Dissecting the Protein–RNA and RNA–RNA Interactions in the Nucleocapsid-mediated Dimerization and Isomerization of HIV-1 Stemloop 1

Abstract

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 adenines 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.

Understanding the Isomerization of the HIV-1 Dimerization Initiation Domain by the Nucleocapsid Protein

“…In the case of ED-obligated assemblies, the predominant 2:2 intermediate formed by stepwise NC losses from higher order assemblies (e. g. , Figure 3b and c) clearly corresponds to the stable 2:2 species formed in solution upon binding to the high affinity sites on the stembulge motifs. 70 In the case of KL-obligated complexes, the fact that NC was readily lost in the gas phase by the 3:2 assembly but not by the more stable 2:2 counterpart (Figure 4c and b, respectively) agrees with the relatively weak binding affinity afforded in solution by the junction site (Figure 1b).…”

“…In previous work, a truncated duplex lacking the stem-bulge motifs was used to demonstrate that these unpaired nucleotides were capable of sustaining effective NC binding in vitro when they formed the small bulge motifs flanking the palindromes of the ED structure (Scheme 1). 70 In contrast, no stable binding could be observed when such nucleotides were part of the hinge region between the kissing hairpins, as shown by the ESI-FTICR analysis of a mixture obtained by titrating a wild-type construct that was heat-denatured and rapidly cooled to obtain predominantly the kinetic KL product (Figure 1a). In this example, the formation of assemblies with a maximum of two protein units per RNA dimer is ascribable to the combination of the tight binding of NC to the hairpins' stem-bulges and the absence of significant interactions with the hinging bases under the selected experimental conditions (see…”

“…In constructs capable of isomerization, however, the same stoichiometry and molecular mass could be shared by corresponding KL and ED assemblies, the latter upon partial saturation of the stem-bulge and flanking-bulge sites (Scheme 1). 70 Considering the possible ambiguities arising from the simultaneous presence of both conformers during isomerization, we have tested tandem mass spectrometry 76, 77 as a possible alternative to distinguish between dimeric forms, which would not rely on the stoichiometry of the assemblies observed in solution. This method involves isolating the complex of interest in the FTICR cell and then activating it by sustained off-resonance irradiation collision-induced dissociation (SORI-CID) 75 to obtain products that are characteristic of the precursor ion structure.…”

“…In the case of wild-type SL1A, the monomeric assemblies contained a maximum of two NC units per RNA, which is consistent with the presence of two high-affinity sites on the apical loop and stem-bulge regions of the hairpin structure (Figure 5a). 70 The dimeric assemblies included up to three protein units, as expected from having G in the junction region. Submitted to mild SORI-CID, these dimers provided dissociation patterns replicating those observed for the KL-obligated mutant (Figure 5b and c), thus confirming the initial conformation of the RNA substrate.…”

“…Indeed, unpaired As can effectively sustain protein binding when they are included in the flanking-bulges of the ED structure, as demonstrated by the detection of 4:2 assemblies for constructs with A in position 272 and 273 (e. g., wild-type SL1B). 70 The different behaviors could be perhaps explained by the fact that the crystal structure of the subtype A kissing dimer presents G273 in a syn conformation, whereas the corresponding A273 adopts an anti conformation in subtype B. 33 In addition, it is possible that the different proton affinities afforded by the two purines may have very distinctive effects on junction dynamics and grip structure, 86 which should be further investigated.…”