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Originally published In Press as doi:10.1074/jbc.M414679200 on March 23, 2005

J. Biol. Chem., Vol. 280, Issue 23, 21987-21996, June 10, 2005
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Peptides Derived from the Reverse Transcriptase of Human Immunodeficiency Virus Type 1 as Novel Inhibitors of the Viral Integrase*{boxs}

Iris Oz Gleenberg{ddagger}§, Orna Avidan{ddagger}, Yehuda Goldgur¶, Alon Herschhorn{ddagger}, and Amnon Hizi{ddagger}||

From the {ddagger}Department of Cell and Developmental Biology, The Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978 and the Department of Chemistry, Ben Gurion University, Beer Sheva 84105, Israel

Received for publication, December 30, 2004 , and in revised form, March 17, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent studies have shown that the integrase (IN) of HIV-1 is inhibited in vitro by HIV-1 reverse transcriptase (RT). We further investigated the specific protein sequences of RT that were involved in this inhibition by screening a complete library of RT-derived peptides for their inhibition of IN activities. Two 20-residue peptides, peptide 4286, derived from the RT DNA polymerase domain, and the one designated 4321, from the RT ribonuclease H domain, inhibit the enzymatic activities of IN in vitro. The former peptide inhibits all three IN-associated activities (3'-end processing, strand transfer, and disintegration), whereas the latter one inhibits primarily the first two functions. We showed the importance of the sequences and peptide length for the effective inhibition of IN activities. Binding assays of the peptides to IN (with no DNA substrate present) indicated that the two inhibitory peptides (as well as several non-inhibitory peptides) interact directly with IN. Moreover, the isolated catalytic core domain of IN also interacted directly with the two inhibitory peptides. Nevertheless, only peptide 4286 can inhibit the disintegration activity associated with the IN core domain, because this activity is the only one exhibited by this domain. This result was expected from the lack of inhibition of disintegration of full-length IN by peptide 4321. The data and the three-dimensional models presented suggested that the inhibition resulted from steric hindrance of the catalytic domain of IN. This information can substantially facilitate the development of novel drugs against HIV INs and thus contribute to the fight against AIDS.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Two viral encoded enzymes play central roles in the early stages of the replication of retroviruses and retrotransposons. The first one, reverse transcriptase (RT),1 converts the single-stranded viral RNA into double-stranded DNA in a relatively complex process, reverse transcription. This step is catalyzed by the two catalytic activities of RT, the DNA polymerase (capable of copying both RNA and DNA into DNA) and the RNase H activity, which concomitantly hydrolyzes the RNA strand in the DNA-RNA heteroduplex formed (1). Subsequently, the RT-produced double-stranded DNA is transported into the nucleus, as part of the nucleoprotein complex (designated the preintegration complex), where it integrates into the genomic target DNA by the second viral enzyme, the IN. IN identifies the ends of the linear viral DNA, trims them (by removing two or three extra nucleotides located 3' to the highly conserved CA 3' termini), and then accompanies the DNA into the nucleus to catalyze integration into the target cellular DNA (1–3). There are several examples for potential linkages between RT and IN. First, the DNA product of RT is the substrate for IN, the next enzyme in the line of the viral replication cycle. Second, both proteins are proteolytic products of the same polyprotein precursor encoded by a single retroviral gene, the pol (1). In some cases, as in avian sarcoma leukosis virus, the IN sequence appears in two forms, one as part of the large {beta}-subunit of the RT and the other as a free IN protein designated pp32 (1, 4). Moreover, PICs, which are capable of performing in vitro integration, contain the viral DNA, IN, RT, and other proteins (5–8). Third, the INs and RTs of HIV-1 and MLV were shown to exhibit physical interactions (9–11). These direct contacts between the IN and RT of HIV-1 were recently confirmed by us by using surface plasmon resonance technology.2 Finally, we and others (12, 13) have shown recently that RT can inhibit in vitro the enzymatic activities of IN, suggesting functional roles for these interactions.

After the completion of reverse transcription in the cytoplasm of the infected cell, there is a significant delay in the process of integration depending on the rate by which the PICs are transported into the nucleus (1). Because all possible catalytic components for integration are likely to be present in the PICs, the viral DNA can serve as donor DNA as well as the target DNA for integration. Such a potential auto-integration process is suicidal for the virus, as it destructs the viral genome. For that reason, it is imperative to understand the mechanisms that regulate the integration of the retroviral genome and learn how to control it. Several cellular proteins are known to be involved in the integration process in HIV-infected cells (14–17). Most interestingly, one of these factors serves also as a barrier to auto-integration in MLV-infected cells (18). Because RT can also inhibit IN activities (12, 13), it is possible that an alternative pathway of inhibiting auto-integration without the involvement of cellular proteins exists as well. This course of action involves the functional interactions between the authentic viral proteins, RTs and INs, prior to integration into the cellular DNA.

Here we have extended our previous in vitro study on the inhibition of IN by RT (12) by studying specific RT-derived sequences that are involved in binding and inhibiting IN. This was done through a systematic in vitro screening of a library of HIV-1 RT-derived peptides for their capacity to inhibit HIV-1 IN activities and to interact directly with IN. We have identified two 20-residue-long peptides that inhibit IN activities in vitro. The first peptide is derived from the DNA polymerase active site, and the second peptide is derived from the RNase H domain of RT. In addition, we have shown by a novel dot-blot analysis that the full-length RT, as well as several RT-derived peptides, bind directly to HIV-1 IN. The localization of the two inhibitory peptides in the three-dimensional structure of the RT and the putative docking (of the DNA polymerase-derived peptide) into IN-CCD suggest that the inhibition of IN activities results from a steric hindrance. These findings can lead to insights into the development of novel peptide-based, specific, and highly potent IN inhibitors. Such inhibitors are expected to interfere with HIV infectivity and, thus, serve as novel prodrugs for the treatment of AIDS.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
The sequences of all studied peptides are given in the Supplemental Material.

