HIV-1 RNA Dimerization Initiation Site Is Structurally Similar to the Ribosomal A Site and Binds Aminoglycoside Antibiotics*,

Eric EnnifarDagger, Jean-Christophe Paillart, Roland Marquet, Bernard Ehresmann, Chantal Ehresmann, Philippe Dumas, and Philippe Walter§

From the UPR9002-Institut de Biologie Moléculaire et Cellulaire du CNRS, 15, rue René Descartes, F-67084 Strasbourg cedex, France

Received for publication, June 10, 2002, and in revised form, November 13, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Human immunodeficiency virus (HIV) genomic RNA is packaged into virions as a dimer. The first step of dimerization is the formation of a kissing-loop complex at the so-called dimerization initiation site (DIS). We found an unexpected and fortuitous resemblance between the HIV-1 DIS kissing-loop complex and the eubacterial 16 S ribosomal aminoacyl-tRNA site (A site), which is the target of aminoglycoside antibiotics. Similarities exist not only at the primary and secondary structure level but also at the tertiary structure level, as revealed by comparison of the respective DIS and A site crystal structures. Gel shift, inhibition of lead-induced cleavage, and footprinting experiments showed that paromomycin and neomycin specifically bind to the kissing-loop complex formed by the DIS, with an affinity and a geometry similar to that observed for the A site. Modeling of the aminoglycoside-DIS complex allowed us to identify antibiotic modifications likely to increase the affinity and/or the specificity for the DIS. This could be a starting point for designing antiviral drugs against HIV-1 RNA dimerization.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

The fight against HIV1 relies essentially on multitherapies targeting two viral enzymes: reverse transcriptase and protease. Due to a highly variable genome leading to rapid selection of mutations that confer resistance to enzymatic inhibitors, there is a strong need for targeting new viral molecules. New potential targets such as viral and cellular proteins involved in viral adsorption, fusion, and integration have been proposed (1). Functional sites within the genomic RNA, such as the Tat-responsive element (2) and the Rev-responsive element (3), have also been described as potential targets for the aminoglycoside antibiotic neomycin B.

Another interesting viral RNA target is the dimerization initiation site (DIS) of HIV-1 genomic RNA. This stem-loop is highly conserved among the different HIV-1 subtypes identified by sequence alignment. It initiates dimerization by forming a kissing-loop complex through base-pairing of a 6-nucleotide self-complementary sequence in each loop (4-8) (Fig. 1). It has been shown that this kissing-loop complex is converted in vitro by the nucleocapsid protein into a more stable complex, assumed to correspond to an extended duplex (9). Stabilization of the RNA dimer was also observed during maturation of the viral particles (10). Genome dimerization facilitates recombination (11) and is required for efficient RNA packaging (12-15) and reverse transcription (13, 16). Alteration of the DIS dramatically reduces viral infectivity (12-15).


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Fig. 1.   Secondary structure of the 1-615 RNA fragment containing the 5'-untranslated region of the HIV-1 RNA genome (adapted from Refs. 39 and 40). The DIS of two viral genomic RNA molecules (boxed in gray) initiates dimerization through formation of a kissing-loop complex. The beige-boxed region in the DIS kissing-loop complex represents the DIS region shown in Fig. 2A. The two molecules engaged in the kissing-loop complex are colored blue and green. The same color code is valid in the following figures. TAR, Tat responsive element; PBS, Primer binding site; SD, Splicing donor site; Psi, Packaging initiation site.

We recently solved the crystal structures of the DIS kissing-loop complexes of HIV-1 subtypes A and B (17). In the present study, we show that, unexpectedly, these structures are very similar to the ribosomal aminoacyl decoding site (A site) complexed with paromomycin (18, 19), an aminoglycoside antibiotic interfering with translation in bacteria. Based on this similarity, we modeled a DIS kissing-loop complex binding two paromomycin molecules. This model is strongly supported by gel shift, inhibition of lead-induced cleavage, and RNA footprinting experiments that demonstrate the ability of the DIS to bind aminoglycosides in a highly sequence-specific manner.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Molecular Modeling-- The model was obtained by superimposing residues involved in antibiotic binding in the paromomycin-A site crystal structure (19) on their counterparts within the DIS kissing-loop complex crystal structure (17). Using the program O (20), the sugar pucker of ribose 271 of the DIS structure was changed from C2'-endo to the C3'-endo conformation observed in the A site to prevent the steric clash with paromomycin. No other modification was required on the kissing-loop DIS crystal structure to obtain a conformation identical to that of the paromomycin-bound A site crystal structure (19).

