title: DDX1 is an RNA-dependent ATPase involved in HIV-1 Rev function and virus replication.
abstract: The human immunodeficiency virus type 1 (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 Escherichia 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.
authors: Department of Molecular Biology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
authors: Stephen P
authors: SP
authors: Edgcomb
authors: Andrew B
authors: AB
authors: Carmel
authors: Souad
authors: S
authors: Naji
authors: Geza
authors: G
authors: Ambrus-Aikelin
authors: Jason R
authors: JR
authors: Reyes
authors: Andrew C S
authors: AC
authors: Saphire
authors: Larry
authors: L
authors: Gerace
authors: James R
authors: JR
authors: Williamson
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism*; D015658:HIV Infections / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D011485:Protein Binding; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication / Q000235:genetics / Q000502:physiology*; D054321:rev Gene Products, Human Immunodeficiency Virus / Q000378:metabolism*
chemical_list: D012333:RNA, Messenger; D012367:RNA, Viral; D011994:Recombinant Proteins; D054321:rev Gene Products, Human Immunodeficiency Virus; C514860:rev protein, Human Immunodeficiency Virus-1; D000251:Adenosine Triphosphatases; C086252:DDX1 protein, human; C027900:RNA-dependent ATPase; D053487:DEAD-box RNA Helicases