hydrophobic plasma membrane, and several viral or nonviral strategies can

Figure 4. Binding curve of the peptide pE to cTAR. The binding of pE to 33 nM Fl-5′-cTAR was monitored through anisotropy changes. The solid line corresponds to the fit of the experimental points (diamonds) by eq 1 (see Methods). The binding constant and number of binding sites are given in the text.

on cTAR, as for NCp7(12−55) and a nonfolded fingerless NC peptide of almost the same size as pE.46 To independently assess the binding of pE to cTAR, gel retardation assays were performed. A progressive decrease in the band corresponding to free cTAR was observed at increasing concentrations of pE, confirming the binding of pE to cTAR (Figure 5A,B). While at low pE concentrations,

Figure 5. Gel retardation assays of pE binding to cTAR DNA. (A) The cTAR 32P-DNA (0.25 μM) was incubated in the absence or the presence of peptides and analyzed by electrophoresis on a 10% polyacrylamide gel, as described in Methods. Lane C1, heat-denatured cTAR DNA. Lane C2, control without peptide; lanes 1−4 and 5−8, peptide to nucleotide molar ratios were 1:8, 1:4, 1:2, and 1:1. Lanes 1−4 and 5−8 were obtained with pE and pZ, respectively. The pE/ cTAR aggregates are indicated by an arrowhead. (B) Fraction of bound cTAR as a function of peptide concentration. Each data point represents the mean value of three experiments. Symbols: filled circles, pE; open triangles, pZ. D


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Figure 6. Quantum yields of 2-AP-labeled cTAR sequences in the absence (gray) and presence (red) of peptide pE. The labeled positions on the X-axis are in red on the cTAR sequence from the Lai strain of HIV-1. The QY values of the labeled cTARs were determined assuming a QY of 0.68 for free 2-AP.

tions, in particular with CPP, are commonly applied.61 We decided to deliver our peptides by complexing them with Pep1, a short amphiphatic CPP known to efficiently deliver a variety of peptides and proteins into a large panel of cell lines with little or no toxicity.62 Peptide toxicity was evaluated by flow cytometry using Annexin V and propidium iodide labeling in order to quantify cell death and apoptosis. No toxicity was revealed for any pE concentration tested in this assay (data not shown). To assess the efficiency of pE transduction by Pep-1, HeLa cells were transduced with Cy3-labeled pE (Cy3-pE) complexed to Pep-1 at different molar ratios. Internalization efficiency was evaluated by confocal microscopy and flow

Table 1. IC50 Dependency on NCp7(11-55) and cTAR Concentrations [cTAR], nM

[NCp7(11−55)], nM

5 20 20 33 50

55 200 600 660 1000

IC50, nMa 64 175 690 500 1000

± ± ± ± ±

10 40 100 120 150

Means ± standard error of the mean for two experiments.

a

be used to circumvent this problem. Among the latter, electroporation, liposomes as well as peptide-based formula-

Figure 7. Cellular localization (A−C) and antiviral activity (D−F) of pE. Cellular uptake of pE and MP72 (A). Cy3-pE and Cy3-MP72 (in red), a positive control for Pep-1 delivery were added to the cells at 1 μM in the absence of Pep-1 (A1 and A3, respectively) or complexed to Pep-1 at a ratio of 1:30 for Cy3-pE (A2) and 1:20 for Cy3-MP72 (A4). Cells nucleus were labeled with DAPI (blue). Cellular localization of pE and NCp7 after transfection (B−C). HeLa cells were transfected with plasmids coding for mRFP (B1), pE-mRFP (B2 and C2), pEminus-mRFP (B3), and NCp7-eGFP (C1). C3 corresponds to the overlap of C1 and C2. Cells were fixed with PFA 4% and observed by confocal microscopy. Scale bars are 20 μm, except for B1 and B2, where it is 10 μm. Quantification of the cellular uptake of Cy3-pE and Cy3-MP72 by flow cytometry (D). Effect of pE on the infectivity of an HIV-1 based lentivector encoding eGFP (E and F). At 24 h postinfection, the percentage of eGFP positive cells was determined by flow cytometry on cells incubated with pE or MP72 in their free forms or complexed to pep-1 (E) or on cells expressing pE after transient transfection (F). The error bars represent the standard deviations for two measurements (>25 000 cells each). E