Lead Probing and Band Shift Assays on the 23-nucleotide DIS Fragment-- Chemically synthesized 23-mer RNA fragments, corresponding to DIS subtypes A, B, and F, as well as A280U and U275C mutants of subtype A (7), were purchased from Dharmacon (Boulder, CO) and purified by ion-exchange chromatography using a Dionex PA-100 column as described (21). Kissing-loop complexes were obtained as follows: RNA was diluted to 6 µM in water, heat-denatured at 90 °C for 5 min, and then immediately cooled on ice for 5 min. Pure DIS extended duplex form was obtained by crystallization as described (21), and crystals were then dissolved in water. For band shift assays, after the addition of the dimerization buffer (final concentration: 2 mM MgCl2, 20 mM sodium cacodylate, 25 mM KCl) and antibiotic at different concentrations, the DIS was incubated at room temperature for 20 min. Alternatively, the antibiotic was added before dimerization buffer with this protocol leading to the same result. Native 15% polyacrylamide gel electrophoresis was performed at 4 °C in TBM buffer (45 mM Tris borate, pH 8.3, 0.1 mM MgCl2). RNA was revealed by staining with ethidium bromide. The U275C mutant, which cannot dimerize as a kissing-loop complex (7), was used as a monomer control. Tested aminoglycosides (purchased from Sigma) are shown in Table I. For lead probing, the DIS kissing-loop complex was incubated in acetate dimerization buffer (2 mM MgOAc2, 20 mM sodium cacodylate, 25 mM KOAc) for 25 min at 30 °C in the presence of 5 mM PbOAc2. The cleavage position was verified using 5'[gamma 32P]ATP-labeled RNA and an RNase T1 ladder (not shown). Products were analyzed by electrophoresis on denaturing 15% polyacrylamide gels and revealed as above.

                              
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Table I
Summary of the effect of tested aminoglycosides (Am, amikacin; KA, kanamycin A; KB, kanamycin B; Gm, gentamicin; Ge, geneticin; Ne, neomycin; Rb, ribostamycin; Pm, paromomycin; Sp, spectinomycin; To, tobramycin) on band shift, DMS probing, and lead-induced cleavage experiments for subtype-A, -B or -F wild-type DIS sequences, or several subtype-A DIS mutants
Lead-induced cleavage can be tested for the wild-type subtype-A DIS only (22,38). The experiments in which the addition of the aminoglycoside had no effect are indicated by (-) and those showing a clear RNA bandshift or a footprint are indicated by (++).

Chemical Footprinting on the HIV-1 5'-leader Region-- Chemical footprinting experiments were performed after the dimerization procedure on either wild-type or mutant 1-615 RNAs (400 nM) as described previously (22). Briefly, RNAs were allowed to dimerize in the dimerization buffer (50 mM sodium cacodylate, pH 7.5, 300 mM KCl, 5 mM MgCl2) for 15 min at 37 °C. Then, antibiotics were added at various concentrations, and incubation was continued for 15 min. Chemical modification was carried out in the presence of 2 µg of Escherichia coli total tRNA by the addition of dimethyl sulfate (Fluka) (0.8 µl, 1:20 dilution in ethanol) followed by incubation at 37 °C for 5 and 10 min. Reactions were stopped by precipitation in ethanol. Chain scission at methylated G-N7 was performed by aniline treatment as described (22). For each reaction, a control without alkylating reagent was treated in parallel. Modified/protected bases were detected by extension with avian myeloblastosis virus reverse transcriptase (Q-biogene) of a 5'[gamma 32P]ATP-labeled primer complementary to residues 294 to 311 of the HIV-1 RNA as described (22). Lead probing was performed in acetate dimerization buffer (50 mM Hepes-KOH, pH 7.5, 300 mM KOAc, 5 mM MgOAc2) by incubating RNA 1-615 for 5 min at 37 °C in the presence of 5 mM PbOAc2.

