HIV-1 Rev Protein Forms a Zinc-Linked Dimer

HIV-1 Rev protein is a small, basic protein of 116 amino acids that assembles reversibly in the presence and absence of its cognate RRE containing RNAs both in vivo and in vitro . The biologically active form of Rev is unclear since studies have shown monomer, dimer, tetramer and higher-order oligomer interactions with various RRE-RNAs. Whilst assembly is essential for its regulatory role in the viral life cycle, it has been a barrier to high resolution structural studies of the whole protein and its complexes. The N-terminal half of Rev has been shown to contain a helix loop helix motif with residues involved both in assembly and RNA binding. The C-terminal half is predicted to contain little secondary structure based on UV-CD spectral analyses, and to contain the leucine rich activation domain (residues 73-83). Early studies had shown the essential part of the C-terminal extends to residue 93 and is required for increased structural stability of the protein and its complexes with RRE-RNAs, and to facilitate the formation of Rev dimers (4, 19). The strong conservation of cysteines at positions 85 and 89, and the less well-conserved histidine residues at 53 or 82 led us to examine Rev-metal interactions. Here we show that Rev binds Zn ²⁺ with a stoichiometry of one equivalent per Rev dimer. Optical spectroscopy of Rev Co ²⁺ complexes revealed that the metal site is composed of four cysteine residues with a tetrahedral coordination geometry. We propose that HIV-1 Rev protein is biologically active as a Zn ²⁺ Cys4-linked dimer.