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capable of displacing NCp7 molecules bound to the genomic RNA. In order to test this hypothesis, we analyzed the ability of pE to inhibit the NCp7 destabilizing activity on cTAR in the presence of different concentrations of cellular RNA. In line with our hypothesis, we observed that cellular RNA at a concentration as low as 0.01 μM can efficiently decrease the inhibitory activity of pE on NCp7 destabilizing activity (Figure 8). As expected from a competition of cellular RNAs and

cytometry. Even in the absence of Pep-1, small quantities of Cy3-pE (Figure 7A1) but not of the control Cy3-MP72 peptide (Figure 7A3) were found within cells. Upon Pep-1 delivery, Cy3-pE was found in dot form and diffused in the cytoplasm (Figure 7A2), while diffuse intracellular localization was observed for Cy3-MP72 (Figure 7A4), used as a positive control cargo for Pep-1 delivery.63 Noticeably, pE/Pep-1 ratios of 1:20 and 1:30 gave similar results, while a 1:40 ratio caused the formation of aggregates outside the cells (data not shown). Taken together, our data show that Pep-1 facilitates the intracellular delivery of pE and that a good proportion of pE is in a diffuse form in the cytoplasm. Flow cytometry (Figure 7D) further revealed that pE can enter in nearly 30% of the cells in the absence of a vector, while MP72 penetrates in less than 3% of the cells. After Pep-1 mediated intracellular delivery, more than 90% of the cells internalized pE, an even higher fraction than for MP72 (82%). In order to evaluate the antiviral activity of pE, we used a lentivirus vector pseudotyped with the vesicular stomatitis virus (VSV) glycoprotein, as a model of the early steps of infection (post-entry to integration). The infection level was monitored by measuring the proportion of eGFP-positive cells which results from eGFP expression after vDNA integration. Cells were processed for peptide transduction immediately prior to infection, and infection was quantified by FACS 24 h postinfection. As shown in Figure 7E, pE was unable to inhibit the infection process, suggesting that it may not have been correctly or sufficiently delivered into the cells to be active and/or may not interact properly with its intracellular target. Since peptide delivery into cells is a challenging issue, we next expressed pE in cells by transfection of plasmids coding pE or the negative control peptide pEminus fused to mRFP. Transfection was performed 24 h prior to infection. Cell infection was checked by FACS 48 h postinfection using the eGFP signal. Using confocal microscopy, pE-mRFP (Figure 7B2) and pEminus-mRFP (Figure 7B3) were observed throughout the cell with a higher concentration in the nucleus and more specifically in the nucleoli. This localization is clearly due to the peptide as mRFP expressed alone (Figure 7B1) is uniformly expressed throughout the cell. Interestingly, the intracellular localization of pE resembled that of overexpressed NCp7,64 with the exception of the nucleoplasm where NCp7 was poorly present (Figure 7C1−3). However, similarly to the negative control pEminus, pE had no incidence on infection by the HIV-based lentivector (Figure 7F). The absence of pE effect on the pseudoparticle infection was not related to the coupling of mRFP to pE, since overexpression of a nonlabeled pE did not show any effect on the infection either (data not shown). As the infection by the pseudoparticles mimics only the early phase of infection, we hypothesized that the viral core might not have been accessible to the peptide early enough to inhibit NCp7. To test the effect of pE on the late phase of viral infection, we expressed the peptide in 293T cells during viral production. Unfortunately, pE did not show any inhibitory activity on viral production either in terms of infectivity or virus titer (data not shown). The negative results of pE in cells are at variance with its strong inhibitory effect on NCp7 activity in vitro. We speculate that the absence of effect on infection may be related to the high concentration of cellular RNAs (ribosomal, transfer, and mRNA) in the cell cytoplasm, which could compete with viral RNA for pE binding. As a result, pE is probably buffered by the cellular RNAs, which decreases the proportion of active pE