UV Melting Studies-- The subtype A RNA (23-mer) was diluted to 1.12 µM in 500 µl of water, heat-denatured at 90 °C for 4 min, and then immediately cooled at 0 °C for 10 min. After 15 min and incubation at 37 °C in 10 mM sodium cacodylate, the antibiotics were added to 5 or 50 µM in a final volume of 540 µl, and the incubation was continued for 15 min at 37 °C. Samples were degassed and transferred in UV spectrometer cells. The fusion and cooling cycles were done on a Biotek UVIKON-XL spectrometer at 260 nm and a Biotek Peltier thermosystem. The heating or cooling rate of 0.5 °C/minute between 15 and 90 °C was driven by the LifePowerTm Junior 1.1 application. The melting curves were processed by using Mathematica 4.1 (Wolfram research). The raw data were processed with a double-step smoothing procedure, allowing us to retrieve more accurate information than by ordinary methods. Essentially, the method is based upon low pass Fourier filtering applied, first, on the melting curve itself (to suppress most of the point-to-point high frequency noise) and, second, on the much less noisy numerical derivative of the smoothed melting curve. Care was also taken to suppress totally the usual "Gibbs phenomenon" affecting both ends of a reconstructed curve by a Fourier series.

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Sequence and Structure Similarities between the DIS and the Ribosomal A Site-- Sequence specificity of aminoglycoside antibiotics (especially paromomycin) binding to the prokaryotic ribosomal A site has been extensively studied by mutagenesis and footprinting analysis (23-25) and by surface plasmon resonance (26). First insights of the A site complexed with antibiotics were obtained by NMR (27, 28). Recently, a more detailed view was provided by the crystal structures of paromomycin bound to the A site of the complete 16 S rRNA (18) or to a small RNA fragment containing the A site (19).

Primary and secondary structure comparisons showed that the ribosomal A site (residues 1406-1410/1490-1495) is surprisingly similar to the dimeric HIV-1 DIS (residues 278-282/270-275). This fortuitous similarity holds true for the kissing-loop complex and duplex forms of the DIS since both topologies lead to identical base-pairing patterns (Fig. 2a). However, some differences are observed depending on the considered HIV-1 subtype: residue 273 is a G (and not an A) in subtype A, and the 275-278* base pair (the asterisk represents the second molecule of the dimer) is either UA or CG for subtypes A and B, respectively, instead of the universally conserved UU mismatch in the A site. Notably, the kissing-loop complex formed by the subtype F DIS, having both A273 and U275 residues, is the closest to the A site (Fig. 2A).


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Fig. 2.   Similarities between the ribosomal A site and the HIV-1 DIS as a kissing-loop complex or an extended duplex. As shown in A, the DIS, either as a kissing-loop complex or as extended duplex, displays sequence similarity with the eubacterial ribosomal A site. Open symbols correspond to the conserved residues involved in direct interaction with paromomycin in the A site (19), and boldface symbols correspond to the residues defining the minimal A site motif. The boxed residues highlight the sequence differences between the A site motif and the DIS. B, stereo view of a superimposition between the paromomycin-bound A site (red and orange) (19) and the DIS kissing-loop complex (blue and green) (17).

In addition to these sequence similarities, the paromomycin-bound ribosomal A site and the HIV-1 DIS kissing-loop complex share an analogous three-dimensional structure essentially characterized by two consecutive bulged-out adenines stacked on each other across from an unpaired adenine stacked inside the helix (17-19). A superimposition of the two structures shows that most of the atomic sites engaged in antibiotic binding are conserved in the DIS with a 0.9-Å root mean square deviation (Fig. 2B). The only steric conflict between the DIS and a bound antibiotic would occur between ribose 271 and the antibiotic ring I (Fig. 2B). This ribose is in a C2'-endo conformation in the DIS but in C3'-endo in its A site counterpart (G1491). Since residue 271 is located at the end of a helical part and followed by two bulged-out residues, its ribose pucker is by no means restricted due to the very low energetic barrier between the two conformations (29) and can thus easily return to the more usual C3'-endo pucker. Supporting this view, there are many examples of mixed C2'-endo-C3'-endo conformations in NMR and crystal structures (e.g. (21, 30). Finally, model building showed that only minor conformational changes of the bulged-out A272-R273 are required for the DIS to adopt an exact A site conformation (Fig. 3a). Due to the difference in topology between the DIS kissing-loop complex and the A site duplex, only two nonspecific contacts between phosphate 1493 with the O4' of ring I and the N3 of ring II of paromomycin are expected to be lost. Similarly, for subtype B, the presence of a C275*-G278 base pair would induce the loss of a direct contact between the O4(U275*) and the N1 of paromomycin ring II (Fig. 3B). However, the presence of a guanine instead of an adenine at position 273 of the subtype A DIS has no influence on a putative antibiotic binding due to the bulged-out conformation of this residue.


