title: Single-molecule studies reveal that DEAD box protein DDX1 promotes oligomerization of HIV-1 Rev on the Rev response element.
abstract: Oligomeric assembly of Rev on the Rev response element (RRE) is essential for the nuclear export of unspliced and singly spliced human immunodeficiency virus type 1 viral mRNA transcripts. Several host factors, including the human DEAD box protein DDX1, are also known to be required for efficient Rev function. In this study, spontaneous assembly and dissociation of individual Rev-RRE complexes in the presence or absence of DDX1 were observed in real time via single-molecule total internal reflection fluorescence microscopy. Binding of up to eight fluorescently labeled Rev monomers to a single RRE molecule was visualized, and the event frequencies and corresponding binding and dissociation rates for the different Rev-RRE stoichiometries were determined. The presence of DDX1 eliminated a second kinetic phase present during the initial Rev binding step, attributed to nonproductive nucleation events, resulting in increased occurrence of higher-order Rev-RRE stoichiometries. This effect was further enhanced upon the addition of a non-hydrolyzable ATP analog (adenylyl-imidophosphate), whereas ADP had no effect beyond that of DDX1 alone. Notably, the first three Rev monomer binding events were accelerated in the presence of DDX1 and adenylyl-imidophosphate, while the dissociation rates remained unchanged. Measurements performed across a range of DDX1 concentrations suggest that DDX1 targets Rev rather than the RRE to promote oligomeric assembly. Moreover, DDX1 is able to restore the oligomerization activity of a Rev mutant that is otherwise unable to assemble on the RRE beyond a monomeric complex. Taken together, these results suggest that DDX1 acts as a cellular cofactor by promoting oligomerization of Rev on the RRE.
authors.affiliation: Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: Rae M
authors.initials: RM
authors.lastname: Robertson-Anderson
authors.forename: Jun
authors.initials: J
authors.lastname: Wang
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Edgcomb
authors.forename: Andrew B
authors.initials: AB
authors.lastname: Carmel
authors.forename: James R
authors.initials: JR
authors.lastname: Williamson
authors.forename: David P
authors.initials: DP
authors.lastname: Millar
mesh_terms: D053487:DEAD-box RNA Helicases / Q000378:metabolism*; D005453:Fluorescence; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D050505:Mutant Proteins; D009841:Oligonucleotides / Q000378:metabolism; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D020218:Response Elements / Q000235:genetics*; D054321:rev Gene Products, Human Immunodeficiency Virus / Q000737:chemistry*
chemical_list: D050505:Mutant Proteins; D009841:Oligonucleotides; D054321:rev Gene Products, Human Immunodeficiency Virus; C086252:DDX1 protein, human; D053487:DEAD-box RNA Helicases