HIV-1 RT-derived Peptides
Two sets of synthetic peptides libraries were generous gifts from the NIH-AIDS Research and Reference Reagent Program. The first set was the HIV-1 HXB2R Pol peptides complete set (catalog number 4358). These peptides are each 20 residues in length with 10 amino acid overlaps between the sequential peptides. All peptides derived from the DNA polymerase domain were prepared to a final concentration of 900 µM, by dissolving in either DDW or in a minimal amount of 100% Me2SO and then diluted with DDW to 6.5% Me2SO, all according to the recommended instructions (www.aidsreagent.org). All peptides derived from the RNase H domain were dissolved in a minimal amount of 100% Me2SO and then diluted with DDW to 6.5% Me2SO (yielding final concentrations of 400 µM). The second peptide set was the complete set of the HIV-1 clade B consensus pol peptides (catalog number 6208). These peptides are each 15 residues in length with 11 amino acid overlaps between the sequential peptides. They were also dissolved as above to a final concentration of 400 µM each in 6.5% Me2SO. All peptides were designated according to the numbering given by the NIH-AIDS Research and Reference Reagent Program (www.aidsreagent.org).

Peptides 4286'-1 (15-mer) and 4321'-1 (14-mer) were custom-synthesized. The first one was diluted to a final concentration of 3.5 mM in DDW and the second one as described above to a final concentration of 5.7 mM in 6.5% Me2SO.

Recombinant Proteins
Bacterial Expression and Purification of HIV-1 IN and HIV-1 RT Proteins—All HIV-1 IN and RT versions used were highly purified as judged from their pattern after analysis by SDS-PAGE (data not shown).

HIV-1 IN—HIV-1 IN from the BH-10 strain of HIV-1, carrying amino-terminal His6 tag, was expressed and purified as described in detail (20).

HIV-1 IN-Catalytic Core Domain (the Double Mutant W131E, F185K)—This large IN fragment (designated IN-CCD) was expressed as an amino-terminal His6-tagged protein and purified as described in detail (21).

HIV-1 RT—The expression and purification of heterodimeric (p66/p51) HIV-1 RT with a His6 tag, attached to the carboxyl-terminal end of the p66, were described in detail previously (22). The expression and purification of a non-tagged heterodimeric HIV-1 RT were described previously (23, 24).

Assays for Enzymatic Activities of HIV-1 IN Oligonucleotides Used
The following gel-purified oligonucleotides were used in the enzymatic assays of HIV-1 IN: A (21-mer), 5'-GTGTGGAAAATCTCTAGCAGT-3'; B (21-mer), 5'-ACTGCTAGAGATTTTCCACAC-3'; C (19-mer), 5'-GTGTGGAAAATCTCTAGCA-3'; D (38-mer), 5-TGCTAGTTCTAGCAGGCCCTTGGGCCGGCGCTTGCGCC-3. Oligonucleotides A–C correspond to the U5 end of the HIV-1 long terminal repeat (25). Boldface letters indicate the highly conserved CA/TG dinucleotide pair. Oligonucleotide C is identical to A, after the removal of the GT dinucleotides from its 3'-end and thus after annealing to oligonucleotide B, creating a dinucleotide overhang at the 5'-end of oligonucleotide B. Oligonucleotide D, termed "dumbbell," folds to form a structure mimicking the integration intermediate. This substrate, used for assaying the disintegration activity, has 5 bp of a viral sequence and 10 bp of a non-viral sequence, as described earlier (26). In order to test the 3'-end processing and the resulting strand transfer activity of IN, the 5'-end-labeled oligonucleotide A, annealed to its complementary strand, oligonucleotide B (both 21 nucleotides long), was used. We have used the duplex of oligonucleotides C and B and for assaying the 3'-end processing activity (12).

5'-End Labeling and Substrate Preparations—Fifty pmol of oligonucleotides A, C, or D were 5'-end-labeled using 1 unit of T4 polynucleotide kinase and 50 µCi of [{gamma}-32P]ATP, in a final volume of 50 µl of the appropriate buffer (supplied by the manufacturer) for 30 min at 37 °C. The samples were then heat-inactivated. The 5'-end-labeled oligonucleotides designated A or C were annealed each to an equimolar amount of oligonucleotide B in 55 mM Tris-HCl (pH 7.5) and 0.27 M NaCl. For the disintegration assay, we have used 5'-end-labeled oligonucleotide D, which forms a dumbbell structure after self-annealing (12).