Figure 8. Effect of cellular RNA on the inhibition by pE of the NCp7(11−55)-induced cTAR destabilization. Increasing concentrations of pE (0.2−1.2 μM) were added to a mixture of cTAR (0.1 μM) and NCp7(11−55) (1 μM) in the presence of different cellular RNA concentrations (0−0.1 μM expressed in cTAR equivalent). After each addition, the percentage of pE inhibition on NCp7(11−55)-induced cTAR destabilization was calculated. As cellular RNA affects NCp7(11−55) chaperone activity, the inhibition in the absence of pE was fixed at 0% for each cellular RNA concentration. Results are expressed as means ± standard error of the mean for two experiments. For the statistical analysis, a one-way ANOVA with a post hoc Tukey HSD test was performed (*padj ≤ 0.05; **padj ≤ 0.01).

cTAR for pE, increasing concentrations of cellular RNAs further reduced the inhibitory effect of pE, clearly indicating that cellular RNAs decreased the amount of pE bound to cTAR. Thus, the lack of pE antiviral activity in cells may be at least partially attributed to its nonspecific binding to cellular RNAs in the cytoplasm, which prevents it from displacing NCp7 molecules bound to the genomic RNA.

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CONCLUSIONS The aim of this study was to design, select, and characterize peptides able to efficiently inhibit the nucleic acid chaperone activity of NCp7 that plays crucial roles in HIV replication.12 To this end, a small peptide library was constructed, based on mimicry with the NCp7 hydrophobic plateau. The rationale was to search for peptides that would compete with NCp7(11−55) for binding to cTAR, a canonical DNA model sequence of HIV-1, in order to prevent NCp7(11− 55) from exerting its chaperone activity. The most active peptide from this screen, pE, exhibited IC50 values as low as 64 nM, indicating that pE could efficiently inhibit the chaperone properties of NCp7 in vitro. This inhibition is the result of the competition of pE with NCp7 for cTAR, as suggested by the relatively high affinity (150 nM) of pE for cTAR, differing by at least 2 orders of magnitude from the affinity of the RG2121 peptide, previously designed to compete with NCp7.43 Though pE and NCp7(11−55) bind with similar affinity to cTAR,46 they exhibit different binding F


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Biochemistry

Taken together, our data reveal that pE efficiently inhibits the NA destabilization activity of NCp7 in vitro, by competing efficiently with NCp7 for its binding sites on NA. This peptide appears thus as a useful tool for in vitro studies of NCp7 properties and functions. However, pE did not show any antiviral activity, likely as a result of its sequestration by cellular RNAs. To obtain pE-based peptides with antiviral activity, additional efforts will be required to improve their specificity against viral NA targets and decrease their affinity for cellular RNAs. To achieve this goal rationally and provide clues for improving peptide design, NMR analysis of pE complexes with viral and non viral RNA sequences will be required to determine the respective roles of charged and hydrophobic residues of pE in these complexes and highlighting differences in the roles of the same residues in the available NMR data of NCp7/oligonucleotide complexes.8−11,49,74 It is likely that the unfolded pE is not able to properly mimic the hydrophobic platform that forms at the top of the folded NCp7 fingers. To reach this objective, one strategy would be to structurally constrain the peptides by cyclization (head-to-tail, head-to-side chain, or side chain-to-side chain cyclization) which would also have the advantage to increase the peptide resistance to cellular proteases. In addition, targeted delivery of these peptides to the NCp7 molecules both in the early and late steps of the viral life cycle will also be instrumental for improving their antiviral activity.

