The HIV-1 Rev response element (RRE) is a highly structured, ~350 nucleotide RNA segment present in the Env coding region of unspliced and partially spliced viral mRNAs. In the presence of the HIV-1 accessory protein Rev, HIV-1 mRNAs that contain the RRE can be exported from the nucleus to the cytoplasm for downstream events such as translation and virion packaging.
RRE and HIV-1 biology
Early phase
The HIV-1 genome contains a single promoter and uses multiple reading frames and alternative splicing to encode 15 proteins from a single pre-mRNA species. Transcription from an integrated HIV-1 provirus generates a single 9 kilobase (kb) pre-mRNA containing multiple splice sites and nuclear retention signals. In the early phase of the viral life cycle, this pre-RNA is completely spliced to RRE-free, 2 kb messages. These smaller messages are then transported from the nucleus to the cytoplasm via standard mRNA nuclear export pathways (see Figure). One of these small, 2kb messages encodes the HIV-1 Rev protein which is imported into the nucleus via its nuclear localization sequence. This phase of the virus life cycle is both Rev and RRE independent.
Late phase
The late phase of the viral life cycle is characterized by the expression of viral proteins that are encoded on the long, unspliced (9kb) or partially spliced (4 kb) messages containing the RRE. Because of their retention and splicing signals, these intron-containing RNAs are initially retained in the nucleus for splicing/degradation. However, after a sufficient level of Rev has been produced by the 2 kb messages, these longer messages can be exported to the cytoplasm via a Rev dependent export pathway. Nuclear export of these RNAs is achieved by a specific, co-operative assembly of multiple Rev molecules on the RRE. Assembly of this Rev-RRE complex is followed by the recruitment of a human protein complex containing the proteins exportin-1 (XPO1/CRM1) and Ran-GTP. Rev recruits this export machinery via a nuclear export sequence (NES) present in Rev. This Rev-RRE-Xpo1/RanGTP complex is then transported to the cytoplasm. In the cytoplasm, these messages are translated to produce all the remaining viral proteins or packaged as genomes for newly budding virions (see Figure).
Secondary Structure and Rev Recognition
The RRE is a highly structured RNA element. Computational predictions, later verified by chemical and enzymatic probing, indicate that RRE contains multiple stem loops and bulges (see Figure). Rev binds to RRE in a sequence specific manner with Rev-RNA recognition mediated by a 17-residue a-helical stretch on Rev, the Arginine-Rich-Motif (ARM).
Stem IIB: a high-affinity binding site
Stem IIB is a site on the RRE which Rev binds with high affinity and specificity. The structure of an isolated stem IIB bound to a peptide corresponding to a Rev-ARM has been solved by NMR. This structure reveals an RNA A-form major groove widened by purine-purine base pairs at the purine-rich bulge to accommodate the a-helical Rev-ARM. Binding is achieved through a combination of base-specific contacts and electrostatic contacts with the phosphate backbone (see figure). More recent studies have identified another region on the RRE, stem IA, that binds Rev in a specific manner, but with a 5-fold weaker affinity than stem IIB.
Co-operative Rev assembly required for RRE function
Although Stems IIB and IA can bind Rev in isolation, a full-length RRE (at least ~250 nt) is required for viral function. Multiple molecules of Rev bind to the full RRE in a specific and co-operative manner through a combination of Rev-RNA and Rev-Rev interactions. It is believed that IIB acts as an "anchor point", with the Rev molecules bound at secondary sites (such as IA) stabilized by protein-protein interactions with other Rev molecules (in addition to the RNA-protein interactions). Biochemical studies on a 242-nucleotide RRE have established a ratio of 6 Rev monomers to each RRE.
In a sense, the RRE acts as a scaffolding platform onto which a specific and co-operative complex of Revs (and eventually cellular export machinery) assembles. This cooperativity that is dictated by RRE structure and sequence is required for the formation of a high affinity, export-competent complex. Current models of Rev assembly on the RRE suggest an initial Rev nucleation event at stem IIB followed by progressive addition of Rev molecules to form the full complex.