Assays of the 3'-End Processing, Strand Transfer (or DNA Joining), and Disintegration Activities—In the strand transfer assays described, the labeled 5'-end substrate employed served as both the target and donor DNA leading to an increase in the molecular size of the substrate, whereas in the 3'-end processing and disintegration assays, we have followed the unique cleavage of the labeled substrates (12, 26). All reactions were performed in 10-µl reaction mixtures with 0.33 pmol of the labeled DNA substrate and the reaction buffer, containing 1 mM HEPES (pH 7.5), 54 mM NaCl, 2.5 mM MnCl2, 50 µM EDTA, 2 mM dithiothreitol, 0.1 mg/ml bovine serum albumin, 0.1 mM spermidine, 25 mM MOPS (pH 7.2), 5% glycerol, and 1% Me2SO (12). We have assayed 250 ng of HIV-1 IN (which equals 4 pmol, assuming IN dimers of the 32-kDa subunits) or 1 µg of HIV-1 IN-CCD (which equals 28 pmol, assuming dimers of the 18-kDa subunits). This difference in the amounts of proteins assayed results from the lower activity of IN-CCD relative to the full-length IN. The two versions of HIV-1 IN were preincubated on ice for 5 min in the presence or the absence of HIV-1 RT or the HIV-1 RT-derived peptides. Reactions were initiated after adding the labeled DNA substrate in the reaction buffer, incubated for 30 min at 37 °C, and then stopped by adding 10 µl of formamide loading buffer (90% formamide, 10 mM EDTA, 1 mg/ml bromphenol blue, 1 mg/ml xylene cyanole). The samples were heat-denatured, cooled on ice, and loaded onto 6 M urea, 14% polyacrylamide denaturing gels, followed by electrophoresis (urea-PAGE). The gels were dried and subjected to autoradiography at –80 °C or at room temperature to obtain close to linear exposures.

Quantitative Analyses of the Inhibition of HIV-1 IN by HIV-1 RT and by RT-derived Peptides
The films were scanned, and the levels of IN activity were calculated using the densitometric software TINA (version 2.07d; Raytest Isotopenmessgeraete, GmbH). The 3'-end processing activity was determined as a percentage of the total 5'-end-labeled DNA oligonucleotides converted to 19-mer DNA. Strand transfer (or DNA joining) activity was calculated as a percentage of the total amount of labeled DNA found in DNA bands of 22 nucleotides or more in length. Disintegration activity was calculated as a percentage of the total 5'-end-labeled 38-mer DNA oligonucleotide converted to 14-mer DNA. All residual activities were relative to the control activity of the IN (with no RT or RT-derived peptides present) and were expressed as percentage of the initial IN activities.

Dot-blot Binding Assay
HIV-1 RT-derived Peptides Bound to HIV-1 IN—Nitrocellulose filters were soaked in DDW followed by transfer buffer, containing 25 mM Tris, 192 mM glycine, and 20% (v/v) methanol, for 5 min and then transferred to a dot-blot apparatus (Bio-Rad). Aliquots of RT-derived peptides or His6-tagged HIV-1 RT (10 µg each) were bound to the filter under vacuum for 15 min. To ascertain the binding of the protein and peptides to the nitrocellulose filter, the blots were stained with Ponceau following by blocking with 5% MTBST (5% milk powder in 30 mM Tris-HCl (pH 8.5), 125 mM NaCl, and 0.1% (v/v) Tween 20) for ~16 h at 4 °C. The experimental blot was then incubated with HIV-1 IN (17 µg/ml in 5% MTBST) for 1 h at room temperature, and the control blot was not incubated with IN. The filters were then washed with a solution similar to MTBST but with no milk powder (TBST). In one set of experiments (set A), the filters were incubated with mouse-anti HIV-1 IN antibodies (diluted 1:1000 in 5% MTBST) for 90 min at room temperature, following by washing with TBST. After that the incubation was performed with horseradish peroxidase-conjugated goat anti-mouse IgG antibodies (from Santa Cruz Biotechnology, diluted 1:500 in 5% MTBST) for 1 h at room temperature and then washed again, followed by an enhanced chemiluminescence reaction by mixing the two stock solutions at 1:1 ratio before use. These solutions are as follows: 25 mM luminol, 400 µM paracoumaric acid in 100 mM Tris-HCl (pH 8.5), and 5.4 mM H2O2 in 100 mM Tris-HCl (pH 8.5). In a second set of experiments (set B), both blots were incubated with anti-His6-horseradish peroxidase antibody (from Sigma, diluted 1:1000 in 5% MTBST) for 90 min followed by an enhanced chemiluminescence reaction.

HIV-1 RT-derived Peptides Bound to HIV-1 IN-CCD—This experiment was identical to the one described above for set B. The HIV-1 RT used here had no tag. The experimental blot was incubated with HIV-1 IN-CCD (20 µg/ml in 5% MTBST) and the other one without IN-CCD.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
We have shown previously (12) that all three enzymatic activities performed by HIV INs, namely the 3'-end processing, the strand transfer (termed together integration), and the reverse process, the disintegration, are inhibited in vitro by RTs. Subsequently, the goal of this study was to identify the sequences in HIV-1 RT that inhibit and interact with the full-length HIV-1 IN, as well as with the IN catalytic core domain. To this end, we have used a large set of synthetic peptides that cover the whole pol gene of HIV-1 (the HXB2R isolate). The peptides were each 20 residues long with 10 amino acids overlaps between the sequential peptides. These peptides were tested for their ability to bind IN and inhibit in vitro the enzymatic activities of HIV-1 IN and of IN-CCD.

