Thermodynamics of Rev–RNA Interactions in HIV-1 Rev–RRE Assembly

Abstract

The HIV-1 protein Rev facilitates the nuclear export of intron-containing viral mRNAs by recognizing a structured RNA site, the Rev-response-element (RRE), contained in an intron. Rev assembles as a homo-oligomer on the RRE using its α-helical arginine-rich-motif (ARM) for RNA recognition. One unique feature of this assembly is the repeated use of the ARM from individual Rev subunits to contact distinct parts of the RRE in different binding modes. How the individual interactions differ and how they contribute toward forming a functional complex is poorly understood. Here we examine the thermodynamics of Rev–ARM peptide binding to two sites, RRE stem IIB, the high-affinity site that nucleates Rev assembly, and stem IA, a potential intermediate site during assembly, using NMR spectroscopy and isothermal titration calorimetry (ITC). NMR data indicate that the Rev–IIB complex forms a stable interface, whereas the Rev–IA interface is highly dynamic. ITC studies show that both interactions are enthalpy-driven, with binding to IIB being 20–30 fold tighter than to IA. Salt-dependent decreases in affinity were similar at both sites and predominantly enthalpic in nature, reflecting the roles of electrostatic interactions with arginines. However, the two interactions display strikingly different partitioning between enthalpy and entropy components, correlating well with the NMR observations. Our results illustrate how the variation in binding modes to different RRE target sites may influence the stability or order of Rev–RRE assembly and disassembly, and consequently its function.

Altered‐specificity mutants of the HIV Rev arginine‐rich motif‐RRE IIB interaction

“…Nonetheless, with the methods used, it is difficult to discriminate quantitatively between specific and nonspecific binding. The relative contributions of enthalpy and entropy of WT‐IIB are roughly similar to what has been reported for a similar interaction in detailed thermodynamic study characterizing a Rev ARM peptide binding to IIB and IA RRE RNAs. Jayaraman et al report dissociation constants ranging from 0.02 nM at 283 K and 100 mM KCl to 16.3 nM at 303 K and 300 mM KCl, approaching the dissociation constant of 22 ± 8 nM reported here for WT‐IIB.…”

“…Other arginines form ionic interactions with the phosphates of the backbone, and Trp45 is not important for binding isolated IIB, but required for multimeric binding of Rev to larger regions of RRE . Importantly, the mutually inducted fit of Rev ARM and IIB binding appears to extend through complex and cooperative binding of multiple Rev proteins to other binding sites in RRE …”

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

“…This oligomeric complex has been reconstituted as a Rev hexamer on the 242‐nt RRE . Although each subunit of the Rev oligomer contains identical ARMs, the mode of RNA recognition at each binding site in the RRE is unique . Structural and thermodynamic studies of the three well‐characterized binding sites (Stem‐loop IIB, Stem IA and Junction IIABC) have revealed that different sets of Rev residues make varied contacts to the RNA in each case (Figure (a)).…”

Oligomeric viral proteins: small in size, large in presence

“…Specific Rev binding was also observed with an isolated stem IA hairpin (Daugherty et al, 2008), which contains a similar asymmetric purine-rich internal loop as in stem IIB, suggesting that at least a portion of binding specificity is dictated by the RNA structure (Daugherty et al, 2008). Stem IA likely forms an intermediate binding site in the overall assembly of the Rev/RRE RNP (Bai et al, 2014; Jayaraman et al, 2015). Interestingly, although each Rev subunit uses a single alpha-helical ARM to bind to the RRE, the mode of RNA recognition is unique to each site (Daugherty et al, 2008; Jayaraman et al, 2015).…”

“…Stem IA likely forms an intermediate binding site in the overall assembly of the Rev/RRE RNP (Bai et al, 2014; Jayaraman et al, 2015). Interestingly, although each Rev subunit uses a single alpha-helical ARM to bind to the RRE, the mode of RNA recognition is unique to each site (Daugherty et al, 2008; Jayaraman et al, 2015). Mutagenesis revealed that Rev utilizes different amino acids to recognize stem IIB and stem IA, in combination with structural studies that corroborated different binding strategies for each site (Daugherty et al, 2008).…”