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Review
. 2020 Jan 11;12(1):86.
doi: 10.3390/v12010086.

Structural Fluidity of the Human Immunodeficiency Virus Rev Response Element

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Free PMC article
Review

Structural Fluidity of the Human Immunodeficiency Virus Rev Response Element

Chringma Sherpa et al. Viruses. .
Free PMC article

Abstract

Nucleocytoplasmic transport of unspliced and partially spliced human immunodeficiency virus (HIV) RNA is mediated in part by the Rev response element (RRE), a ~350 nt cis-acting element located in the envelope coding region of the viral genome. Understanding the interaction of the RRE with the viral Rev protein, cellular co-factors, and its therapeutic potential has been the subject of almost three decades of structural studies, throughout which a recurring discussion theme has been RRE topology, i.e., whether it comprises 4 or 5 stem-loops (SLs) and whether this has biological significance. Moreover, while in vitro mutagenesis allows the construction of 4 SL and 5 SL RRE conformers and testing of their roles in cell culture, it has not been immediately clear if such findings can be translated to a clinical setting. Herein, we review several articles demonstrating remarkable flexibility of the HIV-1 and HIV-2 RREs following initial observations that HIV-1 resistance to trans-dominant Rev therapy was founded in structural rearrangement of its RRE. These observations can be extended not only to cell culture studies demonstrating a growth advantage for the 5 SL RRE conformer but also to evolution in RRE topology in patient isolates. Finally, RRE conformational flexibility provides a target for therapeutic intervention, and we describe high throughput screening approaches to exploit this property.

Keywords: HIV; Rev response element; SHAPE; branched peptides; chemical footprinting; drug discovery.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
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 represent Rev oligomerization domains.
Figure 2
Figure 2
Mutations conferring resistance to trans-dominant RevM10 therapy induce a conformational change in the HIV-1 RRE. (A) Structural probing of the wild-type RRE (left) and RevM10-resistant RRE-61 (right). (C) Control DNA sequencing lane from which nucleotide numbering was derived. Designations − and + refer to untreated and NMIA-treated RNA, respectively. Major alterations in chemical reactivity are indicated by the bar. (B,C) Cartoons depicting SHAPE-derived conformations of wild-type RRE and RRE-61. SL, stem-loop. Positions of RRE point mutations, inducing RevM10 resistance, are indicated by asterisks in (B). Modified from Reference [47].
Figure 3
Figure 3
The HIV-1 RRE exists in a conformational equilibrium. (A) Following extended non-denaturing PAGE, slow and fast migrating RRE conformers were observed. Subjecting these RNAs to in-gel SHAPE defines these as 4 SL (B) and 5 SL conformers (C). Note that, despite their conformational heterogeneity, the topology of SL-II, the primary Rev binding suite, is preserved. Modified from Sherpa et al. [52]. The 232 nt HIV-1 RRE RNAs appended with a 3′ structure cassette were prepared for analysis by in vitro transcription.
Figure 4
Figure 4
Alternative HIV-1 RRE conformers promote different rates of virus replication. (A) Conformer construction (see text). (B) Chemical acylation (SHAPE) confirms a 5 SL conformation of mutant M1 and a 4 SL conformation of mutant M3. (C) Heteroduplex tracking analysis. In both M1/wt and M1/M3 mutant co-infections, the stabilized 5 SL M1 conformer displays a replicative growth advantage. The full experimental background is provided in Reference [52].
Figure 5
Figure 5
Conformational flexibility of HIV-1 RRE SL-I contributes towards assembly of the Rev-mediated export complex. (A) Molecular envelope of the RRE RNA, drawn in mesh and derived by SAXS [32]. The spatial resolution of the envelope is 21 A°. (B) Cartoon representation of the RRE, depicting assembly initiating via a single nucleation point in SL-II for two Rev molecules (blue). (C) Through an SL-I conformational change, “coupling” of SL-I and SL-II Rev-binding sites promote a tetrameric intermediate complex proposed to serve as a specificity checkpoint. Rev and the RRE could thereafter simultaneously sample a number of interaction conformations until an optimal binding state for Crm1 binding and nuclear export is attained.
Figure 6
Figure 6
Patient-derived HIV-1 RREs from early and late time-points post-infection exhibit different secondary structures. Secondary structures of V10-2 RRE (an early isolate, (left) and V20-1 RRE (a late isolate, (right) determined by SHAPE-MaP. (Center) differential migration rate of V10-2 and V20-1 RRE, following non-denaturing PAGE and UV shadowing, is suggestive of alternate conformers/ Adapted from Sherpa et al. [59].
Figure 7
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 (AC, 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].
Figure 8
Figure 8
Screening for small molecules that recognize m6A-modifed HIV-1 RRE SL-IIB. (A) A synthetic RNA fragment harboring the m6A modifications reported in SL-IIB is labeled with Cy5 on its 3′ terminus. (B) Labeled SL-II RNA was flowed over microtiter plates containing covalently immobilized small molecules. Binding of RRE SL-II RNA to candidate ligands was recorded via a fluorescence signal. (C) HTS screening suggests specificity of small molecules for unmethylated (left) and methylated SL-IIB (right). The central panel highlights a ligand that recognizes both SL-II forms.
Figure 9
Figure 9
Restricting RRE conformational flexibility with multivalent branched peptides. (A) Cartoon depicting the branched peptide strategy, i.e., binding in a multivalent fashion to enhance affinity and selectivity toward the RNA target. (B) Structure of branched peptide 4A5. (C) Inhibition of Rev-RRE function in vivo using HEK 293T cells transiently transfected with a Rev-expressing plasmid and a CMV promoter-driven GagPol-RRE plasmid. Modified from Dai et al. [77].

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