modes, as indicated by the inability of pE to modify the fluorescence of 2Ap-labeled cTAR sequences and to destabilize the secondary structure of cTAR. Since pE efficiently inhibits the NCp7(11−55)-induced destabilization of cTAR, pE likely competes with NCp7 for the cTAR binding sites in the lower part of the stem, which is required to initiate the destabilization of cTAR.57 As the contribution of the other binding sites in cTAR destabilization is unknown, a clear conclusion on the ability of pE to compete with NCp7(11−55) for these sites cannot be given. Moreover, using the peptide carrier Pep-1, pE was delivered into HeLa cells, where it did not show any toxicity. However, neither pE delivery nor pE expression after transient cell transfection was able to inhibit cell infection by pseudoparticles modeling the early steps of HIV-1 infection. In addition, pE did not show any effect on pseudovirus production as well. This poor antiviral activity is not intrinsic to NCp7 competitors of peptide nature, since the RB 2121 peptide previously designed as NCp7 competitor, but of smaller size (only 6 a.a.), exhibited antiviral activity by targeting an early step of the viral life cycle.43 Competition experiments revealed that cellular RNAs at sub-micomolar concentrations can efficiently inhibit the effect of pE on the NCp7(11−55)induced destabilization of cTAR, by competing with cTAR for binding to pE. The RNA concentration in the cytoplasm can be estimated to be between 1 and 10 μM taking in account that the HeLa cell volume is around 2600 μm3 and that approximately 105 to 106 molecules of mRNA are found in the cytoplasm and represent 1−5% of the total cellular RNA.65 Therefore, pE either formulated with a CPP or overexpressed in the cell is likely sequestered by cellular RNA in the cytoplasm, so that only a small fraction of pE molecules is available to compete with NCp7 molecules bound to the viral genome. Due to the much lower affinity of RB 2121 for NA (>10 μM) and to the high concentrations of RB 2121 used in the antiviral assays,43 a much higher concentration of this peptide is expected to be available for competition with NCp7 and thus produce an antiviral activity. The poor antiviral effect of pE especially in the early steps of infection may additionally be related to its inability to enter the capsid shell. The HIV-1 capsid which protects the viral components from the cytoplasm components was recently shown to exhibit dynamic and strongly electropositive pores with a maximum diameter of 0.8 nm66 well suited for the transit of the negatively charged dNTPs. Assuming that pE adopts a globular shape, both its predicted diameter of about 2 nm67 and its highly positive charge would prevent it from entering the capsid. However, the exact timing and location of capsid disassembly on the journey of the HIV-1 capsid to the nucleus remains debated.68−72 As in some reports, uncoating of HIV-1 particles was reported to occur only 30 min after membrane fusion,73 the antiviral activity of pE should be limited only by its sequestration by cellular RNAs in this case. Interestingly, a HKWPWW peptide selected to bind specifically to HIV-1 Ψ RNA42 and able to compete with NCp7(11−55) for binding on NAs60 was found to exhibit antiviral activity, even when coupled with the fluorescent protein RFP or a protein transduction domain of 8 a.a.44 As the capsid does not seem to be an obstacle in this case and as this peptide binds less strongly to cTAR than pE, it can be speculated that it is less sequestered than pE by cellular RNAs, allowing it to more efficiently compete with NCp7.

