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- Materials and methods
Monospecific antibodies were raised against a synthetic peptide K159 (SQGVVESMNKELKKIIGQVRDQAEHLKTA) reproducing the segment 147–175 of HIV-1 integrase (IN). Synthesis of substituted and truncated analogs of K159 led us to identify the functional epitope reacting with antibodies within the C-terminal portion 163–175 of K159. Conformational studies combining secondary structure predictions, CD and NMR spectroscopy together with ELISA assays, showed that the greater is the propensity of the epitope for helix formation the higher is the recognition by anti-K159. Both the antibodies and the antigenic peptide K159 exhibited inhibitory activities against IN. In contrast, neither P159, a Pro-containing analog of K159 that presents a kink around proline but with intact epitope conformation, nor the truncated analogs encompassing the epitope, were inhibitors of IN. While the activity of antibodies is restricted to recognition of the sole epitope portion, that of the antigenic K159 likely requires interactions of the peptide with the whole 147–175 segment in the protein [Sourgen F., Maroun, R.G., Frère, V., Bouziane, A., Auclair, C., Troalen, F. & Fermandjian, S. (1996) Eur. J. Biochem. 240, 765–773 ]. Actually, of all tested peptides only K159 was found to fulfill conditions of minimal number of helical heptads to achieve the formation of a stable coiled-coil structure with the IN 147–175 segment. The binding of antibodies and of the antigenic peptide to this segment of IN hampers the binding of IN to its DNA substrates in filter-binding assays. This appears to be the main effect leading to inhibition of integration. Quantitative analysis of filter-binding assay curves indicates that two antibody molecules react with IN implying that the enzyme is dimeric within these experimental conditions. Together, present data provide an insight into the structure–function relationship for the 147–175 peptide domain of the enzyme. They also strongly suggest that the functional enzyme is dimeric. Results could help to assess models for binding of peptide fragments to IN and to develop stronger inhibitors. Moreover, K159 antibodies when expressed in vivo might exhibit useful inhibitory properties.
The human immunodeficiency virus (HIV) replication cycle is initiated by integration of the viral DNA into the cell genome [1, 2]. Integrase (IN) [2–4] encoded by the virus pol gene catalyses integration sequentially through the deletion of two nucleotides at the conserved 3′ extremity of the viral long-terminal repeats (LTRs) (3′ processing) and the covalent insertion of the recessed 3′ viral DNA into the cell genome (strand transfer) [5–10]. Both steps proceed by a one-step transesterification mechanism . IN has no cellular counterpart and may be therefore considered as a specific target for development of anti-HIV therapy .
IN displays three functional domains [13, 14]. The amino-terminal part is highly conserved in retroviral integrases and contains a conserved C,C,H,H motif that binds zinc [15, 16]. This domain is critical for forming a stable complex with viral DNA, its structure is similar to the helix-turn-helix motif of DNA binding proteins and it forms dimers in solution . The carboxy-terminal domain presents a group of basic residues that bind DNA nonspecifically [18–20]. NMR studies performed on fragments of IN have established that the C-terminal domain presents similarities with the SH3 domain and is able to form dimers [21, 22]. Mutational studies have shown that both amino and carboxy-terminal domains are needed for 3′ processing and strand transfer [13, 23, 24]. The central domain is also highly conserved among retroviral INs as well as many transposases; it contains the D,D,E residues forming the catalytic triad of the enzyme [25, 26]. Structural studies of the central domain of HIV-1 and ASV INs have proven the membership of IN to the structurally related superfamily of polynucleotidyl transferases [27–29]. The core of IN displays a dimeric structure both in the crystal and in solution, although it has been proposed that in its active form the entire enzyme is a multimer [6, 27]. Like IN, the central domain IN50–212 is able to catalyze the desintegration reaction [6, 25, 26]. This occurs through liberation of the initial substrate used for strand transfer reaction by cleaving DNA at the CA dinucleotide junction between viral and target DNA .
To catalyze the cleavage and strand transfer reaction, IN interacts with viral and target DNA sequences. Several studies have shown that stable complexes can be formed between IN and viral DNA ends in the presence of divalent ions [30, 31]. Two lysine residues have been recently identified near the catalytic site to be involved in the specific interaction with DNA . A synthetic peptide (K159), reproducing the sequence 147–175 of the IN central domain, displays helical heptads and coiled-coil forming properties together with inhibition effects on the integration activity of IN [33, 34]. A mechanism, involving coiled-coil formation between K159 and its counterpart in the protein, was then considered, particularly as several hydrophobic side-chains of the 147–175 helical heptads have been found oriented outwards from the molecule, at the surface of the protein crystal structure .
