**1. Introduction**

Conformational "fluidity" of RNA allows it to mediate a variety of biological functions, examples of which include (a) catalyzing cleavage by the hammerhead ribozyme of satellite RNAs and viroids [1]; (b) bacterial riboswitches [2]; (c) RNA thermometers [3]; and (d) tRNA-dependent control of specific aminoacylation and translation regulation [4]. In the case of human immunodeficiency virus (HIV), cis-acting sequences encoded in its (+)RNA genome are central to transcription of the integrated provirus, nucleocytoplasmic transport of unspliced and partially spliced RNAs, initiation of reverse transcription, genome dimerization/packaging, and ribosomal frameshifting [5]. A comprehensive understanding of the structural dynamics of these regulatory elements would be predicted to accelerate development of small molecules [6], oligonucleotides [7], peptide nucleic acids [8] evolved RNA recognition motifs [9], and nucleic acid aptamers [10] as novel therapeutic modalities to complement existing anti-HIV agents. With these objectives in mind, the goal of this review was to highlight conformational flexibility of the HIV-1 RRE (and its HIV-2 counterpart) through a series of experiments that extend in vitro structural analysis to their in vivo outcome in cell culture systems and, finally, in sequential viral isolates in a clinical setting.

Figure 1A provides a summary of the functional requirement for the HIV-1 RRE. Productive HIV infection produces three types of viral transcripts, i.e., unspliced, partially spliced, and fully spliced

RNAs (http://hivinsite.ucsf.edu). Early in the viral life cycle, fully spliced viral RNAs encoding the regulatory proteins Rev, Tat, and Nef are exported and translated in the cytoplasm. However, the presence of introns in unspliced and partially spliced viral RNAs results in their nuclear retention by host RNA surveillance mechanisms that normally restrict nucleo-cytoplasmic export of intron-retaining mRNAs [11,12]. Later in the life cycle, Rev, through its nuclear localization signal (NLS), is imported into the nucleus [13,14]. The NLS domain is a basic, arginine-rich motif (ARM) that also serves as an RNA-binding domain (RBD) that binds specifically to the RRE (Figure 1B) [15]. Inside the nucleus, Rev binds cooperatively to the RRE present in all intron-retaining viral RNAs through a process involving both protein–protein and protein–RNA interactions [15–24]. The Rev–RRE complex is recognized by CRM1 and RAN-GTP forming an export competent ribonucleoprotein (RNP) complex [25] allowing unspliced and partially spliced viral RNAs to circumvent host cellular restriction and transit to the cytoplasm, where they are either translated or packaged into assembling virions [26–28].

**Figure 1.** (**A**) Functional requirement of the HIV RRE: Early in the viral lifecycle, fully spliced viral RNAs are exported from the nucleus in a Rev/RRE-independent manner. Among these, Rev mRNA are translated and Rev is imported into the nucleus. In the late phase of the lifecycle, nuclear RRE-containing RNAs recruit Rev and cellular nuclear-export machinery, allowing them to circumvent splicing and transit to the cytoplasm, where they are either translated or packaged into assembling virions (**B**) Organization of the 116 aa HIV-1 Rev and amino acid changes in the trans-dominant M10 variant. NLS; nuclear localization signal, NES, nuclear export signal. Pink areas flanking the NLS

A logical first step in understanding molecular details of the Rev/RRE complex was defining the topology of the RRE, an ~350 nt, RNA comprising multiple stem-loops and bulges. Combining

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represent Rev oligomerization domains.

computational modeling with chemical and enzymatic footprinting led to the proposal of a 5 stem-loop (SL) structure with a central SL-1 branching into SL-II (the primary Rev binding site), SL-III, SL IV, and SL-V [29–31]. The SL-II consists of stem IIA branching out of the central loop and opening into a three-way junction. The junction opens into two stem-loops IIB and IIC [19,24,32–34]. In contrast, a 4 SL structure, differing in rearrangement of SL-III and -IV, has been reported [23,35], wherein SL-III and -IV of the 5 SL RRE combine to form a single SL-III/IV off of the central loop. Despite these differences, the 4 SL and 5 SL RRE conformers preserve SL-II topology. Since the majority of these structures were derived from different in vitro probing methodologies, it cannot be ruled out that such differences reflect subtle alterations in buffer probing conditions and are not truly reflective of the biological system. This review summarizes several papers, including analysis of patient isolates that collectively suggest both that HIV-1 and HIV-2 RRE possess sufficient flexibility to adopt alternative conformations, and that for HIV-1, at least, stabilizing these by in vitro mutagenesis confers a growth advantage for the 5 SL conformer. Lastly, we present data suggesting that RRE conformational flexibility might be exploited therapeutically.
