The HIV-1 Rev protein is essential for the virus because it promotes nuclear export of alternatively-processed mRNAs, and Rev is also linked to translation of viral mRNAs and genome encapsidation. Previously, the human DEAD-box helicase DDX1 was suggested to be involved in Rev functions, but this relationship is not well understood. Biochemical studies of DDX1 and its interactions with Rev and model RNA oligonucleotides were carried out to investigate the molecular basis for association of these components. A combination of gel-filtration chromatography and circular dichroism spectroscopy demonstrated that recombinant DDX1 expressed in E. coli is a well-behaved folded protein. Binding assays using fluorescently-labeled Rev and cell-based immunoprecipitation analysis confirmed a specific RNA-independent DDX1-Rev interaction. Additionally, DDX1 was shown to be an RNA-activated ATPase, wherein Rev-bound RNA was equally effective at stimulating ATPase activity as protein-free RNA. Gel mobility shift assays further demonstrated that DDX1 forms complexes with Rev-bound RNA. RNA silencing of DDX1 provided strong evidence that DDX1 is required for both Rev activity and HIV production from infected cells. Collectively, these studies demonstrate a clear link between DDX1 and HIV-1 Rev in cell based assays of HIV-1 production, and provide the first demonstration that recombinant DDX1 binds Rev and RNA, and has RNA dependent catalytic activity.

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.

SUMMARY Post-transcriptional regulation of gene expression is an important mechanism for modulating protein levels in eukaryotes, especially in developmental pathways. The highly conserved homodimeric STAR/GSG proteins play a key role in regulating translation by binding bipartite consensus sequences in the untranslated regions of target mRNAs, but the exact mechanism remains unknown. Structures of STAR protein RNA binding subdomains have been determined, but structural information is lacking for the homodimerization subdomain. Here, we present the structure of the C. elegans GLD-1 homodimerization domain dimer, determined by a combination of X-ray crystallography and NMR spectroscopy, revealing a helix-turn-helix monomeric fold with the two protomers stacked perpendicularly. Structure-based mutagenesis demonstrates that the dimer interface is not easily disrupted, but the structural integrity of the monomer is crucial for GLD-1 dimerization. Finally, an improved model for STAR-mediated translational regulation of mRNA, based on the GLD-1 homodimerization domain structure, is presented.

Most cellular processes requiring RNA structure rearrangement necessitate the action of Asp-Glu-Ala-Asp (DEAD) proteins. Members of the family, named originally for the conserved DEAD amino acid sequence, are thought to disrupt RNA structure and facilitate its rearrangement by unwinding short stretches of duplex RNA. BstDEAD is a novel 436 amino acid representative of the DEAD protein family from Bacillus stearothermophilus that contains all eight conserved motifs found in DEAD proteins and is homologous with other members of the family. Here, we describe the 1.85 Å resolution structure of the N-terminal domain (residues 1-211) of BstDEAD (BstDEAD-NT). Similar to the corresponding domains of related helicases, BstDEAD-NT adopts a parallel ␣/␤ structure with RecA-like topology. In general, the conserved motifs superimpose on closely related DEAD proteins and on more distantly related helicases such as RecA. This affirms the current belief that the core helicase domains, responsible for mechanistic activity, are structurally similar in DEAD proteins. In contrast, however, the so-called Walker A P-loop, which binds the ␤-and ␥-phosphates of ATP, adopts a rarely seen "closed" conformation that would sterically block ATP binding. The closed conformation may be indicative of a general regulatory feature among DEAD proteins (and RNA helicases) that differs from that used by DNA helicases. BstDEAD also contains a unique extension of ∼ 60 residues at the C terminus that is highly basic, suggesting that it might bind nucleic acids and, in so doing, confer specificity to the helicase activity of the core region.

NXF1-like members of the Nuclear eXport Factor (NXF) family orchestrate bulk nuclear export of mRNA, while functionally distinct NXF variant proteins carry out separate substrate and tissue specific RNA regulation. Metazoan organisms possess at least one NXF1-like gene and one or more NXF variant genes. Heterodimerization of both proteins with the NTF2-related eXporT protein (NXT) is central to NXF family function, but given the multiplicity of NXF/NXT complexes, the specificity and mechanism of heterodimerization remains unclear. Here, we report the structural and functional analysis of the Caenorhabditis elegans NXF variant, ceNXF2, bound to ceNXT1. Contacts crucial for NXF/NXT heterodimer stability and specificity have been identified, including a probable site for phosphoregulation. The ceNXF2 NTF2 domain bears at least two nucleoporin (Nup) binding pockets necessary for colocalization of ceNXF2/ceNXT1 at the nuclear envelope. Unexpectedly, one Nup binding pocket is formed at the heterodimer interface of the ceNXF2/ceNXT1 complex, demonstrating that NXT binding directly regulates NXF function.