To understand better the functionality of the 147–175 helical domain in the protein and to improve our peptide models for the IN inhibition, monospecific antibodies were prepared from rabbits immunized with the synthetic peptide K159 ( Fig. 1). The antibodies recognize injected K159, the whole IN and the central domain (IN). They also efficiently bind P159 a Pro-containing analog of the antigenic K159, as well as C-terminal peptides, proving that the functional epitope resides in the C-terminal part of K159. Moreover, when tested for their ability to inhibit the catalytic activity and the binding capacity of IN to DNA, the antibodies – likewise the antigenic K159 – alter significantly these two properties, therefore highlighting the importance of the segment 147–175 for the IN functionality. Remarkably, two antibody molecules react with IN in filter-binding assays, indicating that IN is dimeric.
Figure 1. The three functional domains of HIV-1 IN. Amino acid sequences of the synthetic peptides K159 reproducing the IN segment 147–175, and of the structural analog P159 as well as truncated analogs IN636, IN637, IN638 and IN642. Peptides were synthesized according to a standard Fmoc procedure as previously described . Each peptide carries a tyrosine residue and an amide group at the C-terminal position (not represented).
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A conformational analysis was carried out by circular dichroism (CD) and 1H NMR (aqueous and trifluoroethanol (TFE)/H2O mixtures) together with secondary structure predictions [35–39]. Data reveal the propensity of K159 for helix formation over its whole length, i.e. over its four heptad sequences. For P159 the helix propensity is still maintained in the C-terminal part. This peptide is recognized by antibodies to the same extent as K159, indicating that the epitope conformation is not affected by the substitution. However, only K159, which exhibits helicity over its full length and a good propensity for coiled-coil formation, presents the double ability to bind antibodies and to inhibit IN.
- Top of page
- Materials and methods
IN is required for integration of viral DNA into cellular genome [1, 2]. It is thus an attractive target for anti-HIV therapy, particularly since no cellular counterpart exists for this protein. During the last few years, considerable effort has been made to study functional and structural relationships in HIV-1 IN. It has been proven that IN is a globular protein requiring a multimeric structure to be functional [13, 14]. IN is known to form a highly stable interaction with viral long-terminal repeat substrate during in vitro integration reactions [13, 14]. Moreover, different antibodies have been raised against various epitopes of the protein [51–54] and several antibody fragments expressed in infected cells have been shown to decrease HIV-1 replication .
Antibodies raised against the peptide K159, reproducing the segment 147–175 located in the catalytic core of IN , present high specific affinity toward not only K159 but also toward IN and the central domain IN50–212. This signifies that K159 or its epitope portion mimics the helical conformation adopted by the segment 147–175 in the protein . Because the antigenic peptide K159 further acts as an inhibitor of IN, the pending question was whether inhibition rested on interaction of the sole epitope portion with the enzyme. This assumption comes from recent observations indicating that proteins such as the human growth hormone–receptor complex  and the erythropoietin–receptor complex  may interact through small portions of the binding epitopes. As a corollary, a cyclic heptapeptide that mimics a small binding epitope, i.e. the CD4 domain 1CC′ loop, has been shown to block the CD4 biological functions . Thus, it appears that small peptide portions reproducing functional epitopes may interfere with the protein interactions and inhibit biological functions, despite the commonly held view that interactions between proteins generally need large surfaces with numerous intermolecular contacts .
Conformational aspects of peptide–antibody recognition
In our previous report , we assumed that the inhibitory activity of K159 relied on the propensity of this peptide to adopt an amphiphilic helix and to form a stable coiled-coil structure with its helix counterpart on the protein leading to inhibition. This was suggested by the fact that the segment 147–175 corresponding to K159 displays an α helix structure at the surface of the crystal structure of IN50–212 and could be easily contacted by ligands such as antibodies, peptides or peptidomimetics. Here, Western blot binding data show that the surface exposure of the IN/IN50–212 segment 147–175 persists in solution. Another point is that antibodies recognize equally well K159 and its analog P159 with a helix-disrupting residue Pro in its middle. This posed the question of both the location of the functional epitope within the antigenic peptide and of how sensitive are antibodies to conformational changes occurring in the epitope. The use of truncated analogs reproducing the N-terminal part and the C-terminal part of K159 provided evidence that the functional epitope lies in the C-terminal portion, i.e. within residues 163 and 175 (GQVRDQAEHLKTA) of IN. As expected, the substitution of Ala by the helix breaker, Pro, in the middle of the epitope decreases significantly the binding affinity of antibodies, highlighting the importance of the epitope conformation. Actually, NMR data, in specifying that the epitope portion has the same propensity for helix stabilization in K159 and P159, explain why these two peptides are identically recognized by the antibodies. At the same time, the peptide gradation provided by the helix stabilization curves (excluding the N-terminal peptide lacking the epitope) parallels the peptide gradation provided by affinity data. These informations clearly indicate the impact of peptides helicity in the binding process. The stronger is the propensity of the C-terminal peptides to adopt an α-helical structure, the higher is the recognition of these peptides by antibodies. At the same time, the flexibility shown by the helix segment 147–175 in the protein  could explain the good cross-reactivity between the antigenic peptide K159 and the entire IN or IN50–212 exhibited by antibodies. Such features have been proven in other systems .
