Oligomeric Assembly of HIV-1 Rev on the Rev Response Element: Role of Cellular Cofactors

Abstract

Rev, a key regulatory protein of HIV-1, activates nuclear export of unspliced and partially spliced viral mRNAs, which encode the viral genome and the genes encoding viral structural proteins, respectively. Initially, a single Rev monomer binds to a highly conserved region, stem IIB located on the Rev Response Element (RRE) of viral mRNA. Following this nucleation step, additional Rev monomers are recruited to the RRE through a combination of RNA-protein and protein-protein interactions, resulting in the formation of a functional nuclear export complex. In addition, several cellular proteins, such as the DEAD box helicases DDX1 and DDX3 are known to be required for efficient Rev function in vivo, although their precise role is unknown. In this study, single-molecule total internal reflection fluorescence (smTIRF) microscopy was used to visualize oligomeric assembly of Rev on the RRE with single monomer resolution. Binding of up to eight fluorescently labeled Rev monomers to a single immobilized RRE molecule was observed in real-time as discrete jumps in fluorescence intensity, and the event frequencies and corresponding binding and dissociation rates for the different Rev-RRE stoichiometries were determined. The smTIRF assay was used to study Rev-RRE assembly in the presence of DDX1, DDX3 and other cellular proteins. The presence of DDX1 promotes oligomeric assembly by accelerating the first few Rev monomer binding steps, suggesting that DDX1 acts as a chaperone of Rev. The smTIRF measurements are being extended to a multi-color format, in order to directly visualize the colocalization of Rev and selected cellular proteins on the same immobilized RRE molecules. These measurements are revealing the precise timing of various protein binding events during ribonucleoprotein assembly. Supported by NIH grant P50 GM082545.