A Novel Short Peptide is a Specific Inhibitor of the Human Immunodeficiency Virus Type 1 Integrase

doi: 10.1016/S0022-2836(02)00033-5 available online at http://www.idealibrary.com on

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J. Mol. Biol. (2002) 318, 45–58

A Novel Short Peptide is a Specific Inhibitor of the Human Immunodeficiency Virus Type 1 Integrase Vaea Richard de Soultrait1,2*, Anne Caumont1,3, Vincent Parissi1,2 Nelly Morellet4, Michel Ventura1,2, Christine Lenoir4, Simon Litvak1,2 Michel Fournier1,2 and Bernard Roques4* 1

IFR 66, “Pathologies Infectieuses: Aspects Biologiques et The´rapeutiques” Bordeaux, France 2 UMR 5097, Universite´ Victor Segalen Bordeaux 2, 146, rue Le´o Saignat, 33076 Bordeaux Cedex, France 3

Laboratoire de Virologie, EA 492, Universite´ Victor Segalen Bordeaux 2, 146, rue Le´o Saignat, 33076 Bordeaux Cedex France 4

De´partement de Pharmacochimie Mole´culaire and Structurale, INSERM U266, CNRS UMR 8600, UFR des Sciences Pharmaceutiques et Biologiques, 4, Avenue de l’Observatoire, 75270 Paris Cedex 06, France *Corresponding authors

The retroviral encoded protein integrase (IN) is required for the insertion of the human immunodeficiency virus type 1 (HIV-1) proviral DNA into the host genome. In spite of the crucial role played by IN in the retroviral life cycle, which makes this enzyme an attractive target for the development of new anti-AIDS agents, very few inhibitors have been described and none seems to have a potential use in anti-HIV therapy. To obtain potent and specific IN inhibitors, we used the two-hybrid system to isolate short peptides. Using HIV-1 IN as a bait and a yeast genomic library as the source of inhibitory peptides (prey), we isolated a 33-mer peptide (I33) that bound tightly to the enzyme. I33 inhibited both in vitro IN activities, i.e. 30 end processing and strand transfer. Further analysis led us to select a shorter peptide, EBR28, corresponding to the N-terminal region of I33. Truncated variants showed that EBR28 interacted with the catalytic domain of IN interfering with the binding of the DNA substrate. Alanine single substitution of each EBR28 residue (alanine scanning) allowed the identification of essential amino acids involved in the inhibition. The EBR28 NMR structure shows that this peptide adopts an a-helical conformation with amphipathic properties. Additionally, EBR28 showed a significant antiviral effect when assayed on HIV-1 infected human cells. Thus, this potentially important short lead peptide may not only be helpful to design new anti-HIV agents, but also could prove very useful in further studies of the structural and functional characteristics of HIV-1 IN. q 2002 Elsevier Science Ltd. All rights reserved

Keywords: HIV-1; integrase; inhibitors; peptides

Introduction HIV-1 integrase (IN) catalyzes the insertion of proviral DNA into the host genome and as such, it is essential for the viral replication cycle.1 Since IN has no cellular counterpart and is necessary for productive infection, identifying specific potent inhibitors of this enzyme should provide novel anti-HIV therapeutic strategies. In contrast to reverse transcriptase (RT) and protease (PR), the other two HIV-1 enzymes currently used as targets Abbreviations used: HIV-1, human immunodeficiency virus type 1; IN, integrase; RT, reverse transcriptase; LTR, long terminal repeat; 3-AT, 3-amino-1,2,4-triazole; ODN, oligodeoxyribonucleotide; TFE, trifluoroethanol; NOE, nuclear Overhauser enhancement; RIP, ribosome inactivating protein(s). E-mail addresses of the corresponding authors: [email protected]; [email protected]

in the multi-therapy strategy, few inhibitors against IN have been described and none of them seems to behave as potential therapeutic agents.2 This lack of IN inhibitors is partly due to the difficulties encountered in structural studies of the whole protein and to insufficient information concerning the biochemical mechanism of proviral integration. Retroviral integration proceeds in two steps: (i) 30 end processing in which two nucleotides are removed from the 30 end of each strand of the linear proviral DNA; (ii) DNA strand transfer, a concerted cleavage – ligation reaction, in which the recessed 30 ends of the viral DNA are covalently joined to the host DNA. The two unpaired nucleotides at the 50 ends of the viral DNA are then removed and the gaps between the viral and the target DNA are repaired. The nature of the DNA polymerase involved in the latter step remains to be established.

0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved

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Characterization of an HIV-1 Integrase Inhibitory Peptide

Figure 1. Effect of I33 and I29 on 30 -end processing and strand-transfer activities. (a) Peptides. (b) 30 -End processing. Reaction mixtures containing 300 nM IN in the presence of increasing concentrations of different peptides, as indicated, were incubated at 37 8C for 20 minutes before the addition of 10 nM substrate and incubated 15 minutes more at 37 8C. Lane S corresponds to the substrate and lane 0 to the complete reaction without peptide. Products were separated on a 12% polyacrylamide gel. (c) Strand transfer. Conditions were identical to those described for 30 -end processing, except for the time of incubation (30 minutes).