DNA Polymerase Domain-derived Peptide 4286 Inhibits in Vitro the Integration Activity of HIV-1 IN—It was already shown by us that HIV INs are inhibited by the p51 subunit of HIV-1 RT, which encompasses the DNA polymerase domain (12). Consequently, we first tested the sequences located within this domain of HIV-1 RT. A total of 42 peptides, designated from 4269 up to 4310 (starting from the amino-terminal peptide, see Supplemental Material), were screened for their ability to inhibit HIV-1 IN. These peptides were first tested in groups, each containing a mixture of three contiguous and partially overlapping peptides (every one at a final concentration of 90 µM). All mixtures were preincubated on ice for 5 min with 0.4 µM purified HIV-1 IN. The enzymatic assays were then initiated by adding the DNA substrate, used for testing the combined 3'-end processing and DNA joining activities of IN (oligonucleotides A and B, see under "Experimental Procedures"), followed by incubation at 37 °C for 30 min. The results obtained show that only the mixture containing peptides 4284, 4285, and 4286 inhibited the integration activity of IN (data not shown). Consequently, each of these peptides was then tested separately (at a final concentration of 270 µM) for its ability to inhibit the integration activity. The results indicated that the inhibition of IN was caused primarily by a single peptide, 4286. This peptide inhibits both IN-mediated strand transfer and 3'-end processing activities (Fig. 1). It is apparent that both activities were substantially inhibited by the peptide at a final concentration of 27 µM. As a result, the extent of inhibition was also quantified to determine the peptide concentrations inhibiting 50% of the initial IN activity (IC50 value, Table I). The value, calculated for the inhibition of the strand transfer, was about 4.5 µM, and the one for the 3'-end processing was ~4.8 µM (Table I).


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  		TABLE I
Inhibition of HIV-1 IN activities by HIV-1 RT-derived peptides

All IC50 values are expressed in µM. All inhibitions of enzymatic assays were performed as described under "Experimental Procedures." Peptides 4285 and 4287 share 10 amino acid overlaps with peptide 4286, and peptides 4320 and 4322 share 10 residues overlaps with peptide 4321. Peptide 4286'-1 is derived from peptide 4286, and peptide 4321'-1 is derived from peptide 4321.

 



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  		FIG. 1.
Effects of the polymerase-derived peptide 4286 (KILEPFRKQNPDIVIYQYMD) on the activities of HIV-1 IN. Increasing concentrations of RT-derived peptide 4286 were preincubated with a fixed amount of HIV-1 IN followed by assaying the IN activities and urea-PAGE analysis (see "Experimental Procedures"). The peptide concentrations are indicated at the bottom of each panel. A, analysis of the strand transfer activity of IN using oligonucleotides C and B. B, analysis of the 3'-end processing activity of IN using oligonucleotides A and B.

 
Because every two adjacent peptides share 10-residue overlaps, we have also calculated the IC50 values for peptides 4285 and 4287 (that share sequences with the inhibitory peptide 4286). The analyses of the reaction products, which led to the calculations of the IC50 values (summarized in Table I), indicate that both 4285 and 4287 were considerably less effective than peptide 4286. Furthermore, we have also checked whether the two peptides 4285 and 4287, when tested together, demonstrate a synergistic effect by increasing their IN inhibiting capacity to a level as efficient as peptide 4286. The results showed that this is not the case, and their combined effect is still far below that of peptide 4286 (data not shown). This indicated that the inhibitory sequence of 4286 can be active only as one continuously linked sequence, which is likely to be structurally different from that of the other two peptides.

Peptide 4321, Derived from the RNase H Domain, Inhibits the Integration Activity of HIV-1 IN—We have found previously that the p51 subunit of HIV-1 RT inhibits the enzymatic activities of HIV INs, without testing the residual RT p15 domain, corresponding to the RNase H domain (12). This does not necessarily exclude the possibility that the p15 by itself can also exhibit inhibitory effects. To address this issue, we have tested all 15 RNase H domain-derived 20-mer peptides (peptide numbers from 4311 up to 4325). In each test, the peptides were analyzed in groups of two adjacent and partially overlapping ones (each at a final concentration of 60 µM). Only the mixture containing peptides 4321 and 4322 inhibited IN activities. Consequently, we have tested each peptide individually, and we found that only peptide 4321 was responsible for inhibiting both the 3'-end processing and strand transfer activities of HIV-1 IN (Fig. 2). The quantitative analyses indicated that the apparent IC50 values calculated for both activities were quite similar (Table I). These values are close to those calculated for peptide 4286. It should be noted that neither peptide 4320 nor peptide 4322 (that share sequences with peptide 4321) inhibited the enzymatic activity of HIV-1 IN in vitro (Table I).

The Complete 20-Residue Sequences of Peptides 4286 and 4321 Are Required to Inhibit HIV-1 IN—To determine the minimal sequence requirements for the inhibition of HIV-1 IN, we have tested short peptides derived from the 20-residue peptides 4286 and 4321. These were the 15-residue-long peptide 4286'-1 and the 14-mer peptide 4321'-1, derived from 4286 and 4321, respectively. As can be seen from the apparent IC50 values (Table I), these shorter peptides were not as effective as the original 20-residue peptides in inhibiting IN enzymatic activities. In addition, we have tested for IN inhibition a battery of 15-mer peptides (with sequences that partially cover those of peptides 4286 and 4321) for their ability to inhibit IN. These peptides were part of a library of 15-mer synthetic peptides that covered the whole pol gene of HIV-1 clade B (see Supplemental Material). Eight peptides (from 5538 to 5545) covered the sequence of 4286, and eight peptides (from 5626 up to 5633) overlapped 4321. Of all 16 peptides tested, only peptide 5628 (related to peptide 4321) moderately inhibited the strand transfer activity of IN with an apparent IC50 value of ~60 µM (as opposed to 5 µM of 4231) without showing a significant inhibition of the 3'-end processing (Table I). These results indicated the importance of the full 20-residue sequences (and potential structures of peptides 4286 and 4321) for the in vitro inhibition of HIV-1 IN.