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Fig. 3.   A, molecular modeling of the subtype A HIV-1 DIS kissing-loop complex with two paromomycin molecules per dimer. Positions that are protected toward chemical modifications upon the addition of antibiotic are shown as orange spheres. The solvent-accessible surface (probe radius of 1.4 Å) is represented in transparency around one paromomycin molecule. Black arrowheads indicate the positions protected toward lead-induced cleavage by antibiotics. B, contacts observed in the subtype A DIS-paromomycin/neomycin model. Positions of the RNA that became protected are circled in orange.

Notably, the three-dimensional similarities described above are not found in a previous NMR structure of the DIS kissing-loop complex (31) due to the non-standard geometry of the interstrand loop-loop helix and the bulged-in unpaired purines (A272, A273) occluding the potential antibiotic binding pocket. Furthermore, the G271-C281 base pair (Fig. 2A), whose homologue (C1409-G1491) is important for antibiotic binding in the A site, is not formed in the NMR structure.

Specific Neomycin and Paromomycin Binding to the DIS Kissing-Loop Complex-- We first checked the binding of a collection of aminoglycosides by band shift experiments on 23-nucleotide fragments representative of minimal DIS of subtypes A, B, and F (Table I). Besides these natural sequences, we introduced two point mutations in the subtype A DIS: U275C, which inhibits kissing-loop complex formation (7), and A280U, since this position corresponds to the A site A1408 and its mutation is expected to prevent antibiotic binding (23) but not dimerization. Among all tested antibiotics, only neomycin and paromomycin, differing only by a 6'NH3+ or 6'OH group (Table I and Fig. 4a), yielded a significant band shift of the DIS kissing-loop complexes (Fig. 4A and Table I). This band shift was observed whether the antibiotic was added before or after dimer formation. That the band shift was partial might possibly result from a dissociation of the complex under electrophoresis conditions. Importantly, binding was restricted to subtypes A and F (Fig. 4A and Table I), indicating a sequence- and ligand-specific interaction. As expected from the model, mutant A280U did not bind neomycin (Fig. 4A). We also checked antibiotic binding on dissolved crystals (21) of the subtype A DIS duplex form. Interestingly, although this form corresponds to the A site topology (Fig. 2A), we did not observe any antibiotic binding, even at millimolar concentrations (Fig. 4A). This is in perfect agreement with two independent observations. First, the crystal structure of the DIS duplex form (21) shows a G273*-A280 base pair (not observed in the kissing-loop complex topology (17)) closing the antibiotic pocket. Second, an A1493G mutation in the A site abolishes antibiotic binding (23), most likely by allowing formation of an A1408-G1493 base pair equivalent to G273*-A280 observed in the DIS duplex form. Finally, both paromomycin and neomycin are unable to dissociate the preformed kissing-loop complex (Fig. 4A).


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Fig. 4.   Paromomycin and neomycin binding to the 23-nucleotide DIS kissing-loop complex. A, band shift assays with increasing concentrations of neomycin B (in µM) on subtype F and B DIS kissing-loop complexes, on A280U mutant of subtype A, and on subtype A extended duplex form (left) or in the presence of 1 mM paromomycin (P), neomycin (N), and tobramycin (T) on subtype A DIS kissing-loop complex (right). The lane labeled M corresponds to a monomer control obtained with the U275C mutant. Lanes labeled 0 are controls without antibiotic. The positions of the dimer (D) and monomer (M) are shown on the left. B, denaturing gels showing lead-induced cleavage on subtype A DIS kissing-loop complex in the presence of increasing concentrations (in µM) of neomycin, paromomycin, and tobramycin. Lanes labeled 0 and - Pb are controls without antibiotic and without lead, respectively.