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METHODS NCp7, Peptides, and Oligonucleotides. NCp7(11−55) (corresponding to a.a. 11 to 55 of NCp7) and unlabeled peptides were synthesized by solid-phase synthesis on a 433 A synthesizer (ABI), as described previously.35 Purification by HPLC was carried out on a C8 column (Uptisphere 300 A, 5 μm; 250 × 10, Interchim) in 0.05% trifluoroacetic acid (TFA) with a linear gradient of 10−70% of acetonitrile for 30 min. Peptide purity was checked by mass spectrometry. The Cy3labeled pE (Cy3-β-ala-KVKFTARRGWGRQMKK) was synthesized by the same approach. A β-alanine residue was added at the N-terminus to form a spacer between the peptide and the fluorescent probe. At the end of the peptide synthesis, 0.05 mmol of the resin was isolated and incubated in 1 mL of Nmethylpyrrolidone containing 0.05 mmol of cyanine3 Nhydroxysuccinimide ester (Lumiprobe, 1 eq., 36.79 mg) and 100 μL of N,N-diisopropylethylamine overnight at room temperature. After filtration and washing with methanol and dichloromethane, the peptidyl-resin was cleaved for 2 h in a TFA solution containing tri(isopropyl)silane (5% v/v) and H2O (2.5% v/v). The peptide was precipitated by ice-cold diethyl ether and then pelleted by centrifugation. The peptide was then purified by reverse-phase HPLC on a C18 column (Nucleosil, 100 Å, 5 μm, 250 × 10, Macherey-Nagel) in a water/acetonitrile mixture containing 0.05% TFA with a linear gradient of 30−80% of acetonitrile for 90 min and monitored at 220 nm. Molecular mass obtained from ion spray mass spectrometry was 2527.5 Da, in agreement with the expected theoretical mass. Prior to use, the peptide was dissolved in distilled water, aliquoted, and stored at −20 °C. The concentration of the labeled pE was determined from the label absorbance, using a molar extinction coefficient of 150 000 M−1 cm−1 at 550 nm. The 29 a.a. peptide Cy3MP72 HHAGPRKRVHERYCSPTAGSAKRRLFGED63 was synthesized through the same approach. Pep-1 synthesis was previously described.75 Pep-1 stock solution (2 mg mL−1, 0.68 G


Article

Biochemistry

of 2-AP-labeled ODNs was calculated using free 2-AP as a reference [0.6858] and an excitation wavelength of 315 nm. Fluorescence anisotropy measurements were performed with a SLM 8000 (Aminco) fluorometer in a T-format. Excitation was at 480 nm. The emission was collected using long-pass filters (>500 nm). In binding experiments, a 33 nM concentration of fluorescein-labeled cTAR (Fl-cTAR) was titrated by increasing concentrations of peptide. The fluorescence anisotropy of FlcTAR was fitted as a function of the peptide concentration using