Peptide 4286 Strongly Inhibits the Disintegration Activity of HIV-1 IN—All studied retroviral INs possess in vitro, in addition to the 3'-end processing and the forward strand transfer activities, the reverse activity of disintegration. The authentic disintegration activity was already studied extensively as a major IN activity (26–28). The disintegration activity involved a specific endonucleolytic excision of the "donor viral" DNA (with the conserved 3'-end "CA" dinucleotide) from its target DNA. Given that HIV-1 RT also inhibited this activity of HIV-1 IN (12), it was of interest to extend the study by testing the inhibitory effects of peptides 4286 and 4321 on the disintegration activity. We have used the 32P 5'-end-labeled 38-nt "dumbbell" substrate (oligonucleotide D, see "Experimental Procedures") and followed the generation of the specific 14-nucleotide DNA product (26). It is apparent that the two RT-derived peptides were capable of inhibiting the disintegration activity of HIV-1 IN (Fig. 3). Peptide 4286 effectively inhibited the disintegration activity with full inhibition obtained at a concentration of 27 µM (Fig. 3A). However, peptide 4321 was less effective, because full inhibition was not accomplished even at a high peptide concentration of 120 µM (Fig. 3B). Accordingly, the quantitative analysis showed that the apparent IC50 values for 4286 and 4321 were ~9.4 and 100 µM, respectively (Table I).



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  		FIG. 2.
Effects of the RNase H-derived peptide 4321 (ELVNQIIEQLIKKEKVYLAW) on the activities of HIV-1 IN. Increasing concentrations of RT-derived peptide 4321were preincubated with a fixed amount of HIV-1 IN followed by assaying the IN activities and urea-PAGE analysis. The peptide concentrations are indicated at the bottom of each panel. A, analysis of the strand transfer activity of IN. B, analysis of the 3'-end processing activity of IN.

 



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  		FIG. 3.
The effect of peptides 4286 and 4321 on the disintegration activity of HIV-1 IN. Increasing concentrations of RT-derived peptides 4286 and 4321 were preincubated with a constant amount of HIV-1 IN, followed by assaying disintegration, using oligonucleotide D ("Experimental Procedures"). The peptides concentrations are indicated at the bottom of each panel. A, the effect of peptide 4286 on the disintegration activity of HIV-1 IN. B, the effect of peptide 4321 on the disintegration activity of HIV-1 IN.

 
HIV-1 RT and RT-derived Peptides Interact Directly with HIV-1 IN—In order to evaluate whether the inhibitory effect of RT-derived peptides 4286 and 4321 involved physical interactions with IN, we have tested all 20-residue peptides that span the whole 560-residue-long RT molecule for their direct IN binding capacity. Because no macromolecules other than IN and the peptides (or RT) were present in these reactions, every binding result was likely to reflect direct physical interactions between these components. To this goal, we have employed a dot-blot binding assay that involved the pre-binding of HIV-1 RT, or the RT-derived peptides, to a nitrocellulose filter, followed by a reaction with HIV-1 IN. Specific anti-IN antibodies were used to detect the bound IN (see "Experimental Procedures"). The observed interaction between the whole HIV-1 RT with HIV-1 IN (Fig. 4A) supported previous findings (9–11). Out of the 57 peptides tested, only 13 have shown a significant binding. The bindings by the positive peptides and two non-binding peptides (peptides 4269 and 4300), along with their test for nonspecific bindings, are shown in Fig. 4A. As three of these peptides (4321, 4316, and 4308) showed some nonspecific binding to the secondary antibodies used in this particular experiment, we have confirmed the specificity of interaction of the inhibitory peptide, 4321 (along with a positive control of peptide 4286) with IN, by using a different set of antibodies (Fig. 4B). Among the peptides shown to bind IN (Fig. 4A and Table II), only three peptides (4286, 4321, and to a lesser extent 4287) also inhibited IN enzymatic activities (Tables I and II). The fact that there are additional RT-derived peptides that bind IN but do not inhibit any of the IN activities indicated that not all peptide-IN interactions necessarily led to inhibition of catalysis. Because we have assumed (see below) that the inhibition was mediated primarily by steric hindrance of the IN catalytic domain, it is likely that these non-inhibitory peptides bound IN sites that were not directly involved in catalysis.


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  		TABLE II
Summary of the RT-derived peptides that bind HIV-1 IN and the inhibition data

The binding was performed by a dot-blot assay (described as set A in "Experimental Procedures"). The inhibition of the strand transfer activity of HIV-1 IN was determined according to "Experimental Procedures." ND, could not be determined due to non-specific deformations of the electrophoresis patterns of the reaction products.

 



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  		FIG. 4.
Dot-blot binding assay of HIV-1 RT or HIV-1 RT-derived peptides to HIV-1 IN. The experiment was conducted as described under "Experimental Procedures." The analysis was performed for the peptides found to be positive in a preliminary binding screening (data not shown) in addition to two non-binding peptides (4269 and 4300), which serve as negative controls. The designations of the tested peptides appear either to the left or to the right of the dots. The antibodies (Ab) used and the orders of their additions are indicated. A, blot 1, binding of full-length HIV-1 RT or RT-derived peptides to HIV-1 IN. Blot 2, control binding to the antibodies with no IN present. B, blot 1, binding of RT-derived peptides 4286 and 4321 to HIV-1 IN. Blot 2, control binding to the horseradish peroxidase (HRP)-conjugated anti-His6 antibodies with no IN.

