Understanding the isomerization of the HIV-1 dimerization initiation domain by the nucleocapsid protein

The specific binding of HIV-1 nucleocapsid (NC) to the hinge region of the kissing-loop (KL) dimer formed by stemloop 1 (SL1) can have significant consequences on its ability to isomerize into the corresponding extended duplex (ED) form. The binding determinants and the effects on the isomerization process were investigated in vitro by a concerted strategy involving ad hoc RNA mutants and electrospray ionization-Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry, which enabled us to characterize the stoichiometry and conformational state of all possible protein-RNA and RNA-RNA assemblies present simultaneously in solution. For the first time, NC-hinge interactions were observed in constructs including at least one unpaired guanine at the 5'-end of the loop-loop duplex, whereas no interactions were detected when the unpaired guanine was placed at its 3'-end. This binding mode is supported by the presence of a grip-like motif described by recent crystal structures, which is formed by the 5'-purines of both hairpins held together by mutual stacking interactions. Using tandem mass spectrometry, hinge interactions were clearly shown to reduce the efficiency of KL/ED isomerization without inducing its complete block. This outcome is consistent with the partial stabilization of the extra-helical grip by the bound protein, which can hamper the purine components from parting ways and initiate the strand exchange process. These findings confirm that the broad binding and chaperone activities of NC induce unique effects that are clearly dependent on the structural context of the cognate nucleic acid substrate. For this reason, the presence of multiple binding sites on the different forms assumed by SL1 can produce seemingly contrasting effects that contribute to a fine modulation of the two-step process of RNA dimerization and isomerization.     

Mechanism of hairpin-duplex conversion for the HIV-1 dimerization initiation site

We have used the dimerization initiation site of HIV-1 genomic RNA as a model to investigate hairpin-duplex interconversion with a combination of fluorescence, UV melting, gel electrophoresis, and x-ray crystallographic techniques. Fluorescence studies with molecular beacons and crystallization experiments with 23-nucleotide dimerization initiation site fragments showed that the ratio of hairpin to duplex formed after annealing in water essentially depends on RNA concentration and not on cooling kinetics. With natural sequences allowing to form the most stable duplex, and thus also the loop-loop complex (or "kissing complex"), concentrations as low as 3 mum in strands are necessary to obtain a majority of the hairpin form. With a mutated sequence preventing kissing complex formation, a majority of hairpins was even obtained at 80 mum in strands. However, this did not prevent an efficient conversion from hairpin to duplex in the presence of salts. Kinetic considerations are in favor of duplex formation from intermediates involving hairpins engaged in cruciform dimers rather than from free strands. The very first step of formation of such a cruciform intermediate could be trapped in a crystal structure. This mechanism might be significant for the dynamics of small RNAs beyond the strict field of HIV-1.     

Solution studies of the dimerization initiation site of HIV-1 genomic RNA.

Dimerization of HIV-1 genomic RNA is an essential step of the viral cycle, initiated at a conserved stem-loop structure which forms a 'kissing complex' involving loop-loop interactions (dimerization initiation site, DIS). A 19mer RNA oligonucleotide (DIS-19) has been synthesized which forms a stable symmetrical dimer in solution at millimolar concentrations. Dimerization does not depend on addition of Mg2+. RNA ligation experiments unambiguously indicate that the formed dimer is a stable kissing complex under the NMR experimental conditions.1H NMR resonance assignments were obtained for DIS-19 at 290 K and pH 6.5. Analysis of the pattern of NOE connectivities reveals that the helix formed by loop-loop base pairing is stacked onto the two terminal stems. Non-canonical base pairs between two essential and conserved adenines are found at the junctions between the two intramolecular and the single intramolecular helices.     