In vitro analyses have shown that only two elements are necessary for integration: the HIV-1 IN and the cis acting DNA sequences at the end of the proviral DNA long terminal repeats (LTRs). Although purified recombinant HIV-1 IN performs all the steps known to be required for end processing and strand transfer on model DNA substrates in vitro, such reactions differ from authentic integration because coordinate joining of the two viral ends remains inefficient. In vivo, the enzyme may attain the expected efficiency by interacting with viral or cellular proteins present in the pre-integration complex (PIC).3 In vivo and in vitro complementation studies suggest that the active HIV-1 IN is a multimer.4,5 This enzyme displays three independent structural and functional domains which are able to form dimers themselves.6 (i) The amino-terminal domain (residues 1 –50) contains the conserved HHCC motif and binds one atom of zinc.7 The structure of the amino-terminal domain has been solved by NMR spectroscopy.8 This region is involved in protein– protein interaction and contributes to the specific recognition of viral DNA

ends.9,10 (ii) The core or catalytic domain (residues 50 –212) contains the highly conserved D,D(35)E motif present in retroviral integrases and retrotransposons. Mutations of any of the three acidic residues Asp64, Asp116 and Glu152 abolish the HIV-1 IN activity.11 – 13 (iii) The carboxy-terminal domain (residues 213– 288) is the least conserved. This region exhibits similarity with an SH3 domain and is involved in non-specific DNA binding and multimerization.14,15 Very recently, the introduction of five point mutations led to the determination of the crystal structure of the IN domain expressing the catalytic core and the carboxy-terminal domain.16 However, the three domains are required for in vitro 30 end processing and DNA strand transfer.17 Despite several efforts to determine the structure of the entire protein, the low solubility of the native integrase has not allowed its determination. Since HIV-1 integrase is essential to reach a productive infection, this enzyme is a key target for antiviral compounds. In an attempt to identify inhibitors against this enzyme, we used the twohybrid system. This technique has been developed

47

Characterization of an HIV-1 Integrase Inhibitory Peptide

Table 1. Sequences and anti-HIV-1 integrase activities of different peptides Peptide

Sequence

IC50 (mM) 30 end processing

The IC50 values are within ^10% of the reported values.

in order to allow identification of potential inhibitors of protein function in the form of small peptides (aptamers) that specifically recognize a protein of interest.18 These high affinity peptides may interfere with the activity of the target protein, either by inhibiting directly the enzyme activity or by blocking its interactions with other proteins or substrates. Peptides interfering selectively with a protein offer several advantages for studying protein functions, since they can be used to target not only specific proteins but also the specific functions of a given protein. In addition, these specific pep-

tides could be expressed in living cells or vectorized into a given cellular compartment. Here we report the identification and characterization of a 33-mer-peptide (I33) able to inhibit the HIV-1 IN activities in vitro. Its inhibitory potential was optimized by using a 12-mer peptide (EBR28) corresponding to the amino-terminal part of I33. This EBR28 peptide was shown to interact with the catalytic core of IN. In the EBR28 NMR structure, all the hydrophobic residues are located on one side of the a-helix and the more hydrophilic ones on the other side. This arrangement is

48

Figure 2. Effect of EBR28 on the catalytic domain of IN. (X) Full length IN (HIS-IN1 – 288), (B) the catalytic domain (HIS-IN50 – 212), or (O) IN deleted in the N-terminal domain (IN50 – 288) were tested in the disintegration reaction. Reaction mixtures containing 300 nM IN in the presence of increasing concentrations of peptide EBR28 were incubated at 37 8C for 20 minutes before the addition of 12.5 nM of dumbbell DNA substrate and further incubated for one hour at 37 8C. Reaction products were separated by electrophoresis on a 12% polyacrylamide gel.

probably important for its interaction with integrase. Various novel EBR28 derivatives were synthesized and assayed in vitro against HIV-1 IN, as well as on human HIV-1 infected cells.

Results Inhibition of in vitro HIV-1 integrase activity In our search for novel inhibitors we used the yeast two-hybrid system to identify peptides able to strongly bind to HIV-1 IN. This screening led us to identify several clones containing IN-interacting inserts. Sequence analyses revealed that 15 inserts belonged to a non-coding region of the mitochondrial genome. They corresponded to two short peptides of different lengths: a 33-mer (I33) and a 29-mer (I29). They were identical except for the four extra amino acid residues in the N-terminal region of the 33-mer. To study the interactions between HIV-1 IN and the peptides corresponding to these inserts, we synthesized I33 and I29 (Figure 1(a)) by solid-phase synthesis. The effect of the synthetic peptides was tested on the in vitro activities of HIV-1 IN: 30 end processing and strand transfer. The 30 processing corresponds to the cleavage of a dinucleotide from

Characterization of an HIV-1 Integrase Inhibitory Peptide

the 30 end of a model DNA mimicking the U5 end of HIV-1 LTR. In the assay shown in Figure 1(b), IN catalyzed the processing of a 21 bp doublestranded oligodeoxyribonucleotide (ODN) giving a 19-mer ODN. In the strand-transfer reaction, the recessed 30 end of a pre-processed 21 bp doublestranded ODN is joined to the 50 end of an INinduced break of a second identical ODN (Figure 1(c)). A dose-dependent inhibition of 30 end processing (Figure 1(b)) and strand transfer (Figure 1(c)) was observed for I33, whereas no inhibition was obtained with a control peptide C35 (Table 1) exhibiting similar size and basic charge (Figure 1(b)). Peptide I33 inhibited the reactions with an IC50 value of 9 mM, while, interestingly, a lower degree of inhibition was obtained with I29 (IC50, 85 mM). The strand transfer reaction was also inhibited by I29 in the same concentration range (data not shown). These results showed that the four amino-terminal residues present in I33 were important for obtaining IN inhibition. To characterize the shortest domain of I33 essential for the inhibition, we tested three synthetic peptides corresponding to the amino-terminal (LCD278C), the central (EBR26) and the carboxyterminal (EBR24) regions of I33. Results in Table 1 show that peptide LCD278C was the best inhibitor amongst the three peptides and had an IC50 value of 21 mM, while lower inhibitions were observed with the central and the carboxy-terminal fragments. These two latter peptides exhibited IC50 values that were at least five times higher than that of I33. Similar results were observed for the strand-transfer activity (data not shown). These results confirm that the inhibitory effect can be correlated with the amino-terminal region of this peptide. The LCD278C inhibitory peptide was further optimized by mimicking the hydrophobic property of I33. We thus introduced an additional tyrosine (Y) on the N-terminal end of LCD278C (Table 1). In spite of its hydrophobicity, the presence of the OH groups on tyrosine residues preserves the aqueous solubility. As shown in Table 1, this new 12mer peptide, which we called EBR28, was even a better inhibitor (IC50, 5 mM) than LCD278C (IC50, 21 mM). The specificity of the inhibition was further examined by determining the effect of EBR28 on different enzymes: Bam HI, a specific restriction endonuclease and T4 polynucleotide kinase, an enzyme catalyzing the transfer of phosphate to a polynucleotide substrate. The activities of these enzymes were assayed under conditions recommended by the manufacturers. EBR28 showed no effect on these enzymes at the concentrations used to inhibit IN (data not shown). We also looked for an enzyme related to HIV-1 IN. In fact, it has been shown that the catalytic domain of HIV-1 IN folds similarly as the HIV-1 and Escherichia coli RNases H, Mu transposase and RuvC.19 Therefore, we tested the effect of EBR28 on the DNA polymerase and the RNase H activities of