Conformational aspects of IN inhibition by peptides and anti-K159
None of the truncated analogs, i.e. neither IN638, which reproduces the N-terminal part of K159, nor IN636 and IN637, which both contain the functional epitopes, are able to block integration. These short peptides are less structured than their counterpart within the full-length peptide K159. Their inability to inhibit integrase could be thus, a priori, assigned to their weaker helix stability. Yet, the inability of P159 – in which the epitope displays same helix stabilization as in K159 – to inhibit IN suggests that the epitope by itself is insufficient to permit good binding to IN.
The ability of K159 both to form stable helix and autoassociate into dimeric structure strongly suggests that inhibition of IN proceeds through formation of a coiled-coil that maintains the peptide fixed to the protein segment 147–175. Actually, a number of at least three or four helical heptads are necessary to permit formation of stable coiled-coil structure, this requiring the cumulative effect of a sufficient number of hydrophobic and electrostatic contacts . Of all the peptides, only antigenic K159 fulfills conditions of dimer formation as it contains the needed four helical heptads, moreover in a correct alignment. In P159, in addition to the local helix destabilization, the Pro residue creates a kink in the middle of the molecule generating a V-shaped conformation rendering P159 unable to line up over its full length and thus inapt to form a stable coiled-coil, as shown by previous cross-linking experiments .
Filter-binding assays showed that inhibition of the binding of IN to its DNA substrates occurs at relatively modest anti-K159 concentrations, supporting the proposition that the IN segment 147–175 is implicated in DNA binding. The latter observation conforms to previous findings indicating (a) that a peptide reproducing the IN segment 139–152 cross-links to viral DNA  and (b) that residues K156 and K159 play a critical role for binding of IN to DNA . Moreover, single-chain variable antibody fragments (SFvs) raised against the domain 145–185 significantly inhibit the HIV-1 replication in human T-lymphoid cells . It can be therefore assumed that anti-K159 inhibits the IN activity by interfering with the association of the enzyme to substrate DNA. A similar mechanism is expected for the antigenic peptide K159, this exhibiting the capacity, like anti-K159, of inhibiting the binding of IN to DNA. Obviously, binding of anti-K159 or antigenic K159 to the target segment 147–175 could also alter the properties of E152, which belongs to the catalytic triad [D, D, E], including access of E152 to the DNA cleavage site.
Finally, an important feature is the stoichiometrical fixation of anti-K159 to IN, observed in the filter-binding assays with DNA substrates. The derived IC50 values indicate that two anti-K159 molecules react with IN, implying that the enzyme is dimeric in our experimental conditions.
The present data demonstrate a relationship between the structure and the function of the peptide domain 147–175 of the catalytic core of IN, critical for integration activity. This domain is exposed at the protein surface, both in crystal and in solution, and encompasses a peptide portion (163–175) that is an important functional epitope. Monospecific antibodies, raised against a synthetic peptide reproducing the peptide domain 147–175, strongly and specifically react with the epitope portion. These antibodies are shown to block the integration/desintegration activity of IN at low concentrations. This is an important result, confirming the importance of the 147–175 domain for the enzyme activity. The peptide 147–175 acts also as an inhibitor of IN, while a truncated peptide mimic of the epitope and a Pro-containing analog of the full peptide, appear too short and distorted, respectively, to insure IN inhibition. Owing to its marked propensity for coiled-coil formation, the full-length peptide is prone to interact with its helical counterpart domain 147–175 of IN according to a coiled-coil structure. This indicates that inhibition of integration proceeds through the binding of the antibody or the antigenic peptide to IN and not to DNA. In filter-binding assays, the complex of IN with the antibody or the peptide is unable to bind DNA, further reflecting the essential role played by the 147–175 domain toward the binding of IN to DNA. Other important information provided by the use of antibodies is that IN displays a dimeric structure in the DNA environment.
In vivo studies on HIV infected cells will tell us whether or not such antibodies are able to react with intracellular IN, the number of IN molecules seemingly being very small. Possibly, antibodies exhibiting high specificity against the 163–175 segment of IN when expressed in vivo will efficiently block the integration process. We also hope that structural information obtained from the conformational analysis of peptides will be helpful for developing binding models and preparation of inhibitors directed against the central domain of HIV-1 IN.