 
Both HIV-1 RT and Peptide 4286 Inhibit the Disintegration Activity of HIV-1 IN-CCD—In addition to the inhibition of the 3'-end processing and forward strand transfer activities, peptide 4286 and to a lesser extent peptide 4321 were also shown to inhibit the disintegration activity of HIV-1 IN (Fig. 3). As the isolated core domain of HIV-1 IN exhibited, in vitro, a disintegration activity (2, 3, 27, 29, 30), it was possible that the two peptides interacted with IN-CCD and interfered with its activity. This recombinant segment of HIV-1 IN was 163 residues in length and was derived from residues 50–212 of the 280-residue full-length HIV-1 IN. The disintegration activity of IN-CCD was known to be substantially lower than that of the full-length enzyme (27, 30).

Because it was never tested whether the complete HIV-1 RT molecule inhibited HIV-1 IN CCD, we have first tested whether RT inhibits the disintegration activity of this IN domain. It is apparent that such an inhibition does exist (Fig. 5A) with full inhibition achieved at molar ratios of RT over IN-CCD between 2:1 and 4:1. The inhibition of IN-CCD-associated activity by peptides 4286 and 4321 was evaluated, and peptide 4286 was found to inhibit the disintegration activity of IN-CCD with an apparent IC50 value of about 22 µM peptide (Fig. 5B). On the other hand, peptide 4321 had no effect on this activity, even at a concentration as high as 270 µM (Fig. 5B).

HIV-1 RT and RT-derived Peptides 4286 and 4321 Physically Interact with IN-CCD—The direct interactions between HIV-1 IN-CCD and the full-length RT or either peptide 4286 or 4321 (along with peptide 4274 as a negative control) were tested. The results show specific binding of IN-CCD to the full-length RT and the two peptides tested (Fig. 6). In all, it is possible that the interactions and, consequently, inhibitions of IN activities observed with RT (or RT-derived peptides) are mediated for the most part through direct binding to the core domain of the IN.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
HIV-1 IN is considered one of the major targets for the development of novel anti-HIV-1 inhibitors. Among these are antibodies (10, 31–34), mono-, di-, and oligonucleotides (25, 35–37), various natural and synthetic compounds (35, 37–40), proteins (41), and peptides. The peptides found to be active against HIV-1 IN are those derived from sequences within IN (42–45), from random sequences identified in synthetic libraries (45–47), from combinatorial libraries of chemically synthesized peptides (45, 48), or from cellular IN-interacting proteins (45, 49, 50). In contrast to many anti-RT drugs used in the treatment of AIDS patients, there is only one group of non-peptide anti-IN drugs that has entered clinical trials (39, 51), and none is used routinely so far in patients.

It was recently reported that the HIV-1 IN activities are inhibited by HIV-1 RT (12, 13). Moreover, we have proposed that this phenomenon has a critical biological significance, as it is possible that the self-destructive process of auto-integration of the viral DNA into itself is blocked in the cytoplasm of infected cells by the observed effect of RT on IN. In the present study, we have defined the precise sequences within HIV-1 RT that inhibit and interact with HIV-1 IN. We show that two peptides inhibit in vitro the enzymatic activities of IN and, in addition to few other peptides, can bind IN directly.

Two inhibitory 20-residue peptides, 4286 and 4321, derived from the RT DNA polymerase and RNase H domains, respectively, were identified in the synthetic peptides library that spans the whole pol protein of HIV-1 (Figs. 1, 2, 3 and Table I). The extent of inhibition of the strand transfer and 3'-end processing activities by both peptides are similar (Table I). On the other hand, the disintegration activity of IN is by far more sensitive to inhibition by peptide 4286 than by peptide 4321. This suggests that the mechanisms of IN inhibition by the two peptides might be different. For that reason, we have checked whether the inhibitory effects of these two peptides are coupled, namely whether their combined effect is stronger than the effect of each separately. The results of this experiment show no such an apparent augmentation (data not shown), suggesting that inhibitions by both peptides might be still interrelated.

We have previously predicted that the inhibition of IN by RT results from direct interactions between the proteins (12). To confirm this, we have conducted the dot-blot experiment, described in Fig. 4. Similar to the whole RT protein, the two peptides 4286 and 4321 interact also with HIV-1 IN. The fact that the in vitro binding assay was a direct one indicates that the observed interactions are not mediated by nucleic acids or other cofactors, further supporting our hypothesis that the inhibition of IN by RT-derived peptides is caused by physical interactions. However, the same assay also shows that non-inhibitory peptides can also bind IN. This may indicate that the regions containing the sequences of these specific peptides (in the context of the RT molecules) also interact with IN, consequently increasing the affinity of the RT toward IN and enhancing the efficiency of the inhibition. This might also explain why the inhibitory peptides 4286 and 4321 are less efficient than the full-length RT in inhibiting IN activities. For that reason, full inhibition of the strand transfer activity of IN by the full-length RT was apparent at around equimolar ratios (12), whereas molar ratios, between IN and peptides 4286 or 4321, of ~1:70 and 1:300, respectively, were required for the complete inhibition of activity (as deduced from Figs. 1A and 2A, respectively). An alternative explanation can result from the potential differences between the folding of free peptides and the structures of the same sequences in the context of the RT protein. It is likely that only a fraction of the free peptide molecules can mimic the folding within the whole protein, and only this portion is effective in inhibition. Moreover, the proper conformation that leads to inhibition by peptides 4286 and 4321 depends on the length of the peptides. It is apparent that the full 20-residue sequences of both inhibitory peptides are crucial for the inhibition of HIV-1 IN, as shorter peptides, derived from sequences of the same peptides (or peptides with partial sequence overlaps with these peptides), exhibit a substantial reduction in the inhibitory effects (Table I).



