Assembly of Ribonucleoproteins Involved in Viral RNA Trafficking

Abstract

Oligomerization of the HIV-1 protein Rev on the Rev Response Element (RRE) regulates nuclear export of genomic viral RNA and partially spliced viral mRNAs encoding for structural proteins. Single-molecule fluorescence imaging has been used to dissect the multi-step assembly pathway of this essential ribonucleoprotein under physiologically relevant conditions, revealing dynamic intermediates and the mechanism of assembly. Assembly is initiated by binding of Rev to a high-affinity site in stem-loop IIB of the RRE and proceeds rapidly by addition of single Rev monomers, facilitated by cooperative Rev-Rev interactions on the RRE. Dwell time analysis of fluorescence trajectories recorded during individual Rev-RRE assembly reactions has revealed the microscopic rate constants for several of the Rev monomer binding and dissociation steps. The high-affinity binding of multiple Rev monomers to the RRE is achieved on a much faster time scale than reported in previous bulk kinetic studies of Rev-RRE association, indicating that oligomerization is an early step in complex assembly. In addition to Rev, a variety of cellular proteins are also required for nuclear export of the viral mRNA. Hence, the single-molecule imaging system has also been used to monitor Rev-RRE complex assembly in the presence of selected cellular cofactors.