As aminoglycosides were reported to modulate ribozyme cleavage by displacing metal ions (for a review, see Ref. 32), we further checked antibiotic binding to the minimal DIS fragment, taking advantage of the strong lead-induced cleavage specifically observed for subtype A DIS (22). In the absence of antibiotic, one strong cleavage was observed in the minimal DIS fragment between A272 and G273 residues (Fig. 4B) in the vicinity of the expected antibiotic binding site (Fig. 3A). Even using a 5 mM Pb2+ concentration, cleavage was strongly reduced upon the addition of 15 µM paromomycin and abolished in the presence of 7.5 µM neomycin (Fig. 4B). These aminoglycoside concentrations are 3 orders of magnitude lower than those required for inhibition of the tRNAPhe lead-induced cleavage (33), indicating a highly specific antibiotic-DIS interaction.

We then tested the DIS-antibiotic interactions in a much larger RNA context (Fig. 1) by using a 1-615 RNA fragment containing the whole 5'-untranslated region of the HIV-1 genome (Fig. 1). First, we verified that paromomycin and neomycin (but not tobramycin) also inhibit lead-induced cleavage of this RNA (Fig. 5a and Fig. 6). Second, we noticed that the ribosomal A site and the HIV-1 DIS kissing-loop complex share a similar pattern of chemical modifications by alkylating agents (DMS and diethylpyrocarbonate) (Fig. 6) (22-24, 34, 35). Such a similar pattern is well accounted for by the DIS kissing-loop crystal structure (17) but not by the corresponding NMR structure (31). Thus, we probed antibiotic binding to 1-615 RNA using chemical footprinting. As expected from the model derived from our crystal structure (Fig. 3A), we did observe strong protections against DMS modification of N1(A280) and N7(G274) positions in subtype A and F DIS upon the addition of neomycin and paromomycin (Figs. 5 (B and C) and 6, and Fig. S1 of the supplementary material). No other significant alteration of the chemical modification pattern was observed in the tested region (nucleotides 150-300) (Fig. S1 of the supplementary material). Interestingly, the minimal antibiotic concentrations necessary for maximal protection are comparable with those necessary for the same protection on the A site within the complete 16 S rRNA (24). In contrast, again as expected from our model, only very weak protections of the subtype B DIS sequence were observed at 100 µM neomycin or paromomycin (Fig. 6, and Fig. S1 of the supplementary material), in agreement with the absence of band shift (Fig. 4A). Furthermore, no footprinting was observed on U275C (not shown) and A278G (Fig. S1 of the supplementary material) DIS mutants that are unable to dimerize (7). The results concerning these two mutants will be discussed later. Finally, no protection was observed on any DIS sequences with the other antibiotics tested in agreement with our band shift experiments (Table I) and with their 10-100-fold weaker affinity for the ribosomal A site (26).


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Fig. 5.   Autoradiographs of footprinting experiments in the presence of antibiotics. A, lead-induced cleavage on subtype A 1-615 RNA in the presence of increasing concentrations (0, 10, 50, and 100 µM) of neomycin, paromomycin, and tobramycin. The lane labeled no lead is a control without lead. B and C, DMS footprinting experiments in the presence of increasing concentrations of antibiotics with subtype A (B) and subtype F (C) 1-615 RNAs. The probing reactions were carried out for 0 (unmodified control), 5, and 10 min at 37 °C in the absence or in the presence of 10, 50, and 100 µM of neomycin, paromomycin, or tobramycin. Positions of nucleotides whose reactivity is changed (protection) upon the addition of antibiotics are indicated. The lanes labeled U, A, C, and G correspond to dideoxy sequencing reactions.


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Fig. 6.   Summary of the chemical modifications of the ribosomal decoding region (A site) and DIS kissing-loop complexes (subtypes A, F, and B). Nucleotides that are modified by DMS are indicated by a circle (position N1-A) or a square (position N7-G). Nucleotides protected from DMS modification by aminoglycoside antibiotics are indicated by open triangles.

