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A new NMR solution structure of the SL1 HIV-1Lai loop–loop dimer

Dimerization of genomic RNA is directly related with the event of encapsidation and maturation of the virion. The initiating sequence of the dimerization is a short autocomplementary region in the hairpin loop SL1. We describe here a new solution structure of the RNA dimerization initiation site (DIS) of HIV-1Lai. NMR pulsed field-gradient spin-echo techniques and multidimensional heteronuclear NMR spectroscopy indicate that this structure is formed by two hairpins linked by six Watson–Crick GC base pairs. Hinges between the stems and the loops are stabilized by intra and intermolecular interactions involving the A8, A9 and A16 adenines. The coaxial alignment of the three A-type helices present in the structure is supported by previous crystallography analysis but the A8 and A9 adenines are found in a bulged in position. These data suggest the existence of an equilibrium between bulged in and bulged out conformations in solution.

Retroviruses are unique in having a diploid genome (1). This diploid feature is directly involved in different stages of the virion life cycle such as recombination and encapsidation (27). Electron microscopy of HIV-1 has shown the RNA genome to consist of two identical genomic RNA strands joined within their 5′ leader, at a 100 bases long region defined as the dimer linkage structure (DLS) (811). In the HIV-1Lai strain, the dimerization initiation site (DIS) is a 6 nt autocomplementary sequence located in the highly conserved 35 bases long sequence stem–loop (SL1) (Figure 1) (12,13). Mutations in the DIS region or deletion of the entire autocomplementary sequence have been shown to affect the yield of dimer formation consequently reducing virion infectivity (1422). Further more this deficit in virulence is closely implicated with defects in encapsidation, indicating a direct involvement between dimerization and formation of the capsid.

In vitro studies with 200 nt RNA sequences containing the DIS region show that HIV-1 dimer formation is initiated via a loop–loop interaction between two RNA molecules (23,24). This metastable complex is converted at 55°C, or at lower temperatures in the presence of the NCp7 protein, into a more stable conformation, an extended dimer (23,2527). Shorter sequences comprising the entire 35 nt SL1 sequence (28) or its extremity of 23 nt (29,30) remain able to dimerize into two different conformations of different stabilities. The structure of the loop–loop dimer or ‘kissing complex’ has been resolved by NMR (31) and crystallography (32). Both studies utilize different RNA sequences. The NMR sequence presents a modification of the base pair order in the stem contrary to the crystallography sequence. Although both studies report identical nucleotide sequences of the loop, discrepancies appear when both proposed structures are compared. The NMR solution structure shows a symmetrical homodimer where the average plane of the loops is roughly perpendicular to the axis of the stems whereas in the crystallographic structure it shows a perfect coaxial alignment with the stems. Unlike the NMR structure where all the bases are inside the RNA molecule, the crystallographic structure presents the A8 and A9 adenines as stacked in a bulged out conformation. The NMR structure shows that the last base pair of the stem (G7-C17) is not paired contrary to the model proposed by crystallography where bases appear paired. Previous NMR findings from our laboratory have confirmed the presence of base pair (G7-C17) in the loop–loop complex in the 23 nt sequence in the wild-type. To clarify these contradictory reports, we solved the 23 nt structure of the SL1 region in the HIV-1Lai wild-type without base pair rearrangement in the stem by multidimensional NMR spectroscopy. We found a new solution structure of the ‘kissing complex’. Its structure is composed by two hairpins, coaxially aligned, linked by their autocomplementary sequence with the loop adenines appearing in a bulged in position.

RNA strands were synthesized by in vitro transcription with T7 RNA polymerase using synthetic templates as described previously (33). Template oligonucleotides (5′-GTTGCCGTGCGCGCTTCAGCAACCTATAGTGAGTCGTATTA and 5′- TAATACACT CACTATAG) were purchased from Eurogentec (Belgium). T7 RNA polymerase was isolated from Escherichia coli strain BL21/PAR1219 as described in Davanloo et al. (34). RNAs were synthesized with unlabelled NTPs (Sigma) or labelled 15N/13C NTPs (Silantes). Following phenol extraction and ethanol precipitation, all RNA samples were purified by PAGE under denaturing conditions with 8 M Urea. The SL1 24 nt RNA (Figure 1) was recovered by diffusion in water. RNA was desalted by reverse phase chromatography on Sep-Pak C18 (Waters) and by gel filtration on DG-10 column (Bio-Rad). A total of 10 µM desalted sample was refolded by heating at 90°C (3 min) and snap-frozen at −20°C. The sample was concentrated by lyophilization. The final concentrations of the unlabelled and labelled SL1 24 nt were 2 mM and 0.6 mM, respectively. Each sample was placed in a Shigemi tube in 170 µl of 90% water, 10% deuterium oxide at pH 5.7, without addition of any salt for the experiments involving exchangeable protons. For the non-exchangeable protons experiments, samples were lyophilized and solved again in 100% deuterium oxide. The NMR experiment was generally run below 12°C to avoid the transition toward the extended structure.