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  		FIG. 5.
The effects of HIV-1 RT or RT-derived peptides (4286 and 4321) on the disintegration activity of IN-CCD. Increasing amounts of purified HIV-1 RT or of RT-derived peptides were preincubated with a constant amount of purified HIV-1 IN-CCD as described under "Experimental Procedures" and Fig. 3. A, the effect of HIV-1 RT on the disintegration activity of IN-CCD. The molar ratios between the heterodimeric (p66/p51) HIV-1 RT and homodimeric (p18/p18) IN-CCD are indicated at the bottom of each panel. B, the effect of peptides 4286 and 4321 on the disintegration activity of IN-CCD. Peptide concentrations are indicated.

 



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  		FIG. 6.
Dot-blot binding assay of HIV-1 RT or peptides 4286 and 4321 to HIV-1 IN-CCD. The experiments were performed as described under "Experimental Procedures." Blot 1, an experimental blot of the binding of HIV-1 RT or the inhibitory peptides (4286, 4321) and 4274, as a negative control, to HIV-1 IN-CCD. Blot 2, a control blot with no IN-CCD present.

 
Peptide 4286 is derived from a sequence within the DNA polymerase active site of HIV-1 RT, which is located in the "palm" subdomain (as parts of {alpha}-helix E and {beta}-sheet 9 (52)). This subdomain contains the highly conserved YXDD motif, typical of polymerases (53). This sequence exists almost entirely in peptide 4286 (except for the last D residue). This may explain our previous findings that HIV-1 and HIV-2 INs are inhibited by both HIV-1 and HIV-2 RTs and by the RT from another retroviral family, MLV RT (12).

Peptide 4321 (residues 516–535 in HIV-1 RT) is derived from the RNase H domain (across {alpha}-helix D' and {beta}-sheet 5' (52)). The fact that this peptide interacts with IN seems not to be in line with a very recent study (54), which suggests that the free RNase H domain of HIV-1 RT (residues 422–560, p15) is not capable of binding IN. This may result from differences in the methodologies used in the two studies. We have tested the binding of merely peptides, employing the dot-blot analysis, a test confirmed by IN inhibition. In contrast, Hehl et al. (54) employed for the p15 protein fragment a pull-down experiment, which might be less sensitive than our dot-blot assay. It is also important to note that, apart from peptide 4321, we have found that five additional RNase H-derived peptides bind directly HIV-1 IN (Fig. 4A and Table II).

An additional potential aspect, relating to the inhibition of IN by the RNase H-derived peptide, can stem from the three-dimensional similarities between IN-CCD and the RNase H of HIV-1 RT and Escherichia coli (55, 56). This can explain why RNase H inhibitors were found to inhibit IN activities as well (57). It is then possible that the RNase H-derived peptide 4321 interacts with IN due to similarities in structure, thereby interfering with the dimerization of IN. A similar finding (that peptides derived from IN-CCD interfere with IN dimerization and, consequently, inhibit IN activity) was already reported (42, 43).



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  		FIG. 7.
The conformations of peptides 4286 and 4321 within the three-dimensional model of HIV-1 RT. A space-filling model of the three-dimensional structure of the p66/p51 heterodimeric HIV-1 RT as found in Protein Data Bank entry 1FK9 [PDB] (58). The structure was displayed using the "MOLMOL" program (66). The p66 subunit of HIV-1 RT is shown in yellow, and the p51 subunit is shown in red. The residues of the DNA polymerase-derived peptide 4286 and of the RNase H-derived peptide 4321 are highlighted in cyan and pink, respectively. Both peptides are located on the surface of RT molecule. The different subdomains of the DNA polymerase domain are defined in the figure. The right-hand three-dimensional structure of the DNA polymerase domain is shown from two angles. Peptide 4286 is located at the base of the palm close to the DNA polymerase active site and penetrates outwards into the backhand surface. A, front view of the back of the right-hand conformation of the RT, with the different subdomains indicated. B, after rotating the molecule, an "inside" view of the palm subdomain of the right-hand p66 RT model.

 
Another issue of interest is to identify the locations of the sequences of peptides 4286 and 4321 in the surroundings of the globular structure of the heterodimeric p66/p51 RT protein. The DNA polymerase domain of the p66 subunit of RT resembles a right-hand structure, with fingers, thumb, and palm subdomain, connected to the RNase H domain by the connection (52, 58). Fig. 7 shows that both peptide segments (peptide 4286 in the p66 and p51 subunits and peptide 4321 in the RNase H domain of p66) are largely facing outwards, as they are on the surface of the protein, thus allowing potential interactions with other proteins. Yet the polymerase-derived peptide also penetrates from the "backhand" into the base of the palm subdomain (in the "forehand"), where the polymerase active site is located (Fig. 7B). This model supports the validity of the findings presented in this work about the inhibitory peptides, as only segments facing outwards of the RT can potentially interact with the IN molecule.