Notably, chemical probing did not reveal any increased accessibility of the DIS loop sequence upon binding of the antibiotics (Figs. 5-6), suggesting that it does not induce dissociation of the kissing-loop complex. Accordingly, both paromomycin and neomycin are unable to dissociate a preformed kissing-loop complex or to prevent dimerization even at 100 µM concentration (Fig. 4A).

Recently, neomycin was reported to bind to a monomeric DIS-containing 176-nucleotide fragment corresponding to the encapsidation region of subtype B HIV-1 genomic RNA (36). The latter study seems to contradict our present results in which we show that neomycin does not bind either to a monomeric DIS mutant (Fig. S1 in the supplementary material) or to a subtype B DIS RNA (Table I and Fig. 4A, and Fig. S1 of the supplementary material). However, the very low salt conditions used in the study by McPike et al. (36) (10 mM Tris-HCl, pH 7.0), and especially the absence of divalent cations, might be responsible for this discrepancy.

Antibiotic Binding and DIS Thermal Stability-- To gain some insight into the effect of antibiotic binding on kissing-complex stability, we performed a preliminary UV melting study of the subtype A DIS (23-mer) either alone or with a specific binder (neomycin or paromomycin). For negative control, we have also used the DIS with an unspecific binder (tobramycin). We did not use magnesium to limit the spontaneous hydrolysis of RNA during the high temperature conditions.

In low salt conditions, adding any of the tested binders resulted in an important shift of the Tm of the DIS from 55.3 °C to higher temperatures (79, 75.6, and 76.2 °C for 5 µM neomycin, paromomycin, and tobramycin, respectively), similar to the shift observed from the addition of 50 µM spermidine (73.5 °C) (not shown). In the presence of 100 mM NaCl, where the Tm of the DIS alone increased to 65.6 °C, we noticed a smaller but fully significant stabilization by 6.4 °C for neomycin, a marginal one (1.7 °C) for paromomycin, and an insignificant one (0.7 °C) for tobramycin (Fig. 7). We interpret the effect of any of these positively charged molecules in low salt conditions as resulting from aspecific ionic interactions, whereas in more stringent salt conditions, only neomycin and, to a lesser extent, paromomycin showed a stabilizing effect. This can be correlated with the fact that neomycin also protects better than paromomycin against lead cleavage, whereas tobramycin does not protect at all (Fig. 4B).


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Fig. 7.   Normalized derivative of the melting curves for the subtype A DIS at 1 µM. Comparison in 100 mM NaCl of the DIS alone or with 5 µM neomycin (A), 5 µM paromomycin (B), and 5 µM tobramycin (C). In all graphs, the curve in dashed line corresponds to the DIS without antibiotics in 100 mM NaCl.

Discussion of the Model-- Taken together, our present results on large RNA fragments strongly support the model (Fig. 3A) that led to the experiments and the relevance of the crystal structure from which it was derived. Indeed, the antibiotic-bound A site crystal structures and the corresponding model for the DIS (Fig. 3A) account for important features. First, that the A278-U275* pair present in subtypes A and F, but not in subtype B, is important for neomycin and paromomycin binding is consistent with the fact that the conserved U1406-U1495 pair in the A site can be changed to AU without affecting antibiotic binding (23, 24, 26). In contrast, a U1495C mutation (thus making the A site closer to the subtype B DIS) led to a loss of antibiotic binding (24). Indeed, our model shows that only U275* (U1495 in the A site) is engaged in antibiotic binding, whereas A278 is not (Fig. 3). Second, since the kissing-loop complex contains two identical copies of the A site motif (Fig. 1), it should bind two antibiotic molecules per kissing-loop complex. The model shows that this is possible without any steric conflict between the two ligands (Fig. 3A). Experimentally, this is strongly supported by almost complete protections against lead-induced cleavage (Fig. 4B) and DMS modification (Figs. 5 (B and C) and 6) upon antibiotic binding.