mM in 2% dimethyl sulfoxide (DMSO) was prepared by adding DMSO first and then H2O, just before sonication in a water bath. All the oligonucleotides (ODN) used in this work were synthesized at a 0.2 μmol scale and purified by reverse-phase HPLC and polyacrylamide gel electrophoresis by IBA GmbH Nucleic Acids Product Supply. In the case of the doubly labeled cTAR species, the 5′ terminus was labeled with carboxytetramethylrhodamine (TMR) or ethyl 2-[3-(ethylamino)-6-ethylimino-2,7-dimethylxanthen-9-yl]benzoate hydrochloride (Rh6G) via an amino-linker with a six carbon spacer arm, while the 3′ terminus was labeled with either 4-(4′dimethylaminophenylazo) benzoic acid (Dabcyl) or 5(and 6)carboxyfluorescein (Fl) using a special solid support with the dye already attached. cTAR species singly labeled by Fl at their 5′ terminus or selectively labeled at different positions with 2′deoxyribosyl-2-aminopurine (2-AP) were synthesized at a 1 μmol scale by IBA GmbH. All cTAR oligonucleotides used in this work correspond to the sequence of the MAL strain of HIV-1, with the exception of the 2-AP-labeled cTAR oligonucleotide. The LAI sequence was selected in this last application due to its richer content in A residues, which facilitated their replacement by 2-AP. Concentrations of all ODNs were calculated from their absorbance using the molar extinction coefficients at 260 nm specified by the supplier. Unless otherwise stated, experiments were performed in 25 mM Tris−HCl (pH 7.5), 30 mM NaCl, and 0.2 mM MgCl2 at 20 °C. Cellular RNA Extraction. Cellular RNA was extracted from 106 HeLa cells using a NucleoSpin RNA plus kit according to kit instructions. Total RNA concentration was expressed as a concentration of cTAR (55 nt) equivalent. cTAR molecular weight was taken as 17786.5 g mol−1. After extraction, total RNA concentration was measured using a NanoDrop One/One spectrophotometer (Thermo Scientific), and the concentration, in cTAR equivalent, was calculated as follows: [RNAcTARequ, μM] = [total RNA, ng mL−1]/17786.5. The same stock at 85 μM was used for all the experiments. Screening Assay for Selecting Inhibitors of NCp7Induced cTAR Destabilization. The assay was performed by mixing 0.1 μM of doubly labeled cTAR (55 nt) and 1 μM NCp7(11−55) into 30 mM NaCl, 0.2 mM MgCl2, and 25 mM Tris-HCl pH 7.4. Fluorescence intensities were measured using FluoroMax3 and FluoroLog spectrofluorometers (Horiba Jobin Yvon) equipped with a temperature-controlled compartment. Excitation and emission wavelengths were at 520 and 550 nm, respectively. The tested peptides were added to the NCp7/cTAR complexes at concentrations of 1 μM and 10 μM, and the fluorescence intensities were measured after 10 min or 1 h. Measurements were taken after 10 and 60 min incubation with the compound to reveal its “immediate” effect and the possible time-dependence of this effect. The relative peptide inhibitory activity (R) was calculated as R = (1 − (If − If0)/(Ifmax − If0)) where If0 is the free cTAR fluorescence; Ifmax is the fluorescence of the NCp7(11−55)/cTAR complex in the absence of the peptide; and If is the fluorescence of NCp7(11−55)/cTAR in the presence of peptide. Steady-State Fluorescence Spectroscopy. Emission spectra were recorded with Fluorolog and FluoroMax3 spectrofluorimeters (JobinYvon Instruments, S.A. Inc.) equipped with a temperature-controlled cell compartment. All spectra were corrected for buffer emission, lamp fluctuations, and instrumental wavelength-dependent bias. Quantum yield

r = r0 − (r0 − rf ) Kd + P / n + D −

(Kd + P /n + D)2 − 4PD/n 2P /n (1)

where D describes the concentration of Fl-cTAR and P the total concentration of peptide, while r0 and rf describe the anisotropy values of the free and the peptide-coated Fl-cTAR, respectively. The parameter n describes the number of peptides that can bind to cTAR. The binding constant (Kd) was determined by fitting the experimental titration curve to eq 1 using the Origin software. As the Fl fluorescence intensity was not altered by the binding of the peptide, no correction for quantum yield changes was necessary. RNA Effect on pE Inhibition of NCp7-Induced cTAR Destabilization Assay. Emission spectra were recorded with an excitation wavelength set at 480 nm. The experiments were performed at 20 °C in Tris buffer (25 mM Tris−HCl (pH 7.5), 30 mM NaCl and 0.2 mM MgCl2). All spectra were corrected from the emission of the buffer and the dilution factor. Emission spectra of 0.1 μM of doubly labeled cTAR (55 nt) were measured before and after addition of a solution of total RNA purified from HeLa cells (concentration from 0 to 5 μM final). NCp7(11−55) (1 μM final) was then added to the mixture, and the emission spectra were recorded before and after addition of increasing concentrations of pE (from 0.2 to 2 μM final). Gel Retardation Assays. Assays were carried out in a final volume of 10 μL. The 5′-end 32P-labeled cTAR DNA (2.5 pmol) at 8 × 103 cpm pmol−1 was dissolved in 6 μL of water, heated at 90 °C for 2 min, and chilled for 2 min on ice. Then, 2 μL of renaturation buffer was added (final concentrations: 30 mM NaCl, 0.2 mM MgCl2, and 25 mM Tris-HCl pH 7.5), and samples were incubated for 15 min at 20 °C in the absence or presence of peptide at various concentrations. Gel loading buffer (final concentrations: 10% w/v glycerol, 0.01% w/v bromophenol blue, 0.01% w/v xylene cyanol) was added, and the samples were analyzed by electrophoresis on a 10% polyacrylamide gel (acrylamide/Bis-acrylamide = 29:1) at 4 °C in 0.5× TBE buffer (45 mM Tris-borate pH 8.3, 1 mM EDTA). After electrophoresis, the gel was fixed, dried, and autoradiographed. Free DNA and peptide/DNA complexes were quantified using a Molecular Dynamics Typhoon imager and ImageQuant software (Molecular Dynamics, GE Healthcare Bio-Sciences Corp.). The fraction of bound cTAR DNA (FR) was determined using i IF zy zz FR = 1 − jjj k IB + IF {