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  		FIG. 8.
Molecular docking of peptide 4286 into the core domain of HIV-1 IN. The global range molecular matching (GRAMM) program (19) was used for docking of peptide 4286 into HIV-1 IN-CCD. A generic high resolution docking was performed with grid step 2.0 Å and grid size 64 Å. Rotation was calculated in 10° intervals. The repulsion score was 40. The 20 highest scored hits were analyzed visually. The structure of HIV-1 IN-CCD used for docking was the one found in Protein Data Bank entry 1BIS [PDB] (21). The peptide conformation was as in Protein Data Bank entry 1REV [PDB] (61). In gray is the space-filling diagram of IN-CCD. The three highly conserved acidic-residues catalytic triad, DX(39–58)DX35E, are indicated in red-orange. HIV-1 RT-derived peptide 4286 is represented by the pink stick and ball diagram and is colored by atom types as follows: carbon, magenta; nitrogen, blue; oxygen, red; and sulfur, orange.

 
The DNA polymerase-derived peptide 4286 effectively interacts with IN-CCD and inhibits its disintegration activity, which is the only enzymatic activity expressed in vitro by this protein (Figs. 5 and 6). So far there are no crystal structures of the full HIV-1 IN, and most structural studies of INs involve the core domain. Therefore, we have docked peptide 4286 into the three-dimensional structure of HIV-1 IN-CCD. Among the published crystal structures of the catalytic core domain of HIV-1 IN, several represent an inactive form of the protein (55, 59), crystal form I (21). Crystal structures of the active core domain (crystal forms II and III (21, 60)) are very similar, with the exception of region 141–148. This region comprises a flexible loop, which is close to the active site. The conformation of this loop is strongly affected by the crystal packing and, presumably, by the binding of the substrate or inhibitor. By taking into account the above considerations, we have used the coordinates of HIV-1 IN-CCD from crystal form II (1.95 Å resolution) with the 141–148 region omitted (21). In the docking model (Fig. 8), we assume that the folding of peptide 4286, by which it interacts with IN, adopts a conformation similar to that one in the structure of HIV-1 RT (61). The peptide position, found in the docking solution by global range molecular matching (energy score 39), is likely to reflect the actual structure of the IN-CCD-peptide complex. Peptide 4286 binds the active site region of IN, close to the flexible active site loop (residues 141–148). Within this segment are residues Tyr-143 and Gln-148, known to be involved in viral DNA binding (62, 63). Tyr-143 was also shown to play a secondary role in catalysis (64). Hence, the binding of peptide 4286 to this region may cause a steric interference with the DNA binding and consequently inhibit IN activities. The proposed docking model (Fig. 8) involves several non-covalent interactions between the peptide and IN-CCD; residue Gln-174 of the peptide (according to the full RT numbering) makes a hydrogen bond with Ser-57 of the IN-CCD. In addition, residues Phe-171, Leu-168, and Ile-178 in the peptide interact by van der Waals interactions with amino acids Val-79, His-114, and Gly-149 in the IN-CCD, respectively. The proposed docking model between the HIV-1 IN-CCD and peptide 4286 suggested here is supported by a similar model that was already found in the crystal structure of avian sarcoma leukosis virus IN-CCD complexed with an HIV-1 IN inhibitor (65). Given that peptide 4321 was not effective in inhibiting the disintegration activity of IN-CCD, a similar docking of this peptide into the catalytic site of the IN is likely not to be valid and hence was not performed.

The study presented here still leaves several unanswered questions. First, little is known about the precise mechanism of inhibition, despite our prediction that it involves steric hindrance. Second, the model proposed for the interaction of peptide 4286 with IN-CCD does not exclude the possibility that RT residues, other than those shown to inhibit IN, are critical for the inhibition. Therefore, it will be important to study the involvements of the RT-derived peptides that bind but do not inhibit IN, as part of the whole RT molecule. Future site-directed mutagenesis of RT should indicate which residues are critical for binding and inhibition and whether the proposed docking model is correct. Likewise, mutagenesis of IN might also help in pinpointing the residues important for molecular interactions with RT. Accordingly, we have recently identified IN-derived peptides that interact with RT. Last of all, our final goal is to see whether the studied peptides and related ones can inhibit integration in HIV-infected cells and, hence, inhibit viral infectivity (and, as such, serves as a novel group of potent and highly specific anti-AIDS drugs). To this goal, the uptake of the peptides by human cells and their effects on viral infectivity will be studied.


   FOOTNOTES
 
* This work was supported by Grant 405/02 from the Israel Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains sequences of all studied peptides. Back

§ Performed this work in partial fulfillment of a Ph.D. thesis at Tel Aviv University. Back

|| Incumbent of The Gregorio and Dora Shapira Chair for the Research of Malignancies. To whom correspondence should be addressed: Dept. of Cell and Developmental Biology, The Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel. Tel.: 972-36409974; Fax: 972-36407432; E-mail: ahizy{at}post.tau.ac.il.

1 The abbreviations used are: RT, reverse transcriptase; IN, integrase; IN-CCD, integrase catalytic-core domain; HIV-1, human immunodeficiency virus, type 1; AIDS, acquired immunodeficiency syndrome;

RNase H, ribonuclease H; PIC, preintegration complex; MLV, murine leukemia virus; DDW, double distilled water; Me2SO, dimethyl sulfoxide; MOPS, 4-morpholinepropanesulfonic acid. Back

2 I. Oz Gleenberg, A. Herschhorn, and A. Hizi, unpublished data. Back


   ACKNOWLEDGMENTS
 
The following reagents were the generous gifts obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National Institutes of Health: HIV-1 HXB2R pol (20-mer) Peptides, Complete Set (catalog number 4358); and HIV-1 Clade B Consensus Pol (15-mer) Peptides, Complete Set (catalog number 6208). We also thank Noa Kirshenboim for purifying HIV-1 RT and Dr. S. Loya (both from our laboratory) for critically reading the manuscript.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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