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 Zn2+ with a stoichiometry of one equivalent per Rev dimer. Optical spectroscopy of Rev Co2+ 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 Zn2+Cys4-linked dimer.