49

Characterization of an HIV-1 Integrase Inhibitory Peptide

peptide is able to interact with the catalytic core of HIV-1 IN. Determination of important residues in the inhibitory peptide

Figure 3. Binding of DNA substrate to HIV-1 IN in the presence of the EBR28 peptide. Binding experiments were carried out using the filter binding assay described in Materials and Methods. Experimental conditions were identical to those described for the 30 end-processing reaction followed by filtering on nitrocellulose. 100% corresponds to the percentage of labeled DNA retained by IN on the filter. (X) EBR28; (B) L3A.

HIV-1 reverse transcriptase in vitro. No inhibition of reverse transcriptase activities was observed (data not shown).

Determination of the IN domain in interaction with the peptide Three different functional domains can be distinguished in HIV-1 IN: N-terminal (residues 1 –50), core (residues 50– 212) and C-terminal domains (residues 212 – 288). Mutational analysis in vitro has shown that full length IN is required for carrying out the 30 end processing and the strand transfer reactions.17 On the other hand, it has been reported that disintegration, which corresponds to the reverse strand transfer reaction of IN,20 can be catalyzed by a truncated IN corresponding to the catalytic core domain.21 To localize the IN domain interacting with the inhibitory peptide, we tested the effect of EBR28 on disintegration by using either a complete His-tagged IN protein (HIS-IN1 – 288) or the His-tagged catalytic core domain (HISIN50 – 212). As shown in Figure 2, when tested on disintegration using a dumbbell substrate,22 these two enzymes were inhibited by EBR28 in the same range of concentrations used for 30 end processing. These results suggest that EBR28 is able to interact with the core domain of IN. To eliminate the possibility that the histidine tag at the N-terminal part of (HIS-IN50 – 212) was involved in the binding to EBR28, we used an IN deleted for the N-terminal domain (IN50 – 288). For this purpose, the histidine tag was released by adding thrombin, as described in Materials and Methods. As shown in Figure 2, the disintegration activity catalyzed by (IN50 – 288) without the histidine tag was also inhibited by EBR28, thus confirming unambiguously that the

To define the EBR28 residues which are critical for the inhibition of IN, we tested Ala-substitution mutants of EBR28 on 30 end processing. We synthesized 11 peptides based on the sequence of EBR28 in which in every individual peptide, a single amino acid was substituted by alanine from positions 2 to 12. The tyrosine (Y) in position 1 was not substituted by an alanine residue because the importance of this amino acid had already been inferred from the difference in IC50 values between LCD278C (21 mM) and EBR28 (5 mM). As shown in Table 1, the substitution of Leu at position 3 by Ala (L3A) severely affected the inhibition, resulting in an IC50 value (165 mM) 30 times higher than that for EBR28. Moreover, substitution of amino acids at positions 5 –9 (I5A, R6A, M7A, I8A, Y9A) gave peptides with higher IC50 values than EBR28, indicating that the central region of this peptide was important for the inhibitory effect. By contrast, Ala substitution in other positions, Q2A, L4A, K10A, N11A and I12A, showed little effect on inhibition. Truncated mutants of EBR28 also revealed the important contribution of amino acids 3– 9. Deletion of amino acids at positions 10 – 12 (LCE41) had no significant effect on inhibition (IC50, 5 mM), whereas deletion of amino acids at positions 9 –12 (LCE40) increased the IC50 value to 120 mM (Table 1). Furthermore, an additional deletion of tyrosine in position 1 (LCD278B) completely abolished the inhibition of HIV-1 IN. Effect of the inhibitory peptide on DNA binding Binding of both viral DNA and host chromosomal DNA are critical steps in IN-catalyzed reactions. To better understand the inhibition of HIV-1 IN activity by the peptide, we analyzed DNA binding to IN in the presence of EBR28. IN was incubated with the same substrate and under the experimental conditions used for 30 end processing. The reaction products were filtered on nitrocellulose membranes and the radioactivity retained on the filters and corresponding to the IN-labeled DNA complex was quantified. Under these conditions, IN retained about 30– 35% of labeled DNA on the filter. This value was considered as 100% of binding. When IN was pre-incubated with increasing concentrations of EBR28 before the addition of the DNA substrate, a significant decrease of labeled DNA bound to the enzyme was observed (Figure 3). The L3A peptide which exhibited an IC50 value 30 times higher than that of EBR28 (Table 1) for 30 end processing, did not affect the binding of the DNA substrate to IN. Thus, the interaction of IN with EBR28 which prevented

50

Characterization of an HIV-1 Integrase Inhibitory Peptide

Figure 4. 2D 1H – 1H NOESY 400 MHz spectrum of the EBR28 (left) and Pro7EBR28 (right) peptides recorded in 30% TFE/70% H2O at 293 K. The HN-aliphatic region of the NOESY spectrum shows some differences between the chemical shifts of the Ile5 and Ile8 d proton resonances.

