A structurally plastic ribonucleoprotein complex mediates post-transcriptional gene regulation in HIV-1

Abstract

HIV replication requires the nuclear export of essential, intron-containing viral RNAs. To facilitate export, HIV encodes the viral accessory protein Rev which binds unspliced and partially spliced viral RNAs and creates a ribonucleoprotein complex that recruits the cellular Chromosome maintenance factor 1 export machinery. Exporting RNAs in this manner bypasses the necessity for complete splicing as a prerequisite for mRNA export, and allows intron-containing RNAs to reach the cytoplasm intact for translation and virus packaging. Recent structural studies have revealed that this entire complex exhibits remarkable plasticity at many levels of organization, including RNA folding, protein-RNA recognition, multimer formation, and host factor recruitment. In this review, we explore each aspect of plasticity from structural, functional, and possible therapeutic viewpoints. WIREs RNA 2016, 7:470-486. doi: 10.1002/wrna.1342 For further resources related to this article, please visit the WIREs website.

FIGURE 1

Rev transports partially spliced and unspliced viral RNAs from the nucleus to the cytoplasm. (1) HIV-1 transcription produces a single 9-kb RNA from its promoter; (2) The RNA is spliced into 2-kb mRNAs that are exported to the cytoplasm via the nuclear pore; (3) The Rev-encoding 2-kb mRNAs are translated and Rev is imported into the nucleus via its nuclear localization sequence; (4) Multiple Rev molecules bind the RRE (Rev-Response Element) before splicing occurs; (5) The Rev-RRE complex recruits a Crm1 (Chromosome maintenance factor 1)/RanGTP dimer to the unspliced or incompletely RNAs; (6) The RNAs are exported through the nuclear pore to the cytoplasm.

FIGURE 2

Domain Organization of Rev and Plastic Assembly of the Export RNP. (a) Domain organization of Rev. Partial crystal structures of Rev from PDB codes 3LPH and 3NBZ are shown and colored accordingly to the labeled domains: OD, oligomerization domain; ARM, arginine-rich motif; NES, nuclear export sequence. (b) Points of Rev-RRE-Crm1 export complex assembly with demonstrated plasticity. The RRE (Rev-Response Element) can adopt multiple conformations through alternative secondary or tertiary structures or mutation. The Rev oligomer recognizes these scaffolds using multiple modes of RNA recognition and the entire oligomer can rearrange as directed by the RNA scaffold. Finally, the Rev-RRE complex recruits a Crm1 (Chromosome maintenance factor 1) dimer using multiple Rev NESs from its flexible C-terminal region.

FIGURE 3

Secondary structures of the RRE (Rev-Response Element). The RRE predominantly adopts either a five stem-loop structure (pictured) or a four stem-loop structure (the Stem III/IV rearrangement is highlighted in green). The three well-characterized Rev-binding sites are indicated (Stem IA: cyan, Stem IIB: blue, Junction IIABC: purple). Regions that show the greatest protection upon addition of Rev in SHAPE experiments are circled in black. Two mutations found in RRE61 which change the RRE secondary structure and rescue function in the context of a Rev dominant negative nuclear export sequence (NES) mutant (M10) are shown in red. The portion of Stem I dispensable for Rev activity in reporter assays is to the left of the dashed line.

FIGURE 4

Rev-RRE (Rev-Response Element) interactions of the arginine-rich motif (ARM). (a) Plasticity in Rev-RNA recognition. The ∝-helical Rev ARM is shown with each of three well-characterized binding sites. The structures of complexes with IIB and IIABC have been solved by NMR and crystallography,^, while the IA-binding region is inferred from mutational data. Each site uses a different set of residues to contact the RNA and buries different faces and sections of the ARM helix. Other sites yet to be identified may utilize other portions of the helix, which is surrounded with arginine residues, for binding. (b) Models for order of assembly. A small angle x-ray scattering (SAXS) model of the RRE juxtaposes the IIB- and IA-binding sites such that a Rev dimer (subunits 1 and 2 in the upper diagram) might bridge the two sites to initiate oligomer assembly. SHAPE and biochemical data and the cocrystal structure of a dimer-RNA complex suggest that dimer binding of the IIB and IIABC sites nucleates Rev oligomerization across the bridge of the 'A'-shaped RNA structure. (Reprinted with permission from Refs 25 and . Copyright 2014 Creative Commons Attribution License)

FIGURE 5

Plasticity of the Rev oligomer. Structures of the core dimer and multimer surfaces of Rev^, reveal different crossing angles. In addition to the different states of unbound Rev, the core dimer surface rearranges upon RNA binding by pivoting around hydrophobic residues in the dimer interface, likely one of many possible angles depending on the structure of the RNA as well as the energetic stability of the bound dimer. The multimer surface may also rearrange to accommodate RNA binding but this complex has not yet been observed. Each dimer arrangement may orient the helical subunit arginine-rich motifs (ARMs) to bind RNA in slightly different ways during oligomer assembly.

FIGURE 6

The Rev nuclear export sequence mimics host cargoes to bind a Crm1 (Chromosome maintenance factor 1) dimer. (a) The spacing of hydrophobic residues (Φ) in an NES within the Crm1-binding pocket is shown for Rev (green) and protein kinase A inhibitor (PKI; purple), which has a canonical NES. The Rev NES establishes similar hydrophobic contacts with Crm1 (yellow) but uses a shorter and less-ordered peptide (PDB 3NBZ) than the PKI NES (PDB 3NBY). (b) Crm1 forms a dimer interface that enhances Rev-RRE (Rev-Response Element) complex formation. Two Crm1 monomers were unambiguously fit into the EM density (gray) with an additional, weak density that corresponds to the position of the Rev-RRE complex (red) and sits between the binding sites for the Rev NES (green). The location of the Rev-RRE differs from the known binding site of Snurportin1 (purple). (c) The interface of the Crm1 dimer was observed as a crystal contact in the structure of human Crm1. The residues (annotated) in this interface that differ between the human and murine proteins were previously recognized to be important for Rev-RRE nuclear export. (Reprinted with permission from Refs 25 and . Copyright 2014 Creative Commons Attribution License)

FIGURE 7

Model for how Rev-RRE (Rev-Response Element) plasticity can lead to different export outputs. Multiple conformations of the Rev oligomer are seen in existing crystal structures and in principle can be mixed and matched to generate different arrangements of the oligomer. The RRE scaffold, and its possible different conformers, can present the Rev nuclear export sequences (NESs) in different ways to the Crm1 (Chromosome maintenance factor 1) dimer and thereby influence nuclear export activity, most likely by increasing the number of viable configurations for Crm1 binding. (Reprinted with permission from Refs 25 and . Copyright 2014 Creative Commons Attribution License)