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 HIV-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, was 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 non-productive 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 (AMP-PNP), 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 AMP-PNP, 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.

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

“…DEAD-box proteins are involved in many aspects of RNA metabolism, including ribosome biogenesis, RNA splicing, translation and RNA degradation (20,21), though their mechanistic role in these processes remain largely elusive. In a previous single-molecule study, we provided evidence that DDX1 promotes Rev-RRE assembly in vitro (12). However, understanding the role of DDX1 during Rev-RRE assembly is complicated by the fact that DDX1 can physically interact with the Rev protein or the RRE RNA (17).…”

“…Assembly is initiated by the binding of a single Rev monomer to a high affinity site in stem IIB (Figure 1A) (5-7) and proceeds by incorporation of additional Rev monomers into the ribonucleoprotein complex (8)(9)(10), which are recruited one at a time through a combination of protein-RNA and protein-protein interactions (11). Kinetic parameters describing each step of Rev-RRE assembly or disassembly have been determined through singlemolecule studies (11,12). A secondary Rev binding site was identified in stem IA of the RRE (10) and a model for the three-dimensional architecture of the RRE has been proposed on the basis of small-angle X-ray scattering studies (13).…”

HIV-1 Nef-associated Factor 1 Enhances Viral Production by Interacting with CRM1 to Promote Nuclear Export of Unspliced HIV-1 gag mRNA

“…Several other cellular factors have also been identified as Rev co-factors that are required for efficient Rev function, such as the RNA binding motif protein 14, the single-stranded nucleic acid-binding protein Pur-alpha, eukaryotic initiation factor-5A, and the DEAD (Asp-Glu-Ala-Asp) box RNA helicase DDX1, DDX3, and DDX5 (23)(24)(25)(26)(27)(28)(29)(30)(31). However, some host factors recruited by Rev can interfere with its function.…”

RNA helicases in infection and disease

“…57 It interacts with the oligomerization domain of HIV-1 Rev, 58 and promotes Revoligomerization on the RRE. 59 The binding of DDX1 to Rev may target DDX1 to incompletely spliced transcripts. 60 DDX1 has also been associated with the lifecycle of polyoma and coronaviruses, 61,62 and of HCV.…”