the binding of DNA, strongly suggests that EBR28 and DNA bind to the same region of IN. EBR28 does not interact directly with DNA, since when this peptide was incubated with the labeled ODN in the absence of IN, no radioactivity was retained in the membrane. Secondary structure prediction We used secondary structure prediction algorithms23 in order to assess the secondary structure in which EBR28 could fold. This peptide tends to form an a-helix. To confirm this prediction we replaced methionine by proline in the middle of EBR28 (compound Pro7EBR28 in Table 1), since the latter amino acid is known to disrupt the ahelical structure. Then the effect of Pro7EBR28 was assayed on the 30 end processing activity of HIV-1 IN. Our results showed that this single amino acid substitution abolished the inhibitory effect observed with EBR28 (Table 1), thus confirming the secondary structure prediction. NMR study of the EBR28 and Pro7EBR28 peptides In order to understand the mechanism of action for the EBR28 peptide we have determined the structures of the EBR28 and Pro7EBR28 peptides. Initially, we have tried to analyze the 1H NMR spectra of the two peptides in 100% H2O at pH 5.5 and pH 3.5. Due to their shortness and flexibility

it was difficult to determine any structure under these conditions. The solvent conditions chosen for these studies were pH 3.5, addition 30% (v/v) TFE at 293 K. In the first experiment all the proton resonances were attributed using the procedures described by Wu¨thrich,24 and the 1H chemical shifts of the two peptides were compared using the same conditions of pH and temperature (Figure 4). Some differences between the chemical shifts of these two peptides were observed, indicating that they very likely adopt different conformations. In Figure 4 we can see that the Ile5 and Ile8 d proton resonances are down and up-field shifted, respectively, with regard to the same protons of the Pro7EBR28. In the presence of 30% TFE the EBR28 peptide showed NOEs indicating the presence of a-helix conformation: Ha3/NH6, Ha4/NH7, Ha7/NH10, NH5/NH7. One medium NOE involving sidechains was also observed: Hd5/Hd1,299. Other NOEs characteristic of this secondary structure have not been observed due to resonance overlap. In the case of Pro7EBR28 the Ha3/NH6 and Ha8/NH10 NOEs were also present, but Hd5/Hd1,29 and Ha7/ NH10 NOEs were not observed.

NMR derived three-dimensional structures of EBR28 and Pro7EBR28 The structures were calculated by restrained simulated annealing, and subsequently refined using restrained molecular dynamics and energy

51

Characterization of an HIV-1 Integrase Inhibitory Peptide

Figure 5. Stereo view of the (a) EBR28 and (b) Pro7EBR28 structures, showing the amphipathic property of the a-helix (a). The hydrophobic residues are colored in red and the hydrophilic residues are blue and dark blue.

minimization. A total of 65 and 53 interproton distance restraints were used in the structure calculations of the EBR28 and Pro7EBR28 peptides, respectively. Among the distance restraints, 25 and 23 were intra-residual, 35 and 28 sequential, and five and two medium-range for the two peptides, respectively. From 50 structures, ten structures with the lowest total energy for EBR28 and Pro7EBR28, were selected and used for structural analysis. Figure 5 shows a comparison of the best structure of the two peptides. The main structural motif for EBR28 is a well defined a-helix that extends from Gln2 to Asn11 (Figure 5(a)), when only a turn between Pro7 and Lys10 is observed in Pro7EBR28 (Figure 5(b)). In Figure 5 the amino acid side-chains have been classified into two categories according to their preference for aqueous or non-polar environment. The EBR28 peptide presents the structural characteristics of an amphipathic helix, since it shows two faces of opposite polarity. The hydrophobic face on the one hand is formed by the following amino acids: Leu3, Leu4, Ile5, Met7, and Ile8. The hydrophilic face on the other hand is formed by Tyr1, Gln2, Arg6, Tyr9, and Lys10 amino acid side-chains (Figure 5). In

Pro7EBR28 these hydrophobic and hydrophilic arrangements do not exist. Effect of the I33 peptide on HIV-1 IN-induced yeast lethality We have previously described a system where the expression of active IN in yeast leads to a lethal phenotype caused by yeast genomic DNA damage.25 To analyze the effect of the peptides on the lethal phenotype, the yeast strain AB2 containing the IN gene was co-transformed with pACTII(His)I33, which expressed I33 fused to the GAL4 activation domain (Figure 6). The yeast lethal phenotype after IN expression is shown in Figure 6(a), line b. As expected, when IN was co-expressed with the inhibitory peptide, the lethal phenotype was abolished (Figure 6(a), line e). Various control experiments were performed to show the specificity of the inhibition. Thus, neither the expression of the vector pBS24-1 without IN (line a) nor the expression of the peptide in the yeast strain transformed by the latter vector affected yeast growth (line c). Moreover, the

52

Figure 6. (a) Effect of I33 expression on yeast lethality. The drop test was performed as described in Materials and Methods. The AB2 yeast strain was transformed with: a, pBS24-1 (control without IN); b, pHIV1SF2IN (expression of IN); c, pBS24-1 and pACTII(His)I33; d, pHIV1SF2IN and pACTII(His); e, pHIV1SF2IN and pACTII(His)I33. (b) Expression of HIV-1 IN. Protein extracted as described for the purification procedure was analyzed on SDS-12% PAGE. Western blot analysis63 was performed with monoclonal antibodies directed to the C-terminal domain of HIV-1 IN (kindly provided by Dr S.H. Hughes, Maryland, USA). Western blot analysis of proteins from the yeast strain AB2 transformed by pHIV1SF2IN (lane 1), cotransformed by pHIV1SF2IN and pACTII(His) (lane 2), or by pHIV1SF2IN and pACTII(His)I33 (lane 3) using monoclonal antibodies against IN.

expression of the GAL4 activation domain pACTII(His) had no effect on yeast lethality (line d). To ascertain whether IN was expressed in the strain co-transformed by pHIV1SF2IN and pACTII(His)I33, total proteins were extracted using the IN purification procedure (see Materials and Methods). Western blot analysis with a monoclonal antibody against IN (Figure 6(b)) showed that IN expression was not modified in the presence of the inhibitory peptide (Figure 6(b), lane 3), nor in the presence of the GAL4 activation domain (Figure 6(b), lane 2). These results indicated that the abolishment of lethality was not due to the absence of IN expression in yeast, but to a direct inhibitory effect of the peptide on IN activity. Also these results demonstrated the importance of the interaction between IN and the inhibitory peptide within a cellular context.

