**6. Conformational Changes Underlying "Maturation" of the HIV-2 RRE**

An intriguing issue is whether observations and models suggesting structural fluidity are unique to the HIV-1 RRE or whether its' HIV-2 counterpart is likewise conformationally heterogeneous. Early mutational studies of the HIV-2 RRE [63] indicated that (i) the interaction with its cognate Rev was more dependent on maintenance of secondary structure than primary nucleotide sequence and (ii) HIV-2 RRE structures permitting interaction with HIV-1 Rev, while coinciding with those required for HIV-2 Rev binding, were dissimilar in structure and nucleotide sequence. Prior to performing HIV-2 RRE characterization by SHAPE, data from Figure 7A raised a formidable challenge, since in the absence of any binding partner this too displayed unexpected conformational flexibility. Although denaturing polyacrylamide gel electrophoresis indicated a single RNA species following in vitro transcription, subsequent non-denaturing electrophoresis identified three conformers that gradually "coalesced" into a single species upon prolonged renaturation [64]. Since SHAPE requires that the target RNA adopt a uniform structure in solution [65], understanding this unexpected stepwise HIV-2 RRE folding required a mathematical model to be developed that extracted the contributions of individual conformers from ensemble chemical reactivity values. The model makes two assumptions. Firstly, it assumes that each ensemble SHAPE reactivity value obtained from a population of RNA conformers equals the sum of reactivity values of the contributing conformers, weighted according to their fractional contribution to the total RNA population. Secondly, if ensemble reactivity values and fractional contributions of individual conformations changed with differing conditions (e.g., folding time), and these values could be determined for a number of conditions equal to or greater than the total conformer content of the mixture, specific chemical reactivity values for each conformer could be mathematically derived. By adopting this strategy, the HIV-2 RRE folding program depicted in Figure 7B was proposed.

The most significant differences among these structures involved the central junction, changes within which affect positioning of the substructures relative to each other. Transition from the open to intermediate conformation requires several concerted substructure translations/rotations, most notably rotation of SL-IIB/IIC, caused by base pair formation in S-L IIA. Pairing of the bridge helix defines the subsequent transition from the intermediate to closed conformer, which also involves inward rotation of the SL-IV/V substructure, folding of the SL-IIB apical loop toward the central junction and formation of mutually stabilizing contacts between SL-IIB and SL-V. The SL-IIB is arranged orthogonally to SL-IIC and coaxially with SL-I, consistent with assembly models, whereby Rev initially binds to high affinity sites on SL-IIB and SL-I, then multimerizes linearly along an SL-IIB/SL-I axis. Given the close contact between SL-IIB and SL-V in the closed conformer, rotational/translational flexibility of the SL-IV/V substructure would be required to create space for Rev binding to SL-IIB.

**Figure 7.** Time-dependent conformational rearrangement of the HIV-2 RRE. Analyses were performed on a 216 nt RRE derived from HIV-2ROD by in vitro transcription. (**A**) Native gel electrophoresis as a function of incubation time indicates the HIV-2 RRE comprises a mixture of "open", "intermediate", and "closed" conformers (**A**–**C**, respectively) at short incubation times and whose ratio varies with time, with the closed conformer ultimately predominating. (**B**) SHAPE-derived conformations of the open, intermediate, and closed HIV-2 RRE forms, respectively. Secondary structural motifs are indicated and color-coded as follows: SL-I, red; S-IIA, dark green, SL-IIB, -IIC and adjacent connecting loops, magenta; SL-III, yellow; SL-IV, blue; SL-V, orange. Modified from Reference [64].
