Implications of the HIV-1 Rev dimer structure at 3.2 Å resolution for multimeric binding to the Rev response element

Abstract

HIV-1 Rev is a small regulatory protein that mediates the nuclear export of viral mRNAs, an essential step in the HIV replication cycle. In this process Rev oligomerizes in association with a highly structured RNA motif, the Rev response element. Crystallographic studies of Rev have been hampered by the protein's tendency to aggregate, but Rev has now been found to form a stable soluble equimolar complex with a specifically engineered monoclonal Fab fragment. We have determined the structure of this complex at 3.2 Å resolution. It reveals a molecular dimer of Rev, bound on either side by a Fab, where the ordered portion of each Rev monomer (residues 9-65) contains two coplanar α-helices arranged in hairpin fashion. Subunits dimerize through overlapping of the hairpin prongs. Mating of hydrophobic patches on the outer surface of the dimer is likely to promote higher order interactions, suggesting a model for Rev oligomerization onto the viral RNA.nuclear export | ribonucleoprotein structure | Fab cocrystallization | posttranscriptional regulation | crystal structure T he unspliced and partially spliced viral mRNAs whose nuclear export is promoted by the 13 kDa HIV-1 Rev protein (1, 2) are used both as genomic RNA for packaging into assembling virions and for translation of many of the viral proteins (3), thereby eliciting the transition from early to late phase infection (1). Rev targets viral mRNAs by recognizing a ∼350 nt, highly structured region within an env intron known as the Rev response element (RRE) (4). Rev then oligomerizes onto the RRE to form a ribonucleoprotein (RNP) complex of 200-300 kDa containing 8-10 Rev molecules (5, 6) that binds Crm-1 (exportin-1), GTP-bound Ran, and other host cell proteins via the Rev nuclear export sequence (NES), to promote nuclear export (7). Rev contains an arginine-rich RNA-binding motif (ARM) that specifically binds to a purine-rich bulge within stem loop IIb of the RRE (2, 8) with high affinity to nucleate assembly of the Rev-RRE complex (9, 10). Subsequently, 7-9 additional Rev molecules are recruited (4-6, 11), one at a time, in a highly cooperative process mediated primarily through Rev-Rev interactions (5,(12)(13)(14). As befits their biological importance, the interactions whereby Rev recognizes RRE-containing RNAs have been intensively studied, as has Rev's propensity to oligomerize, both in the absence of RNA and in the context of the RRE (9, 10, 15, 16).Knowledge of Rev structure is essential to understanding its cooperative binding to the RRE and for the development of antiviral drugs that interfere with Rev's essential functions. So far, detailed structural information has been limited to an NMR structure of a 22-residue synthetic polypeptide corresponding to the ARM in complex with stem loop IIb (8). Rev's propensity to oligomerize into filaments (16) has hindered crystallization. Using a Fab engineered to inhibit Rev polymerization, we have been able to purify and crystallize a Rev-Fab complex (17). Here we report the structure determination of this com...

Chemical Synthesis and Expression of the HIV‐1 Rev Protein

“…[53] The purified protein is stable (it does not aggregate) at concentrations up to 200 mm in the absence of stabilizing additives. Higher concentrations were reached when crystallization of the protein in complex was studied; [9] however, our purification protocol is the first for obtaining high concentrations of free native soluble Rev in solution with no additives. The recombinant protein was also essential in the development of our optimal chemical synthesis of Rev, and enabled us to improve the synthesis and to develop a method for transferring the chemically synthesized protein to a buffer suitable for biological experiments.…”

“…[6,[10][11][12] Only very recently was the X-ray structure of this protein solved; however, it was complexed with a specifically engineered monoclonal Fab (fragment antigen-binding) antibody that served as the solubilizing agent. [9] This structure revealed that only the central part of the protein (residues 9-65) is structured, while the rest is intrinsically disordered. Motivated by the important role of Rev in the HIV replication cycle, and its potential as a drug target, [11,13,14] we developed two efficient synthetic and recombinant methods that allow the production of sufficient amounts of highly pure Rev, and provide the flexibility to prepare novel synthetic analogues of Rev for future studies to elucidate its structure-function relationship with spatial and temporal control.…”

“…[3][4][5][6] Although Rev was discovered nearly three decades ago, [7] biochemical and structural studies aimed at understanding the structural and functional aspects of the Rev protein are still limited because of its strong tendency to oligomerize, aggregate, and precipitate. [4,8,9] As a result, the majority of the Rev studies have been performed on peptides derived from the protein sequence, and on Rev conjugates such as Rev-GFP. [6,[10][11][12] Only very recently was the X-ray structure of this protein solved; however, it was complexed with a specifically engineered monoclonal Fab (fragment antigen-binding) antibody that served as the solubilizing agent.…”

Analysis of the Relationship between the Structure and Function of the HIV-1 Rev Response Element (RRE)

“…By modeling the 4 SL secondary structure of the RRE on the SAXS envelope, the authors deduced a SAXS structural model where the distance between the Rev binding sites on stem loop IIB (the primary Rev binding site) and stem loop I (the secondary Rev binding site) is ~55 Å. This distance matches the previously shown distance between the two RRE binding motifs in a Rev dimer (52,62). However, the study did not attempt to fit the 5 SL RRE structure into the SAXS envelope; thus, it sheds no light on the basis for the replication differences observed here.…”

“…Next, more Rev molecules (about 6-12) bind co-operatively along the other regions of the RRE in a process that involves protein-protein as well as protein-RNA interactions (41,46,51,52,62,104,149,155,249,250). Rev binding to the primary Rev binding site and Rev oligomerization on the RRE are both essential for Rev-RRE function (47,149,168,249).…”