Effect of peptides on viral infectivity To investigate the potentiality of these peptides as antiviral agents, we used an HIV-1 infectivity

Characterization of an HIV-1 Integrase Inhibitory Peptide

Figure 7. Effect of peptides on HIV-1 infection. The peptide properties were assayed on HeLa CD4-b-Gal cells infected by HIV-1 as described in Materials and Methods. Cells exposed to 200 ml of viral suspension in the presence of increasing concentrations of (O) EBR28, (B) L3A, (X) LCE41 were cultured for 24 hours at 37 8C and then the b-galactosidase was measured. Results are expressed as the percentage of infectivity and are representative of three independent experiments, each one in duplicate.

assay. In this bioassay the expression of b-galactosidase in P4 indicator cells (HeLa-CD4þ, LTRLacZ) is strictly inducible by the HIV transactivator protein Tat, thereby allowing a precise quantification of HIV-1 infectivity based on a single cycle of infection.26 This assay reflects the ability of viruses to infect, integrate the proviral DNA and express the viral transactivator Tat, and it has been used to determine the dose response of drugs after viral infection.27,28 Target cells were infected by HIVLAI in the presence of the peptide for 24 hours, after which cells were washed, subsequently lysed and the b-galactosidase activity measured as described in Materials and Methods. The infectivity for each peptide concentration was compared to that in the absence of peptide. The peptide EBR28 was able to inhibit HIV-1 infectivity with an IC50 value of 40 mM (Figure 7). The 11 substituted peptides obtained from alanine scanning were also tested in this assay. As shown in Figure 7, the mutant with a substitution at position 3 (compound L3A) gave no inhibition with concentrations up to 100 mM. The same result was observed for mutants L4A, I5A, R6A, I8A and K10A. The other Ala substitutions moderately affected the inhibition of infectivity, with 50% inhibition for concentrations around 100 mM (data not shown). Assays of inhibition of infectivity by HIV-1 were also performed with the three truncated peptides. LCE41 exhibited an IC50 value of 55 mM (Figure 7), whereas no inhibition was observed for LCE40 and LCD278B at 100 mM. Cell viability estimated by the MTT assay was decreased by about 5% at peptide concentrations higher than 100 mM (data not shown).

Characterization of an HIV-1 Integrase Inhibitory Peptide

Discussion HIV-1 IN is a potential target for therapeutic intervention, given the essential role of proviral integration in the retroviral life cycle. The lack of any known human enzyme activity analogous to retroviral IN raises the hope that integration inhibitors might be relatively non-cytotoxic. Progress made in the understanding of the integration process has led to the discovery of compounds able to inhibit IN activity in vitro. Inhibitors of this enzyme have been described to act either on the viral DNA or by direct interaction with IN, thereby preventing DNA binding or IN-oligomerization. However, most of them exhibit a high degree of toxicity and little or no specific inhibition in vivo. Among these agents, monoclonal antibodies,29 – 31 single-chain variable antibody fragments against a distinct domain of IN32,33, peptide-based inhibitors such as the hexapeptide HCKFWW,34 a 30-mer peptide derived from the IN catalytic core,35 and ribosome inactivating proteins (RIPs) such as MAP30, GAP3136 or luffin and saporin37 have been described. They inhibit IN activity in vitro in the mM range. The hexapeptide and the 30-mer peptide interact with the catalytic core of IN, whereas for MAP30 and GAP31 the inhibition mechanism remains to be elucidated. Our laboratory has used the two-hybrid system to study HIV-1 IN with two aims. First, the isolation and identification of cellular proteins able to interact with HIV-1 IN, which may be partners of this enzyme in the pre-integration complex (results to be published elsewhere). Second, the search for high affinity peptide ligands which may inhibit the retroviral enzyme activities. Using this system, we identified a 33-mer peptide (I33) that tightly binds HIV-1 IN. In vitro studies showed that I33 inhibits the IN activities, 30 end processing and strand transfer. The size of this peptide was optimized to a 12-mer, EBR28, corresponding to the N terminus of I33. EBR28 inhibited IN by interfering with the binding of the enzyme to the DNA substrate. Moreover, EBR28 was able to inhibit the in vitro disintegration activity mediated by the catalytic core (HIS-IN50 – 212), suggesting that this short peptide interacts with the catalytic domain of IN. This domain is characterized by the presence of a triad of conserved amino acids D,D(35),E which are essential for catalysis and are evolutionary conserved. The triad coordinates the divalent metal ion required for catalysis.38 This domain is also involved in the specific recognition and binding of the viral DNA end.39,40 Thus, we speculate that EBR28, by interacting with this region, could inhibit the binding of viral DNA. Recently, Chen and co-workers generated the first multidomain HIV-1 IN crystal that incorporates the catalytic core and the C-terminal domains.16 This structure shows a contiguous strip of positive amino acid residues beginning at the active site of one monomer and extending along the linking helix to

