The kinetics of interaction between the human immunodeficiency
virus-1 Rev protein and its RNA target,
Rev response element (RRE) RNA was determined in
vitro using a biosensor technique. Our results showed
that the primary Rev binding site is a core stem-loop
RNA molecule of 30 nucleotides that bound Rev at a 1:1
ratio, whereas the 244-nucleotide full-length RRE bound
four Rev monomers. At high Rev concentrations, additional
binding of Rev to RRE was observed with ratios of
more than 10:1. Because RRE mutants that lacked the
core binding site and were inactive in vivo bound Rev
nonspecifically at these concentrations, the real stoichiometric
ratio of Rev-RRE is probably closer to 4:1. Binding
affinity of Rev for RRE was approximately 10210 M,
whereas the affinity for the core RNA was about 10211 M,
the difference being due to the contribution of low affinity
binding sites on the RRE. Mathematical analysis
suggested cooperativity of Rev binding, probably mediated
by the Rev oligomerization domains. C-terminal
deletions of Rev had no effect on RRE binding, but truncation
of the N terminus by as few as 11 residues significantly
reduced binding specificity. This method was
also useful to rapidly evaluate the potential of aminoglycoside
antibiotics, to inhibit the Rev-RRE interaction.
