UCSF

Replication of HIV requires the nuclear export of unspliced viral RNAs for the translation of structural proteins and viral packaging. Crucial to this process is oligomeric binding of the regulatory protein Rev to the Rev Response Element (RRE) within the viral RNA, and subsequent export through the nuclear pore complex. Despite two decades of intense scrutiny, the mechanistic importance of Rev oligomerization in RNA export was unknown. More strikingly, the molecular structures of the Rev protein and Rev-RRE complexes were unknown, in large part due to difficulties with aggregation and oligomerization of the Rev protein. Described here are my efforts to use biochemical, biophysical and structural techniques to understand the manner in which Rev oligomerizes on RNA and the role of assembly of the Rev-RRE complex in viral replication. We first established that Rev monomers bind cooperatively to multiple discrete RNA sites within the RRE using an adaptable protein-RNA interface, forming a homooligomeric complex with 500-fold higher affinity than the tightest single interaction. Importantly, proper multimeric Rev-RRE assembly is strongly correlated with RNA export activity, indicating that oligomerization mediates high affinity complex formation and is thus required for in vivo function. With a better understanding of defined Rev-RNA complexes, we next sought to enhance the solubility and homogeneity of these complexes for structural studies. Using these techniques, we obtained low resolution structural information for a Rev dimer in complex with an essential portion of the RRE, as well as with a hexamer of Rev in complex with the RRE. This work thus provides the first structural glimpse of a functional Rev-RRE complex. As a final effort to understand Rev oligomerization, we crystallized the Rev dimer and solved the structure at 2.8 Å resolution. After nearly twenty years, this is the first molecular structure of the Rev protein and reveals the basis for monomeric folding, dimeric stability and cooperative RNA binding.