Abstract
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 10(-10) M, whereas the affinity for the core RNA was about 10(-11) 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.
Highlights
The genome of HIV-11 is transcribed as a full-length 9-kilobase mRNA that can follow two different “pathways” (1– 4)
A number of groups have demonstrated that a single Rev monomer bound to a high affinity site on the Rev response element (RRE), after which additional Rev molecules were recruited through protein-RNA and protein-protein interactions (20, 58, 60, 61), others have shown that Rev bound to its target RNA in an oligomeric form (62, 63)
Rev-RRE interaction has been extensively studied, a number of fundamental questions remain to be answered. These include whether Rev acts solely to transport RRE-containing RNA to the cytoplasm or actively inhibits splicing, the precise number of Rev monomers bound to each RRE, whether Rev monomers bind sequentially or in oligomeric form and whether this binding is cooperative, and precise knowledge of the various rate constants of Rev-RRE binding
Summary
Vol 274, No 25, Issue of June 18, pp. 17452–17463, 1999 Printed in U.S.A. DEFINITION OF MINIMAL BINDING SITES ON RNA AND PROTEIN AND STOICHIOMETRIC ANALYSIS*. Later in the virus lifecycle, unspliced or partially spliced RNAs are exported from the nucleus to the cytosol, where the unspliced RNA is packaged into virus particles; the different partially spliced mRNAs are translated into Gag, Pol, Env, and smaller proteins, such as vif, vpR, and vpU (4, 6 –10) This temporal switch in the HIV RNA complexity and the coding potential is mediated by virus coded Rev protein (11–19), an approximately 16-kDa basic RNA-binding protein with two functional domains (Fig. 1A). Notwithstanding the functional definition of the minimal Rev-responsive sequence, Mann et al (59) have reported that a larger RRE-RNA structure of 351 nucleotides is required for complete biological activity. This method can be used to rapidly and conveniently investigate RNA-protein interactions in general and was quite useful in rapidly evaluating the effects RNA and protein mutations on the binding, as well as inhibitors of these interactions
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