A structure-based approach for targeting the HIV-1 genomic RNA dimerization initiation site

Dimerization of the genomic RNA is an important step of the HIV-1 replication cycle. The Dimerization Initiation Site (DIS) promotes dimerization of the viral genome by forming a loop-loop complex between two DIS hairpins. Crystal structures of the DIS loop-loop complex revealed an unexpected and strong similitude with the bacterial 16S ribosomal aminoacyl-tRNA site (A site), which is the target of aminoglycoside antibiotics. As a consequence of these structural and sequence similarities, the HIV-1 DIS also binds some aminoglycosides, not only in vitro, but also ex vivo, in lymphoid cells and in viral particles. Crystal structures of the DIS loop-loop in complex with several aminoglycoside antibiotics provide a detailed-view of the DIS/drug interaction and reveal some hints about possible modifications to increase the drug affinity and/or specificity.     

Molecular determinants of HIV-1 NCp7 chaperone activity in maturation of the HIV-1 dimerization initiation site

Human immunodeficiency virus genome dimerization is initiated through an RNA–RNA kissing interaction formed via the dimerization initiation site (DIS) loop sequence, which has been proposed to be converted to a more thermodynamically stable linkage by the viral p7 form of the nucleocapsid protein (NC). Here, we systematically probed the role of specific amino acids of NCp7 in its chaperone activity in the DIS conversion using 2-aminopurine (2-AP) fluorescence and nuclear magnetic resonance spectroscopy. Through comparative analysis of NCp7 mutants, the presence of positively charged residues in the N-terminus was found to be essential for both helix destabilization and strand transfer functions. It was also observed that the presence and type of the Zn finger is important for NCp7 chaperone activity, but not the order of the Zn fingers. Swapping single aromatic residues between Zn fingers had a significant effect on NCp7 activity; however, these mutants did not exhibit the same activity as mutants in which the order of the Zn fingers was changed, indicating a functional role for other flanking residues. RNA chaperone activity is further correlated with NCp7 structure and interaction with RNA through comparative analysis of nuclear magnetic resonance spectra of NCp7 variants, and complexes of these proteins with the DIS dimer.     

Cation-dependent cleavage of the duplex form of the subtype-B HIV-1 RNA dimerization initiation site

The crystal structure of subtype-B HIV-1 genomic RNA Dimerization Initiation Site duplex revealed chain cleavage at a specific position resulting in 3′-phosphate and 5′-hydroxyl termini. A crystallographic analysis showed that Ba²⁺, Mn²⁺, Co²⁺ and Zn²⁺ bind specifically on a guanine base close to the cleaved position. The crystal structures also point to a necessary conformational change to induce an ‘in-line’ geometry at the cleavage site. In solution, divalent cations increased the rate of cleavage with pH/pKa compensation, indicating that a cation-bound hydroxide anion is responsible for the cleavage. We propose a ‘Trojan horse’ mechanism, possibly of general interest, wherein a doubly charged cation hosted near the cleavage site as a ‘harmless’ species is further transformed in situ into an ‘aggressive’ species carrying a hydroxide anion.     

A high affinity binding site for the HIV-1 nucleocapsid protein.

The nucleocapsid protein (NC) of HIV-1 is a small zinc finger protein that contributes to multiple steps of the viral life cycle, including the proper encapsidation of HIV RNA. This is accomplished through an interaction between NC and a region at the 5'-end of the RNA, defined as the Psi element. However, the specificity of NC for Psi or for RNA in general is not well understood. To study this problem, we used SELEX to identify high affinity RNA ligands that bind to NC. A 'winner' molecule (SelPsi), as well as a subregion of Psi RNA, were further characterized to understand the interaction between NC and SelPsi and its relationship to the interaction between NC and Psi. The comparison makes predictions about the sequence and structure of a high affinity binding site within the HIV-1 Psi element.     

Crystal structures of coaxially stacked kissing complexes of the HIV-1 RNA dimerization initiation site

