A Dead-Box Protein Acts through RNA to Promote HIV-1 Rev-RRE Assembly

Abstract

The HIV-1 Rev (Regulator of Expression of Virion) protein activates nuclear export of unspliced and partially spliced viral mRNAs, which encode the viral genome and the genes encoding viral structural proteins. Rev interacts with a highly conserved region, the Rev Response Element (RRE), located within the viral mRNA. In order to activate nuclear export, multiple Rev proteins must assemble on the RRE. The host DEAD box protein 1 (DDX1) enhances the RNA export activity of Rev through an unknown mechanism. We used a single-molecule assembly assay utilizing immobilized full length RRE and fluorophore-labeled Rev to monitor each step of Rev-RRE assembly, in the presence or absence of DDX1. More Rev monomers were observed to bind to the immobilized RRE in the presence of DDX1, indicating that DDX1 promotes oligomeric Rev-RRE assembly. Further experiments using specific DDX1 mutants that are defective in either Rev binding or RNA binding indicate that DDX1 must be capable of associating with RNA in order to promote assembly of the Rev-RRE complex. Single-molecule Förster resonance energy transfer (smFRET) experiments show that DDX1 transiently interacts with the RRE and that both DDX1 and Rev can occupy the same RRE molecule. Taken together, these results suggest that DDX1 acts as an RNA chaperone, folding the RRE into a conformation that is pre-organized to bind the first Rev monomer, thereby promoting the overall Rev-RRE assembly process. Supported by NIH grant GM082545.