|Título||Quantitative mass spectrometry of DENV-2 RNA-interacting proteins reveals that the DEAD-box RNA helicase DDX6 binds the DB1 and DB2 3' UTR structures.|
|Publication Type||Journal Article|
|Year of Publication||2011|
|Autores||Ward, AM, Bidet, K, Yinglin, A, Ler, SG, Hogue, K, Blackstock, W, Gunaratne, J, García-Blanco, MA|
|Date Published||2011 Nov-Dec|
|Palabras clave||3' Untranslated Regions, 5' Untranslated Regions, Base Sequence, Binding Sites, Blotting, Western, Carcinoma, Hepatocellular, Cell Line, Tumor, Cytoplasmic Granules, DEAD-box RNA Helicases, Dengue Virus, HeLa Cells, Humans, Liver Neoplasms, Mass Spectrometry, Microscopy, Fluorescence, Molecular Sequence Data, Nucleic Acid Conformation, Proto-Oncogene Proteins, RNA Interference, RNA, Viral, RNA-Binding Proteins, Virus Assembly, Virus Replication|
Dengue virus (DENV) is a rapidly re-emerging flavivirus that causes dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), diseases for which there are no available therapies or vaccines. The DENV-2 positive-strand RNA genome contains 5' and 3' untranslated regions (UTRs) that have been shown to form secondary structures required for virus replication and interaction with host cell proteins. In order to comprehensively identify host cell factors that bind the DENV-2 UTRs, we performed RNA chromatography, using the DENV-2 5' and 3' UTRs as "bait", combined with quantitative mass spectrometry. We identified several proteins, including DDX6, G3BP1, G3BP2, Caprin1, and USP10, implicated in P body (PB) and stress granule (SG) function, and not previously known to bind DENV RNAs. Indirect immunofluorescence microscopy showed these proteins to colocalize with the DENV replication complex. Moreover, DDX6 knockdown resulted in reduced amounts of infectious particles and viral RNA in tissue culture supernatants following DENV infection. DDX6 interacted with DENV RNA in vivo during infection and in vitro this interaction was mediated by the DB1 and DB2 structures in the 3' UTR, possibly by formation of a pseudoknot structure. Additional experiments demonstrate that, in contrast to DDX6, the SG proteins G3BP1, G3BP2, Caprin1 and USP10 bind to the variable region (VR) in the 3' UTR. These results suggest that the DENV-2 3' UTR is a site for assembly of PB and SG proteins and, for DDX6, assembly on the 3' UTR is required for DENV replication.
|Alternate Journal||RNA Biol|