We describe the crystal structures of the RNA dimerization initiation sites (DIS) of HIV-1 subtypes A and B. Both molecules adopt a hairpin conformation, with loop sequences consisting of 272-AGGUGCACA-280 and 272-AAGCGCGCA-280, respectively. This includes a six-base self-complementary stretch (underlined) that allows homodimerization through the formation of a loop-loop, or 'kissing-loop', complex. The DISs for the two sequences have identical conformations, and the two interacting hairpins show a perfect coaxial alignment. The conserved purines, A272 and R273, are stacked in a bulged-out conformation and symmetrically join the upward and downward strands of each hairpin by crossing the helix major groove. There is good agreement between these structures and previous results from chemical probing in solution, as well as with differences in magnesium dependence for dimerization. The overall shape of the kissing-loop complex is very similar to that of the previously determined subtype A DIS duplex form. Unexpectedly, the purine R273 is the only base seen at a different position and is responsible for the difference in topology between the two forms. We propose that the transition from kissing-loop duplex could occur by a recombination mechanism based on symmetrical chain cleavage at R273 of each hairpin and subsequent cross-religation, rather than by base-pair melting and subsequent reannealing.     

Solution RNA structures of the HIV-1 dimerization initiation site in the kissing-loop and extended-duplex dimers

Dimer formation of HIV-1 genomic RNA through its dimerization initiation site (DIS) is crucial to maintaining infectivity. Two types of dimers, the initially generated kissing-loop dimer and the subsequent product of the extended-duplex dimer, are formed in the stem-bulge-stem region with a loop including a self-complementary sequence. NMR chemical shift analysis of a 39mer RNA corresponding to DIS, DIS39, in the kissing-loop and extended-duplex dimers revealed that the three dimensional structures of the stem-bulge-stem region are extremely similar between the two types of dimers. Therefore, we designed two shorter RNA molecules, loop25 and bulge34, corresponding to the loop-stem region and the stem-bulge-stem region of DIS39, respectively. Based upon the chemical shift analysis, the conformation of the loop region of loop25 is identical to that of DIS39 for each of the two types of dimers. The conformation of bulge34 was also found to be the same as that of the corresponding region of DIS39. Thus, we determined the solution structures of loop25 in each of the two types of dimers as well as that of bulge34. Finally, the solution structures of DIS39 in the kissing-loop and extended-duplex dimers were determined by combining the parts of the structures. The solution structures of the two types of dimers were similar to each other in general with a difference found only in the A16 residue. The elucidation of the structures of DIS39 is important to understanding the molecular mechanism of the conformational dynamics of viral RNA molecules.     

HIV-1 RNA dimerization initiation site is structurally similar to the ribosomal A site and binds aminoglycoside antibiotics

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.     

Targeting the dimerization initiation site of HIV-1 RNA with aminoglycosides: from crystal to cell

The kissing-loop complex that initiates dimerization of genomic RNA is crucial for Human Immunodeficiency Virus Type 1 (HIV-1) replication. We showed that owing to its strong similitude with the bacterial ribosomal A site it can be targeted by aminoglycosides. Here, we present its crystal structure in complex with neamine, ribostamycin, neomycin and lividomycin. These structures explain the specificity for 4,5-disubstituted 2-deoxystreptamine (DOS) derivatives and for subtype A and subtype F kissing-loop complexes, and provide a strong basis for rational drug design. As a consequence of the different topologies of the kissing-loop complex and the A site, these aminoglycosides establish more contacts with HIV-1 RNA than with 16S RNA. Together with biochemical experiments, they showed that while rings I, II and III confer binding specificity, rings IV and V are important for affinity. Binding of neomycin, paromomycin and lividomycin strongly stabilized the kissing-loop complex by bridging the two HIV-1 RNA molecules. Furthermore, in situ footprinting showed that the dimerization initiation site (DIS) of HIV-1 genomic RNA could be targeted by these aminoglycosides in infected cells and virions, demonstrating its accessibility.     

NMR analysis of intra- and inter-molecular stems in the dimerization initiation site of the HIV-1 genome

Two positive-strand HIV-1 genomic RNAs form a dimer in virion particles through interaction of the dimerization initiation sites (DIS). The DIS RNA fragment spontaneously formed a "loose-dimer" and was converted into a "tight-dimer" by supplementation with nucleocapsid protein NCp7. This two-step dimerization reaction requires the whole DIS sequence [Takahashi et al. (2000) RNA 6, 96-102]. In the present study, we measured imino proton resonances to investigate the secondary structures of the two types of dimers in a 39-mer RNA covering the entire DIS (DIS39), including discrimination between intra- and inter-molecular base pairing. Both the presence and absence of inter-molecular NOE between (15)N-labeled and unlabeled DIS39 were unambiguously detected in an equimolar mixture of (15)N-labeled and unlabeled DIS39. The stem-bulge-stem structures in both dimers were confirmed and found to be very close to each other from clear superimposition of the NMR spectra in the two dimeric states. Nevertheless, the modes of base pairing in the stems of the loose- and tight-dimers were intra- and inter-molecular, respectively. Our results suggest a large structural alteration of genomic RNA occurs during virion maturation.     