H

(2) DOI: 10.1021/acs.biochem.8b00527 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry

505 nm long-pass filter associated with a 610/20 band-pass filter. The eGFP was excited using a 488 nm laser and detected using a 505 nm long-pass filter with a 525/50 band-pass filter. For each sample, 25 000 cells were counted. Positive cells were determined according to a signal threshold fixed by using nontransduced and noninfected cells. Nonreplicative Pseudotyped Lentivector (LV) Production. Stocks of VSV glycoprotein (VSV-G) pseudotyped pseudoparticles were produced in 293T cells. Briefly, 5 × 106 293T cells were seeded in a 100 mm Petri dish in high glucose complete DMEM (Fisher ref: 31966). A total of 21 μg of plasmid was transfected 24 h later: 3 μg of pMD2.G (Addgene plasmid # 12259, a gift from Didier Trono), 6 μg of pCMVdR8.91 and 12 μg of pSicoR (Addgene plasmid # 11579, a gift from Tyler Jacks) using the standard Jet PEI transfection protocol (Polyplus). Cell medium was changed 24 h posttransfection and cell supernatants were collected 24 h later. Supernatants were filtered on 0.45 μm cellulose acetate filters (Millipore SLHV033RS), and p24 antigen concentration was determined using an ELISA assay (Innotest HIV, Innogenetics, 80563). The viral stock was stored at −80 °C. Infection Inhibition Tests. For inhibition after transfection, 3.5 × 104 HeLa cells in complete DMEM were seeded in a 12-well plate 24 h prior to transfection with 1.5 μg plasmid using jetPEI. For inhibition after peptide transduction, 18 × 104 HeLa cells in complete DMEM were seeded in a six-well plate 24 h prior to transduction. At 24 h after transfection or 1 h after peptide transduction, the medium was removed, and cells were infected with 50 μL of LV supernatant (equivalent to 11−15 pg of p24) in 1 mL of DMEM per well containing 8 μg/mL polybrene (hexadimethrine bromide − Sigma No. H9268). Cell supernatants were replaced with fresh medium 24 h later. Cells were prepared for cytometry quantification 48 h post infection.

