Recent crystal structures of Rev dimers29,30
reveal a hydrophobic core that mediates Rev-Rev interactions, with the ARMs pointing away from the core and available to contact the RNA. The structures, in combination with results from detailed biochemistry that indicate a stoichiometry of 6 Rev monomers on a ~250 nucleotide RRE,31
have led to the proposal of a “jellyfish” model for the Rev-RRE complex29
(). Formation of the complex presumably involves nucleation of Rev assembly at stem IIB followed by sequential addition of Rev monomers,9,27
as supported by kinetic studies of Rev-RRE assembly.32
Figure 4. Schematic representation of how an export-competent Rev-RRE complex might form. Rev molecules assemble onto the RRE scaffold to form an oligomeric assembly. In the “jellyfish” model, the jellyfish head comprises Rev oligomers (more ...)
Rev oligomerization and Rev-RNA interactions contribute synergistically toward formation of a high-affinity cooperative assembly that is directly correlated with its export competence. Studies with chimeric Rev and RRE (Rev fused to MS2 coat-protein and RRE stem IIB replaced with the MS2-RNA)33-35
and with polyvalent Rev-binding elements (e.g., stem IIB concatemers)36,37
have shown that although high affinity correlates with function, affinity alone is unable to recapitulate full export activity of the Rev-RRE complex.
These data suggest a model in which the RRE serves as an architectural scaffold that orchestrates the specific assembly of a Rev oligomer using a combination of affinities derived from RNA binding and oligomeric interactions. This complex in turns influences the structure, positioning, and stoichiometry of the larger Rev-RRE/Crm1/RanGTP export complex.
This model is reminiscent of other biological scaffolding strategies: scaffold proteins are central to organizing and coordinating signal transduction cascades in cells.38
Integral to the functioning of macromolecular machines such as the ribosome,39
and signal recognition particle42
are scaffold RNAs that provide the structural framework to position the different components and in some cases to organize their catalytic abilities.
The RRE is an example of a unique RNA scaffold, providing the framework for assembling a homo-oligomeric complex. The protein-binding sites it presents recruit multiple Rev molecules through diverse sets of interactions with specific positional and orientation requirements. Viral evolution has thus served as a selection experiment—identifying RNA-binding partners for Rev and arranging them structurally to derive maximal functional efficiency from such a complex, even under additional constraints imposed by an overlapping protein-coding reading frame. By deriving specificity from three dimensional restraints imposed by oligomer formation, the virus is able to maintain enhanced specificity of Rev for RRE over the pool of cellular RNAs without relying solely on high-affinity sequence recognition.
At least two significant observations support this model of viral evolution selection. Biochemical studies from our lab identified stem IA to be a secondary Rev-binding site26
with very different structural and thermodynamic modes of Rev recognition compared with stem IIB (), as might have been identified by an in vitro selection experiment. More evidence for this model comes from an intriguing study with a dominant negative Rev mutant, RevM10, that harbors a mutation in its nuclear export sequence (NES).43
RevM10 assembles on the RRE like wild-type Rev but fails to trigger nuclear export of RRE-containing RNAs. Viral resistance selection experiments resulted in two silent mutations in the RRE that altered the structure of RRE in stems III/IV/V with no detectable change in binding to Rev or RevM10.44