HIV-1 nucleocapsid protein binds to the viral DNA initiation sequences and chaperones their kissing interactions

The chaperone properties of the human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein (NC) are required for the two obligatory strand transfer reactions occurring during viral DNA synthesis. The second strand transfer relies on the destabilization and the subsequent annealing of the primer binding site sequences (PBS) at the 3' end of the (-) and (+) DNA strands. To characterize the binding and chaperone properties of NC on the (-)PBS and (+)PBS sequences, we monitored by steady-state and time-resolved fluorescence spectroscopy as well as by fluorescence correlation spectroscopy the interaction of NC with wild type and mutant oligonucleotides corresponding to the (-)PBS and (+)PBS hairpins. NC was found to bind with high affinity to the loop, the stem and the single-stranded protruding sequence of both PBS sequences. NC induces only a limited destabilization of the secondary structure of both sequences, activating the transient melting of the stem only during its "breathing" period. This probably results from the high stability of the PBS due to the four G-C pairs in the stem. In contrast, NC directs the formation of "kissing" homodimers efficiently for both (-)PBS and (+)PBS sequences. Salt-induced dimerization and mutations in the (-)PBS sequence suggest that these homodimers may be stabilized by two intermolecular G-C Watson-Crick base-pairs between the partly self-complementary loops. The propensity of NC to promote the dimerization of partly complementary sequences may favor secondary contacts between viral sequences and thus, recombination and viral diversity.     

Convergence of natural and artificial evolution on an RNA loop-loop interaction: the HIV-1 dimerization initiation site

Loop-loop interactions among nucleic acids constitute an important form of molecular recognition in a variety of biological systems. In HIV-1, genomic dimerization involves an intermolecular RNA loop-loop interaction at the dimerization initiation site (DIS), a hairpin located in the 5' noncoding region that contains an autocomplementary sequence in the loop. Only two major DIS loop sequence variants are observed among natural viral isolates. To investigate sequence and structural constraints on genomic RNA dimerization as well as loop-loop interactions in general, we randomized several or all of the nucleotides in the DIS loop and selected in vitro for dimerization-competent sequences. Surprisingly, increasing interloop complementarity above a threshold of 6 bp did not enhance dimerization, although the combinations of nucleotides forming the theoretically most stable hexanucleotide duplexes were selected. Noncanonical interactions contributed significantly to the stability and/or specificity of the dimeric complexes as demonstrated by the overwhelming bias for noncanonical base pairs closing the loop and covariations between flanking and central loop nucleotides. Degeneration of the entire loop yielded a complex population of dimerization-competent sequences whose consensus sequence resembles that of wild-type HIV-1. We conclude from these findings that the DIS has evolved to satisfy simultaneous constraints for optimal dimerization affinity and the capacity for homodimerization. Furthermore, the most constrained features of the DIS identified by our experiments could be the basis for the rational design of DIS-targeted antiviral compounds.     

Aminoglycoside binding to the HIV-1 RNA dimerization initiation site: thermodynamics and effect on the kissing-loop to duplex conversion