53

the C-terminal domain of the other monomer. This strip has been proposed to be the binding site of viral DNA. Therefore, an alternative mechanism to explain EBR28 inhibition of viral DNA binding is the impairment of the dimerization of the two IN monomers. Studies with EBR28 mutants showed that Ala substitutions of residues L3 to Y9 have a drastic effect on the inhibition, especially L3A, thus providing additional evidence for the specificity of the peptide. These amino acids important for inhibition are localized in the central region of EBR28 and are essentially aliphatic. Although the structural basis for EBR28 binding to IN is unknown, the present evidence suggests that EBR28 binds the catalytic core through hydrophobic interactions. In order to determine how these interactions could happen, the solution structure of two synthetic peptides, EBR28 and Pro7EBR28, were determined knowing that the first peptide interacts with IN and not the second one. Due to the shortness and then the flexibility of these peptides, their analyses were done in the presence of 30% TFE. TFE is known to decrease hydrogen bonding of amide protons to water and consequently to favor intramolecular hydrogen bonds thus enhancing a-helical conformations in structured regions. It is very important to note that TFE stabilize a-helices in regions where a high propensity for helix formation exists.41 – 46 The secondary structure prediction algorithms confirm that EBR28 tends to form an a-helix. Its interaction with the IN probably induces this structuration, then it was biological relevant to use TFE. Under these conditions characteristic NOEs of a a-helix conformation have been found all along the sequence of EBR28. We observed chemical shift variations of some of the aliphatic protons (Ile5 and Ile8) between the two peptides, suggesting that these residues are in the vicinity of Phe9 in EBR28, and not in Pro7EBR28. It seems that EBR28 adopts a more defined secondary structure (a-helix) than Pro7EBR28. Proline residues are more involved in reverse turn, than in a-helix. Their backbone at Pro residues within a polypeptide chain has no amide hydrogen atom for participation in hydrogen bonding. In the EBR28 NMR structure all the hydrophobic residues are located on the same side, and the more hydrophilic ones on the other. Pro7EBR28 does not exhibit such arrangement of its residues, and then it cannot interact with integrase. As the Leu3 hydrophobic residue has been shown to be very important for the interaction of EBR28 with integrase, we think that the hydrophobic face of the a-helix is implicated in this interaction. To determine whether a possible interaction between HIV-1 IN and the peptides could take place within a cell, we first investigated the effect of I33 on IN activity in vivo using the lethal assay.25 The co-expression of IN and the I33 peptide in yeast led to the abolishment of the lethal

54

Characterization of an HIV-1 Integrase Inhibitory Peptide

phenotype. Furthermore, as I33 was not toxic for yeast cells and no modification of yeast growth was observed when expressing it, we can conclude that I33 specifically interacted with IN to inhibit the lethal phenotype without interfering with the activity of other yeast proteins. To confirm and extend the results obtained with the yeast cell, we studied the effect of EBR28 on the integration process during a single HIV-1 replication cycle. This assay reflects the ability of viruses to infect cells, to integrate the proviral DNA and to express the viral transactivator Tat. The EBR28 peptide inhibited viral infectivity with an IC50 value of 40 mM. This relatively high IC50 value is probably due to the lack of efficient peptide cell delivery. The use of a carrier system to overcome the poor entry of EBR28, as already shown with other peptide vectorizing agents,47 should certainly improve its antiviral activity. Nevertheless these results are not unambiguous proof that IN is the intracellular target of the peptide. Our bioassay implies three different steps of virus replication: entry, reverse transcription and integration. We have previously shown that EBR28 has practically no effect on the DNA polymerase activity of HIV-1 reverse transcriptase in vitro, making this enzyme an unlikely target of the peptide. Moreover, preliminary experiments performed by pre-incubating P4 cells with EBR28, followed by several washing steps before infection, showed that the inhibitory peptide kept its anti-viral activity (data not shown), thus strongly suggesting that EBR28 does not interfere with viral entry. All these observations therefore reinforce the assumption that IN is the target of EBR28 in a single cycle assay. Experiments are in progress to confirm the latter effect by the analysis of viral infectivity in the presence of EBR28 following replication in Tcell lines. The two-hybrid system has allowed us to isolate, identify and optimize a lead peptide able to inhibit HIV-1 IN activity, both in vitro and in human infected cells. Work is underway to define the precise mechanism of inhibition of HIV-1 IN by the 12-mer peptide, its effect on viral infection, and to increase the bioavailability and resistance of EBR28 against cellular proteases by synthesizing chemically modified derivatives. Moreover, inhibitory peptides designed to have high affinity contacts with HIV-1 IN may be used to gain further knowledge of the structure– function relationship of this enzyme and help in attempts to crystallize the entire protein.

The yeast strains used in the two-hybrid screening were HF7c and Y187 (Clontech). For the yeast lethal assay the AB2 strain was used.25

Materials and Methods Strains and media Yeast JSC310, a yeast strain deficient for several proteases was used for the expression and purification of IN.48

Bacteria The E. coli strain DH5a was used for plasmid amplification, and BL21(DE3) for expression of His-tagged IN. Luria Beriani medium containing 50 mg/l of ampicillin was used for E. coli strains DH5a and BL21(DE3). Kanamycin (10 mg/l) was added as required. Peptide synthesis Peptides were synthesized by the solid-phase method, using fluorenylmethoxycarbonyl strategy.49 Plasmids Untagged HIV-1 IN purified from yeast cells The pBS24-1 was used for the construction of the expression plasmid pHIV1SF2IN containing the HIV-1 IN gene.25 His-tagged integrase pET-15b(HIS-IN1 – 288), pET-15b(HIS-IN50 – 212), pET15b(HIS-IN50 – 288) expression vectors encoding Histagged truncated INs were a generous gift from J.F. Mouscadet (UMR-CNRS 8532, Villejuif, France). Integrase purification Untagged HIV-1 IN purified from yeast cells Protein extraction and purification were performed as described.48 His-tagged integrase HIS-INs were purified as described by Leh et al.50 with minor modifications: all buffers contained 7 mM Chaps and ZnSO4 was omitted during elution. Proteins were dialyzed overnight against 20 mM Tris – HCl (pH 8), 0.5 M NaCl, 2 mM dithiothreitol (DTT) and 10% (v/v) glycerol. Chaps (7 mM) was added to fractions containing IN. After elution, the HIS-IN50 – 288 fusion protein was cleaved using biotinylated thrombin (Novagen) and dialyzed as described above. Thrombin was then captured by incubation with streptavidin magnesphere paramagnetic particles (Promega). In vitro integrase assays Substrates used to assay the 30 end processing and strand transfer reactions were oligonucleotides (ODN) of different sizes containing sequences derived from the U5 end of the HIV-1 LTR. Processing This was performed with 10 nM 50 labeled ODN-1 (50 GTGTGGAAAATCTCTAGCAGT-30 ) annealed to its complementary strand ODN-3 (50 -ACTGCTAGAGATTTTCCACAC-30 ).

