Rev is a small HIV regulatory protein essential for viral replication whose well-known function is to export unspliced and partially spliced viral RNAs from the nucleus to the cytoplasm. The full-length mRNAs encode the viral structural proteins and supply the genomic RNA used in viral packaging. Rev binds as an oligomer to the RRE RNA within the Env coding region using an alpha-helical arginine-rich motif (ARM) for RNA recognition. Six Rev subunits bind to a 240-nt version of the RRE, with different subunits docking to different regions of the RNA in distinct binding modes. We recently solved a high-resolution crystal structure of the Rev dimer, a co-crystal structure of the Rev dimer bound to RRE RNA, and have assembled well-defined nuclear export complexes with Crm1/RanGTP. These studies revealed that Rev is a highly modular protein that utilizes plastic RNA-binding and oligomerization surfaces for interaction and that Crm1 forms a unique dimer with the Rev-RRE complex bound across the Crm1 subunits. Chemical mapping experiments further revealed a previously unknown interacting region within the full-length 350-nt RRE that helps form a scaffold to direct binding of the Rev oligomer. In addition, Rev appears to interact with additional cytoplasmic host partners, which we hypothesize may reorganize the Rev-RRE oligomer. The genomic architecture of HIV in which the reading frame of Rev overlaps with those of Tat and Env, and where the RRE also overlaps with Env, imposes extra constraints on the evolution of Rev and RRE structure. In this renewal application, we will define the dynamic behavior of Rev and the RRE at the structural and functional levels by: (1) determining the roles of alternative RNA conformations in assembling Rev-RRE export complexes; (2) characterizing non-export functions and novel host protein interactions with Rev; (3) examining the evolution of Rev-Env and RRE-Env overlapping reading frames; and (4) examining RRE RNA structure in vivo by 3D mapping. These experiments will lead to further structural advances aimed at understanding the molecular basis for Rev-RRE function and defining potential new therapeutic targets.