NOESY, DQF-COSY, TOCSY, 15N and 13C HSQC, HNN-COSY, HMBC, NMR pulsed field-gradient spin-echo experiments (35) were run on a Varian 600 MHz UNITY INOVA spectrometer. 15N and 13C double-half-filter NOESY experiments were run on a Varian 600 MHz and on an 800 MHz UNITY INOVA spectrometer.

Pf1 phage was purchased from ASLA Ltd (Riga, Latvia). Phage concentrations were adjusted by weighing appropriate amounts of a 50 mg/ml stock solution in H2O. Phage alignment was verified by measuring the splitting of the HOD signal in 90% H2O/10% D2O. Resonance assignments are reported relative to DSS.

The structure of the loop–loop complex was calculated using torsion angle molecular dynamics (TAMD) as described by Stallings and Moore (36). First, one hundred random structures were generated using CNS 1.0 (37). The basic started structures were subjected to a simulated annealing-restrained molecular dynamics protocol in torsion angle space. TAMD consisted of a high-temperature torsion angle dynamics for a total of 15 ps at 20 000 K with a Van der Waals scale factor of 0.1. The molecules were then cooled to 300 K in 4000 steps for 60 ps of torsion angle dynamics with a Van der Waals scale factor increased from 0.1 to 1. This was followed by another simulated annealing step in Cartesian space from 2000 K to 300 K in 3000 steps for 15 ps where the Van der Waals scale factor increased from 1 to 4, finalized by 2000 steps of Powell minimization with full electrostatic and Van der Waals terms. The structures with zero violation on NOE distance (0.2 Å), dihedral (5°) and with the lowest energies were selected. Molecular graphics image were visualized with the MOLMOL program (38). All helical parameters were determined using CURVES (39,40).

The coordinates of the eighteenth structures have been deposited in the Protein Data Bank (PDB ID code is 2F4X).

The sequential assignment of the imino and amino protons of the dimer was obtained by using a 80 ms mixing time NOESY spectrum recorded at 10°C. The three guanine imino protons of the loop were linked by hydrogen bonds to cytosines C11, C13, C15 and a sequential assignment G10-G14-G12 was observed. These data were confirmed by using the 15N HSQC and HNN-COSY spectra. 15N HSQC discriminated the 15NH belonging to T, G or U by their 15N chemical shifts (41). HNN-COSY (42) allowed the direct observation of hydrogen coupling by a cross hydrogen scalar coupling between the imino 15N1 of the donor guanines with the hydrogen bond acceptor 15N4 atom of the complementary cytosines, in the Watson–Crick base pairs. The pairing of G7 and of C17 bases, which is essential for the modelling, has been shown by several spectra. First, the connectivity 15N1-1H of G7 clearly appears on the 15N HSQC spectrum, demonstrating the pairing of G17. Second, the observation of two NH2-H5 connectivities on the 80 ms NOESY spectrum showed a rotation hindrance of the amino group of C17 and strongly suggested its pairing. Lastly, weak connectivity appeared between 15N1(G7) and the 15N4 (C17) on the HNN-COSY.

All the H6/8—H1′ connectivities, except the sequential way between H8(A9) and H1′(G10), were directly assigned on the NOESY spectra at 80 ms mixing time in H2O (Figure 2). H2 assignment of A8, A9 and A16 were discriminated from the corresponding H8 resonances by using their 13C2 and 13C8 chemical shifts on the HSQC spectra at 10°C. The assignments were confirmed using NOESY-HSQC, HCCH-TOCSY to connect H1′ to H2′, H1′ to H6 or H8 in HCN (43) as well as H8 to H2 of adenines in HMBC (44).

The diffusion coefficient was obtained by NMR pulsed field-gradient spin-echo techniques (35). The size of the loose complex was determined by comparing this coefficient with the extended complex and the mutated SL1-A12. These two structures were already characterized as an extended duplex of 2 × 24 nucleotides (28,45) and as a stem–loop monomer of 23 nt (46), respectively. At 10°C, the diffusion coefficients of the extended duplex and those of the loose complex were the same (0.52 ± 0.05 10−6 and 0.51 ± 0.05 10−6 cm2/s), showing a similar size (2 × 24mer). This result confirms the data of Muriaux et al. (27) showing that the loose duplex migration is similar to the extended duplex on non-denaturing PAGE on longer DIS sequences.

To discriminate between the presence of the loop–loop complex and that of the extended complex we performed NOESY WATERGATE. This was done without 15N decoupling during t1 and t2 at 80 ms mixing time on a 1:1 mixture of a labelled 13C15N SL1-24mer and an unlabelled SL1-24mer. As shown by Takashashi et al. (28), the N3H(U6)-N1H(G18) NOESY connectivity is predicted to show quite different patterns for the extended duplex and the loop–loop complex due to non existent 14NH-15NH connectivities in the loop complex. Figure 3 shows a clear pattern with a central connectivity 14N1H-14N3H surrounded by four smallest cross-peaks 15N1H-15N3H with J(15N,1H) = 95 Hz, in agreement with a loop–loop association. Confirmation of our NH assignments in the ‘kissing complex’ was obtained by 15N double-half-filter NOESY experiment on a mixing (1:1) of unlabelled SL1 and 13C15N labelled SL1 (47).

