A DEAD-box protein acts through RNA to promote HIV-1 Rev-RRE assembly

The HIV-1 Rev protein activates nuclear export of unspliced and partially spliced viral RNA transcripts, which encode the viral genome and the genes encoding viral structural proteins, by binding to and oligomerizing on the Rev Response Element (RRE). The human DEAD-box protein 1 (DDX1) enhances the...

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Bibliographic Details
Published in:Nucleic acids research 2017-05, Vol.45 (8), p.4632-4641
Main Authors: Lamichhane, Rajan, Hammond, John A, Pauszek, 3rd, Raymond F, Anderson, Rae M, Pedron, Ingemar, van der Schans, Edwin, Williamson, James R, Millar, David P
Format: Article
Language:English
Subjects:
Binding Sites
Biological Transport
Carbocyanines - chemistry
DEAD-box RNA Helicases - genetics
DEAD-box RNA Helicases - metabolism
Fluorescence Resonance Energy Transfer
Fluorescent Dyes - chemistry
Gene Expression
Genes, env
HIV-1 - genetics
HIV-1 - growth & development
HIV-1 - metabolism
Host-Pathogen Interactions
Humans
Molecular Biology
Nucleic Acid Conformation
Protein Binding
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - metabolism
Rhodamines - chemistry
RNA, Messenger - chemistry
RNA, Messenger - genetics
RNA, Messenger - metabolism
RNA, Viral - chemistry
RNA, Viral - genetics
RNA, Viral - metabolism
Single Molecule Imaging
Staining and Labeling
Sulfonic Acids - chemistry
Virus Assembly - genetics
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Summary:The HIV-1 Rev protein activates nuclear export of unspliced and partially spliced viral RNA transcripts, which encode the viral genome and the genes encoding viral structural proteins, by binding to and oligomerizing on the Rev Response Element (RRE). The human DEAD-box protein 1 (DDX1) enhances the RNA export activity of Rev through an unknown mechanism. Using a single-molecule assembly assay and various DDX1 mutants, we show that DDX1 acts through the RRE RNA to specifically accelerate the nucleation step of the Rev-RRE assembly process. Single-molecule Förster resonance energy transfer (smFRET) experiments using donor-labeled Rev and acceptor-labeled DDX1 show that both proteins can associate with a single RRE molecule. However, simultaneous interaction is only observed in a subset of binding events and does not explain the extent to which DDX1 promotes the nucleation step of Rev-RRE assembly. Together, these results are consistent with a model wherein DDX1 acts as an RNA chaperone, remodeling the RRE into a conformation that is pre-organized to bind the first Rev monomer, thereby promoting the overall Rev-RRE assembly process.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/gkx206