Biochemical characterization of binding of multiple HIV-1 Rev monomeric proteins to the Rev responsive element

Abstract

Recombinant HIV-1 Rev protein was overexpressed in Escherichia coli using translational coupling to the beta-glucuronidase gene and demonstrated to interact with high affinity and specificity with the Rev responsive element (RRE). A complex Rev-dependent binding pattern was observed using the gel shift assay which could be simplified to one or two primary bands in the presence of stoichiometric concentrations of RRE. Competition of these bands with a series of homopolymer RNA species demonstrated that Rev is essentially a poly-G binding protein, although poly-I was also shown to compete for specific RRE binding. The stoichiometry of the Rev-dependent gel shift complexes was determined using 125I-labeled Rev. The stable, lowest mobility complex was determined to possess a ratio of between 7 and 8 Rev molecules per RRE containing RNA fragment while the two fastest migrating complexes contained ratios of one and two Rev molecules per RRE, respectively. Using the Hill equation as a model for cooperative interactions, a Hill coefficient of n(app) = 2 was obtained from fitting of direct nitrocellulose filter binding assays, reflecting cooperatively bound Rev molecules on the RRE under equilibrium binding conditions. An increase in ionic strength from 0.0 to 0.3 M NaCl reduced cooperative Rev binding to the RRE, but specificity of Rev for the RRE relative to antisense RNA was increased 100,000-fold. At molar ratios of Rev to RRE above 2, Rev dissociated from the RRE with a T1/2 of approximately 20-25 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of CD83 Is Regulated by HuR via a Novel cis-Active Coding Region RNA Element

“…RNA gel retardation assays were performed using in vitro transcribed 32 P-labeled CD83-derived probes, MS2 competitor RNA, and GST fusion protein as described previously (46), except that RNA-protein complexes were resolved on 4 or 6% native polyacrylamide gels. Supershift assays were performed using anti-HuR (clone 19F12, Invitrogen) and anti-FLAG (Sigma) monoclonal antibodies.…”

The Human T-cell leukemia virus Rex protein

“…Although the RxRE harbors a single high affinity binding site for Rex, it has been suggested that multiple Rex proteins bind to a single mRNA molecule prior to export via cooperative protein-protein and protein-RNA interactions (76,77). There are two regions in both Rex-1 and Rex-2 that serve as multimerization domains (54,64,78).…”

Structural and Functional Characterization of RNA-binding Site of Rev and Rev-Response-Element RNA Complex via All Atom Molecular Dynamics Simulations

“…Then the export of RNA is carried out by Crm1 nuclear export pathway [17]. First Rev oligomerizes onto the RRE to form a ribonucleoprotein (RNP) complex of 200-300 kDa containing 8-10 Rev molecules [18][19], that binds Crm-1 (exportin-1), GTP-bound Ran, and other host cell proteins via the Rev nuclear export sequence (NES), to promote nuclear export [20]. Structure of REV contain 116 a.a in a single chain [21].…”