55

Characterization of an HIV-1 Integrase Inhibitory Peptide

Strand transfer 0

0

This was done using 10 nM 5 labeled ODN-2 (5 -GTGTGGAAAATCTCTAGCA-30 ) annealed to ODN-3. Disintegration ODN 4 (50 -TGCTAGTTCTAGCAGGCCCTTGGGCCGGCGCTTGCGCC-30 ) forming a “dumbbell” substrate (12.5 nM) was used.22 In all assays the reaction mixture contained 20 mM Hepes (pH 7.5), 10 mM DTT, 7.5 mM MnCl2, 10 mM NaCl, 0.05% (v/v) Nonidet-P 40 and 300 nM IN in a final volume of 20 ml. Different 50 -labeled ODNs were added depending on the assay, incubated for additional lengths of time and stopped by adding 5 ml of loading buffer (95% (v/v) formamide, 20 mM EDTA, 0.005% (w/v) bromophenol blue) followed by heating at 90 8C for five minutes. The reaction products were submitted to electrophoresis in 12% (w/v) polyacrylamide – 7 M urea gels in TBE (Tris – borate – EDTA (pH 7.6)) and autoradiographed. Gels were analyzed using NIH-image equipment. Inhibition experiments Inhibition tests were done as described above for IN assays. Peptides were dissolved (1023 M final concentration) and kept in dimethylsulfoxide (DMSO). Controls were done in the presence of the same final concentration of DMSO. Preincubation for 20 minutes at 37 8C of 300 nM IN and different concentrations of peptides was performed to optimize the inhibition in the standard reaction mixture containing a final concentration of 1% (v/v) DMSO. The degree of inhibition (%) was calculated as follows: 100 £ ½1 2 ðD 2 CÞ=ðN 2 CÞ where C, N and D are the fractions of 30 end processing products or strand-transfer products for DNA alone (C), DNA plus IN (N), and IN plus inhibitor (D). The IC50 value was determined by plotting the inhibitor concentration versus the percentage of inhibition and determining the concentration which produced 50% inhibition. DNA binding assay Nitrocellulose filters (0.45 mm, Whatman) were treated with 0.4 M KOH, rinsed with distilled water and soaked in prewash buffer (20 mM Hepes (pH 7.5), 10 mM MnCl2, 10 mM NaCl and 100 mg/ml calf thymus DNA). IN was incubated under the same conditions used for 30 end processing reactions. One ml of prewash buffer was added and reactions were filtered. Filters were then washed twice with 1 ml of prewash solution and twice with 4 ml of wash solution (20 mM Hepes (pH 7.5), 10 mM MnCl2, 30 mM NaCl). The radioactivity retained on the filter was determined. Infectivity assays The effect on viral infection was assayed on P4 cells expressing CD4 receptors and the lacZ gene under the control of the HIV-1 LTR.27 P4 cells were plated using 100 ml of DMEM medium (Boehringer) plus 10% (v/v) fetal calf serum, gentamycin (45 mg/ml) and geneticin (200 mg/ml) in 96 plate multiwells at 12,500 cells per well. After overnight incubation at 37 8C, the supernatant was discarded and 200 ml of fresh medium containing HIVLAI

in the presence or in the absence of peptides was added. The supernatant was discarded 24 hours later and the wells were washed three times with a solution of 0.9% (w/v) NaCl. Each well was refilled with 200 ml of a reaction buffer containing 50 mM Tris–HCl (pH 8.0), 100 mM b-mercaptoethanol, 0.05% Triton X-100 and 5 mM of 4-methylumbelliferyl-D -galactoside (4-MUG). After three hours at 37 8C, the fluorescent reaction products were measured in a fluorescence microplate reader (Cytofluor II) with 360/460 nm Ex/Em filters. The cytotoxicity of peptides was investigated by treating the P4 cells with increasing concentrations of peptides (1 – 100 mM) and cell proliferation was measured over 24 hours. Cytotoxicity was evaluated using the MTT assay in the presence of 3-(4,5-dimethylthiazol-24)-2,5-diphenyl tetrazolium bromide, after removing the cell culture medium and replacing it with saline phosphate buffer containing 5 mg/ml of MTT.51 Two-hybrid system YPD medium (1% (w/v) yeast extract, 1% (w/v) tryptone and 2% (w/v) glucose) was used for cell growth and mating. SD (0.67% (w/v) yeast nitrogen base without amino acids and 2% glucose) was used as a selective medium, where tryptophane (200 mg/l), leucine (1 g/l), histidine (200 mg/l) and 3 mM 3-amino-1,2,4-triazole (3-AT) were added as required. The yeast genomic DNA library FRYL (kindly provided by M. Fromont-Racine and P. Legrain, Institut Pasteur, Paris) has been described.52 This library contains 5 £ 106 different E. coli clones with yeast genomic DNA fragments cloned into the pACTII multicopy vector (Clontech), which carries the yeast LEU2 gene and the GAL4 transcription activation domain (AD) (prey plasmid). The IN hybrid expression plasmid pDB-IN (bait plasmid) was constructed by fusing HIV-1 IN to the GAL4 DNA binding domain (amino acid residues 1 – 147) in the pGBT9 vector, which carries the yeast TRP1 gene (Clontech). The plasmid pHIV1SF2IN encoding HIV-1 IN was cleaved by Nco I, blunted by treating with T7 DNA polymerase and digested by Sal I. The IN gene was inserted into the Sma I– Sal I digested pGBT9 vector. Y187 (MATa) and HF7c (MATa) cells were transformed by prey and bait plasmids, respectively, using the lithium acetate TRAFO method.53 The mating strategy was performed as described by Bendixen et al.54 Diploid cells containing both plasmids were then plated on selective medium and grown at 30 8C. After three to six days of incubation, the HIS3 reporter system was assayed in drop tests by testing the ability of diploids (about 2 £ 105 cells) to grow on agar plates containing selective medium (SD) lacking tryptophane, leucine and histidine, in the presence of 3 mM 3-AT. The liquid b-galactosidase assay was performed as described by Kippert.55 Out of approximately 1.7 £ 107 screened diploid cells, 70 were able to grow in the absence of histidine and expressed b-galactosidase activity. No interactions were observed between candidate interacting partners and the vector pGBT9, used as control. Sequence analysis IN-interacting plasmids were rescued from yeast cells as described.56 The DNA insert and the different constructions were sequenced using the Thermo Sequenasee radiolabeled terminator cycle sequencing kit (Amersham) with the 33P-labeled terminator pack.