Notably, binding of either neomycin or paromomycin to subtype A DIS should displace a magnesium cation that is bound through water molecules to the 275UGCA278 sequence in the loop-loop helix and that is important for a stable dimerization (17). On the contrary, the subtype B DIS, which does not require magnesium for dimerization, does not bind magnesium on the corresponding 275CGCG278 sequence (17). It thus appears that the presence of an H-bond donor amino group on C275, instead of an O4 H-bond acceptor as on U275, explains the lack of binding of both antibiotic and magnesium to the subtype B DIS. Finally, as residue 278 seems neutral for antibiotic binding, the lack of footprinting on the monomeric A278G DIS mutant suggests that neomycin and paromomycin binding is restricted to dimeric species.

The DIS as a New Target for Aminoglycoside-like molecules?-- We have shown that, due to an unexpected resemblance to the prokaryotic ribosomal A site, subtypes A and F DIS kissing-loop complexes bind neomycin and paromomycin, likely displacing a magnesium ion. The binding of these aminoglycosides does not prevent dimerization and even provides further stabilization of the dimer. Since neomycin shows micromolar binding affinity to the DIS, neomycin-based aminoglycosides can be viewed as lead molecules for targeting the DIS in view of overstabilizing the dimer. One might anticipate that DIS binders might interfere with recognition of the DIS by the RNA encapsidation machinery and with the late stages of reverse transcription. However, it is necessary to test whether neomycin or paromomycin actually affect viral replication through their interaction at the level of the DIS. Experiments are in progress to address this point. Notably, neomycin has already been shown to inhibit the viral production of HIV-1 up to 85% in a dose-dependent fashion in U1 cells, although this was interpreted as the result of the inhibition of the Rev-RRE interaction by this aminoglycoside (3). Another important issue, not addressed in this study, is a possible adverse effect of the basic nucleocapsid protein on antibiotic binding.

Molecular modeling of the DIS-antibiotic complex, based on solid facts and very minor assumptions, suggests guiding lines for a rational drug design aimed at improving ligand affinity or modifying its specificity. In particular, one would expect viral escape mutants to arise by adopting a subtype B DIS self-complementary sequence, which is insensitive to drugs tested in this study. This concern could be addressed by targeting the subtype B DIS sequence, which would require, for instance, the substitution of the hydroxyl group for the ammonium N1 group of ring II (Fig. 3B). Another obvious improvement suggested by our model would be the use of symmetrical molecules, obtained by covalently linking the two drugs in interaction with the kissing-loop complex (Fig. 3A). The link should involve the N1 (ring II) groups of each molecule, which are only 5.6 Å apart. As only rings I and II appear to be essential for binding, the size of such a symmetric drug could be reduced by suppressing rings III and IV, i.e. using a neamine core for symmetrization. Novel antibiotics that bind to the ribosomal A site were recently designed using such a neamine core and a drug design strategy (37), thus showing that a rational approach for modifying and optimizing aminoglycosides is feasible. We are well aware, however, that these optimistic considerations should be moderated by keeping in mind the difficulties always encountered on the way from a potential antiviral molecule to a drug amenable to therapeutic use.

    ACKNOWLEDGEMENTS

We thank E. Westhof and Q. Vicens for fruitful discussions.

    FOOTNOTES

* This work was supported by the "Agence Nationale de Recherche sur le SIDA" (ANRS).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplementary data showing audioradiographs of dimethyl sulfate (DMS) footprinting experiments in the presence of increasing concentrations of neomycin with 1-615 RNA fragments. Left, subtype A; middle, subtype A; and right, mutant A278G. The probing reactions were carried out for 0 (unmodified control), 5, and 10 min in 37 °C in the absence (left panel) or in the presence of 10, 50, and 100 µM of neomycin. The position of the nucleotide whose reactivity is changed (protection) upon the addition of antibiotic is indicated (position N1-A280 for subtype A RNA only). The lanes labeled U, A, C, and G correspond to dideoxy sequencing reactions.

Dagger A fellow of ANRS.

§ To whom correspondence should be addressed. Tel.: 33-388417052; Fax: 33-388602218; E-mail: p.walter@ibmc.u-strasbg.fr.

Published, JBC Papers in Press, November 14, 2002, DOI 10.1074/jbc.M205726200

    ABBREVIATIONS

The abbreviations used are: HIV, human immunodeficiency virus; HIV-1, HIV type 1; DIS, dimerization initiation site; DMS, dimethyl sulfate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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