Role of Dead Box Helicases in HIV-1 Rev Function: a Single-Molecule Approach

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. Initially, a single Rev monomer binds to stem loop IIB of the RRE, whereupon 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 host proteins, such as the DEAD box helicases DDX1 and DDX21 are known to be required for efficient Rev function in vivo, although their precise role is unknown. In this study, a variety of single-molecule fluorescence spectroscopic methods were used to dissect the role of DDX1 and DDX21 during assembly of Rev-RRE complexes. To facilitate these studies, the large DDX1 and DDX21 proteins were enzymatically labeled with bright and photostable dyes, while Rev was labeled at a single cysteine by maleimide chemistry. Single-color TIRF measurements with labeled Rev were used to monitor individual Rev monomer binding steps during oligomeric Rev-RRE assembly. Moreover, single-color experiments with labeled DDX1 or DDX21 reveal that each helicase is capable of binding directly to the RRE. Two-color colocalization and FRET measurements were used to monitor the simultaneous binding of both Rev and DDX1 to the RRE, revealing the temporal correlation between individual protein binding steps. Together, these studies are revealing how DEAD box helicases are able to promote the oligomeric assembly of Rev on the RRE, thereby acting as cellular cofactors of HIV-1. Supported by NIH P50 grant GM082545.