where IF and IB are the band intensities of free and bound cTAR DNAs, respectively. Plasmids and Cells. HeLa cells (ATCC CCL-2) were cultured at 37 °C in a 5% CO2 atmosphere in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), antibiotics (penicillin 100 IU mL−1, streptomycin 100 IU mL−1). Cells were passed at subconfluency every 3−4 days. pE-mRFP and pEminus-mRFP coding plasmids were obtained by insertion of the same complementary DNA (cDNA) coding pE in sense or antisense, respectively, in BamHI in frame and in 5′ of the mRFP cDNA previously inserted in BamHI-EcoRI in a pcDNA3.1 (+) backbone (Thermo Fischer). Cell Transfection. 5 ×104 HeLa cells in complete DMEM were seeded 24 h prior to transfection in a 12-well plate containing an ethanol sterilized 35 mm glass coverslip. The day of transfection, the medium was changed with 1 mL of fresh complete DMEM, and 1 μg of plasmid was transfected with jetPEI (PolyPlus) according to supplier’s recommendations. At 24 h post transfection, cells were processed for imaging. Cell Transduction with Pep-1/Cy3-pE Complexes. Cy3-pE solution (2 μM) was prepared in sterile phosphate buffer saline (PBS, 154 mM NaCl, 1 mM KH2PO4, 5.6 mM Na2HPO4). Three Pep-1 solutions were prepared at 40, 60, and 80 μM in sterile PBS in order to achieve Pep-1/Cy3-pE ratio of 20:1, 30:1, and 40:1. 100 μL of Cy3-pE solution was added to 100 μL Pep-1 solution, mixed gently, and finally incubated at 37 °C for 30 min. 5 ×105 HeLa cells in complete DMEM were seeded in a sixwell plate 24 h prior to transduction. For confocal imaging, a 35 mm glass coverslip was introduced into the wells, sterilized with 100% EtOH, and washed twice with PBS. The day of transduction, the medium was removed, and cells were washed twice with sterile PBS prior to being overlaid with 200 μL of Pep-1/Cy3-pE complex solution. Cells were placed back at 37 °C in a 5% CO2 atmosphere for 30 min. Then, 1 mL of complete DMEM was added, and cells were incubated again for 30 min. Cells were then treated for imaging or cytometry analysis. Confocal Imaging. After transduction, the DMEM medium containing the complexes was removed, and cells were washed twice with sterile PBS. Cells were fixed for 15 min with 4% paraformaldehyde (PFA) in PBS. Glass coverslips were mounted with Prolong Gold Antifade mounting medium (Invitrogen, 36930) or Dapi fluoromount-G (Southern Biotech, 0100−20). Confocal images were taken using a confocal microscope (SPC UV1 AOBS, Leica) equipped with HCX PL APO CS 63× oil immersion and HXC PL APO 20× objectives and an Ar/Kr laser. The eGFP images were obtained by scanning the cells with a 488 nm laser line and filtering the emission with a 500−550 nm band-pass filter. For the mRFP images, a 568 nm laser line was used in combination with a 580−700 nm band-pass filter. Cytometry Measurements. DMEM medium containing the complexes was removed, and cells were washed twice with sterile PBS. 500 μL of trypsin-versene (Lonza BE02−007E) 1× in PBS was added to the cells for 3 min at 37 °C. Cells were resuspended in 2 mL complete DMEM, washed twice with PBS, and fixed for 15 min with 4% PFA in PBS. After two washes with PBS, cells were resuspended in 300 μL of PBS. The samples were analyzed on a BDFACS Aria II. mRFP and Cy3 were excited using a laser at 561 nm and detected using a

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ASSOCIATED CONTENT

S Supporting Information *

This material is available free of charge via the Internet The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.8b00527.

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Table S1 giving the list of all tested peptides (PDF)

AUTHOR INFORMATION

Corresponding Authors

*(E.R.) E-mail: [email protected]. *(Y.M.) E-mail: [email protected]. ORCID

May C. Morris: 0000-0001-8106-9728 Eléonore Réal: 0000-0002-5523-4408 Yves Mély: 0000-0001-7328-8269 Author Contributions #

V.S., S.Z., and B.B. contributed equally to this work.

Funding

This work, V.S. and B.B. were supported by the European TRIOH Consortium and the Agence Nationale de la Recherche sur le SIDA (ANRS). S.Z. was supported by a fellowship from Ministère de l’Enseignement Supérieur et de la Recherche. Notes

The authors declare no competing financial interest. I


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ACKNOWLEDGMENTS The authors thank C. Ebel, head of the cytometry flux service at the IGBMC (Illkirch), for her help.

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