As expected for a loop–loop complex, the 15N→15N or the 14N→14N sub spectra showed only the connectivities between residues belonging to the stem while the 15N→14N or the 14N→15N sub spectra showed the connectivities NH(G10)-NH(G12) and NH(G12)-NH(G14) as well as the corresponding NH-NH2 connectivities.

Several unique features were noticed on the NOESY, COSY or TOCSY spectra. First, A8 and A9 were not found in anti conformation and their sugar in north conformation. Second, the H2 proton of A16 showed medium or strong connectivities with the H2 proton of A9 and with the H8 and H1′ protons of G10. Third, the imino proton of G10 showed connectivities with the H1′, H2 and H8 protons of A16.

The part of the dimer corresponding to the stems (G0 to G7 and C17 to C23) and to the loops (G10 to C15) showed a typical double helix A-type conformation and no ambiguity with the intra and intermolecular connectivities assignment. The A8 and A9 nucleotides adopt a different conformation accommodating the transition from an A-RNA conformation to a loop–loop association with a minimum of constraints. They present original connectivities whose discrimination between inter-strand and intra-strand is not trivial. In order to choose between intra and inter connectivities, four [1H, 1H]NOESY spectra were obtained using a 13C(ω12) double-half-filter where each half-filter was applied alternatively. A linear combination of these four spectra gave separately the 13C→13C, 13C→12C and 12C→12C connectivities (48). The strong NOE connectivities H8(A8)-H1′(A8) and H8(A9)-H1′(A9) were found intra-strand and H1′(G10)-H2(A16), H8(G10)-H2(A16), H2(A8)-H1′(A9) inter-strand. We attempted to obtain supplementary information concerning the relative orientation of the loop and the stem by measuring residual dipolar coupling. This experiment, which requires partial orientation of the molecule, produced a transition of the ‘kissing complex’ toward the extended dimer conformation in presence of phages. Classical A–helix connectivities were observed for G1 to G7, G10 to C15 and C17 to C23. For these nucleotides, the following dihedral restraints were used: α = −85 ± 50°, β = −165 ± 50°, γ = 53 ± 50°, ɛ = 180 ± 50° and ς = −60 ± 50° (Table 1). Cross-validation of our structure was obtained by removing 25% of the restraints involving G7, A8, A9, G10, A16 and C17. The eighteen converged structures showed no violation of distance restraints >0.2 Å. A very weak connectivity H2(A9)-H1′(G10) was observed at 300 ms mixing time, corresponding to a distance of 6.2 Å in our structures. It can be explained by the spin diffusion way H2(A9)→H2(A16)→H1′(G10).

The solution structure of the loose complex of HIV-1Lai showed a symmetric homodimer fully paired on the stems and the loops (Figure 4). These two loops, which are hydrogen bonded by six central GC base pairs, were roughly located in the axe of the stems. The overall phosphodiester backbone was similar to the canonical A-type geometry, except for the residues A8 and A9 whose sugars were in south conformation and positioned syn.

The molecular edifice was stabilized by stacking of A16 with G7-C17 of the other strand, in agreement with the observed upfield shift resonance of H1′(C17) (5.1 p.p.m.).

The solution structure presented in our NMR study is very similar to the crystallographic structure (32) (Figure 5), however significant differences emerged when comparisons were made with the NMR structure obtained in the study by Mujeeb et al. (31). The orientation of the loops versus the stems represented a major difference between these two NMR structures (Figure 5). A valid explanation accounting for this difference lies with the discrepancies in obtaining the restraints for the structures in both studies (Table 2). The overall conformation of our ‘kissing complex’ presented similarities with the crystallographic structure (23), particularly in the co-linearity of the three helices as well as the stacking of A16 with G7-C17. However, several elements involving the A8, A9 and A16 nucleotides were different. A9 was found in proximity of A16 in agreement with a medium NOE between H2(A9) and H2(A16) (Table 2). In addition, the sequential way between A8-A9 indicated the proximity of A8 to A9 of the same strand. These data did not support the conformational model where A8 and A9 are found in the bulged out position as observed in the crystallographic structure. It is worth noting that in our structure, the relative positions of the four adenines A8 and A9 of the two strands were such that their H2 and H8 resonances were exposed to small ring current effect supported by their chemical shift (Figure 6). On the contrary, in the crystallographic structure A8 and A9 were found stacked. The bulged out conformation of A8 and A9 generated a ‘hole’ between G7-C17 and A16 (C1′(A16)-C1′(C17) = 15 Å), which did not exist in our solution structure (C1′(A16)-C1′(C17) = 5 Å) (Figure 5). The bulging out phenomenon was a destabilizing factor, which can be compensated by the interdimer stacking of the adenines in the crystalline form. In solution, the absence of such a self-association could explain the bulged in conformation where the corresponding hole is filled by A8 and A9.