56

Characterization of an HIV-1 Integrase Inhibitory Peptide

Sequences were compared to the full yeast genome at the SGD web site (Saccharomyces Genome Database†) with the BLAST software.57 Sequence analysis revealed that 15 inserts corresponded to the same non-coding region of the mitochondrial genome.

that had the lowest total energy and number NOE restraint violations were used for the final structural analysis. Calculations were performed using the NMRchitect software (Biosym/MSI).

Yeast lethal assay The effect of HIV-1 IN on yeast growth was determined using the drop test.25 Yeast selective media YNB corresponded to 0.67% yeast nitrogen base without amino acids and 5.6% glucose lacking uracil, leucine and histidine. Amino acids and bases (20– 30 mg/l) were added as required. The vector pACTII(His)I33 was derived from pACTIII33, by converting the selection gene marker LEU2 in HIS3. The Y187 strain was cotransformed with pACTIII33 cleaved with Cla I within the LEU2 gene and with the pLH7 plasmid carrying the yeast HIS3 and kanamycin resistance gene58 cut with Xho I and Hpa I. The AB2 yeast diploid strain already containing either the pHIV1SF2IN or pBS24.1 (a control plasmid without the IN gene) was transformed by pACTII(His)I33 by using the lithium acetate method.59 The transformants were selected on medium lacking uracil and histidine. The effect of pACTII(His)I33 on the yeast lethality mediated by IN expression was determined by visual observation on medium lacking uracil, histidine and leucine plates. 1

H NMR experiments

Due to their high hydrophobicity the EBR28 and Pro7EBR28 peptides were dissolved in the presence of 30% (v/v) TFE to give a final concentration of about 1 mM at pH 3.5. Two-dimensional phase-sensitive 1H Clean-TOCSY with a 70 ms spin lock60 and NOESY at 200 ms mixing time61 experiments, using time-proportional phase incrementation (TPPI)62 were recorded at 293 K on advance Bruker spectrometer operating at 400.13 MHz without sample spinning with 2 K real points in t2 with a spectral width of 6000 Hz and 512 t1 increments. The transmitter frequency was set to the water signal, which was suppressed by irradiation during the relaxation delay of 1.6 seconds between FIDs. The data were processed using UXNMR software (Bru¨ker). A p/2 phase-shifted sine square bell window function was applied prior to Fourier transformation in both dimensions (t1 and t2). Computational procedures NOE cross-peak volumes (NOESY, 250 ms mixing time, 293 K) were converted into distances semi-quantitatively, by counting contour levels. Using the bH geminal protons as the calibration peak, NOE signals were classified into six categories with upper distance ˚ to 5 A ˚ . Pseudo-atom correclimits ranging from 2.5 A tions were added when necessary. Calculations were performed with the Discover/NMRchitect software package from MSI with the Amber forcefield using a dielectric constant 1 ¼ 4r, in order to diminish in vacuum electrostatic effects. Fifty structures were generated for the two peptides using simulated annealing, followed by energy minimization until a maximum gradient ˚ was obtained. The structures value of 0.1 kcal/mol per A † http://genome-www.stanford.edu/Saccharomyces

Acknowledgments We thank S.H. Hughes (NCI, Frederick, Maryland, USA) for kindly providing HIV-1 IN antibodies, J.F. Mouscadet (UMR-CNRS 8532, Villejuif, France) for the generous gift of the histidine-tagged HIV-1 IN constructions and P. Durrens for his help with the two-hybrid assay. We acknowledge M.L. Andreola (UMR-CNRS 5097, Bordeaux, France) for fruitful discussions and pertinent suggestions and L. Tarrago-Litvak (UMR-CNRS 5097, Bordeaux, France) for critical comments on the manuscript. The excellent technical assistance of Ms C. Calmels is acknowledged. This work was supported by the Agence Nationale de Recherche contre le SIDA (ANRS), the CNRS, the University Victor Segalen Bordeaux 2 and the Poˆle Medicament Aquitaine. V.R. de S. was supported by Sidaction and V.P. by a MNERT pre-doctoral fellowship.

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Edited by M. Yaniv (Received 11 September 2001; received in revised form 21 January 2002; accepted 29 January 2002)