Owing to a striking, and most likely fortuitous, structural and sequence similarity with the bacterial 16 S ribosomal A site, the RNA kissing-loop complex formed by the HIV-1 genomic RNA dimerization initiation site (DIS) specifically binds 4,5-disubstituted 2-deoxystreptamine (2-DOS) aminoglycoside antibiotics. We used chemical probing, molecular modeling, isothermal titration calorimetry (ITC) and UV melting to investigate aminoglycoside binding to the DIS loop–loop complex. We showed that apramycin, an aminoglycoside containing a bicyclic moiety, also binds the DIS, but in a different way than 4,5-disubstituted 2-DOS aminoglycosides. The determination of thermodynamic parameters for various aminoglycosides revealed the role of the different rings in the drug–RNA interaction. Surprisingly, we found that the affinity of lividomycin and neomycin for the DIS (K_d ~ 30 nM) is significantly higher than that obtained in the same experimental conditions for their natural target, the bacterial A site (K_d ~ 1.6 µM). In good agreement with their respective affinity, aminoglycoside increase the melting temperature of the loop–loop interaction and also block the conversion from kissing-loop complex to extended duplex. Taken together, our data might be useful for selecting new molecules with improved specificity and affinity toward the HIV-1 DIS RNA.     

Dissecting the protein-RNA and RNA-RNA interactions in the nucleocapsid-mediated dimerization and isomerization of HIV-1 stemloop 1

The specific binding of HIV-1 nucleocapsid protein (NC) to the different forms assumed in vitro by the stemloop 1 (Lai variant) of the genome's packaging signal has been investigated using electrospray ionization-Fourier transform mass spectrometry (ESI-FTMS). The simultaneous observation of protein-RNA and RNA-RNA interactions in solution has provided direct information about the role of NC in the two-step model of RNA dimerization and isomerization. In particular, two distinct binding sites have been identified on the monomeric stemloop structure, corresponding to the apical loop and stem-bulge motifs. These sites share similar binding affinities that are intermediate between those of stemloop 3 (SL3) and the putative stemloop 4 (SL4) of the packaging signal. Binding to the apical loop, which contains the dimerization initiation site (DIS), competes directly with the annealing of self-complementary sequences to form a metastable kissing-loop (KL) dimer. In contrast, binding to the stem-bulge affects indirectly the monomer-dimer equilibrium by promoting the rearrangement of KL into the more stable extended duplex (ED) conformer. This process is mediated by the duplex-melting activity of NC, which destabilizes the intramolecular base-pairs surrounding the KL stem-bulges and enables their exchange to form the inter-strand pairs that define the ED structure. In this conformer, high-affinity binding takes place at stem-bulge sites that are identical to those present in the monomeric and KL forms. In this case, however, the NC-induced "breathing" does not result in dissociation of the double-stranded structure because of the large number of intermolecular base-pairs. The different binding modes manifested by conformer-specific mutants have shown that NC can also provide low affinity interactions with the bulged-out adenine bases flanking the DIS region of the ED conformer, thus supporting the hypothesis that these exposed nucleotides may constitute "base-grips" for protein contacts during the late stages of the viral lifecycle.     

The crystal structure of the dimerization initiation site of genomic HIV-1 RNA reveals an extended duplex with two adenine bulges

BACKGROUND: An important step in retroviral replication is dimerization of the genomic RNA prior to encapsidation. Dimerization is initiated by the formation of a transient 'kissing-loop complex' that is thought to be subsequently matured into an extended duplex by the nucleocapsid protein (NCp). Although chemical probing and nuclear magnetic resonance spectroscopy have provided insight into the structure of the kissing-loop structure, no structural information concerning the extended-duplex state is available so far. RESULTS: The structure of a minimal HIV-1 RNA dimerization initiation site has been solved at 2.3 A resolution in two different space groups. It reveals a 22 base pair extended duplex with two noncanonical Watson-Crick-like G-A mismatches, each adjacent to a bulged-out adenine. The structure shows significant asymmetry in deep groove width and G-A base-pair conformations. A network of eight magnesium cations was clearly identified, one being unusually chelated by the 3' phosphate of each bulge across an extremely narrowed deep major groove. CONCLUSIONS: These crystal structures represent the putative matured form of the initial kissing-loop complex. They show the ability of this self-complementary RNA hairpin loop to acquire a more stable extended duplex structure. Both bulged adenines form a striking 'base grip' that could be a recognition signal, either in cis for another viral RNA sequence, or in trans for a protein, possibly the NCp. Magnesium binding might be important to promote and stabilize the observed extrahelical conformation of these bulges.