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

Alex Michael Ward, Katell Bidet, Ang Yinglin, Siok Ghee Ler, Kelly Hogue, Walter Blackstock, Jayantha Gunaratne, Mariano Garcia-Blanco

Research output: Contribution to journalArticle

99 Citations (Scopus)

Abstract

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 positivestrand 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 USP 10, 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 USP 10 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.

Original languageEnglish (US)
JournalRNA Biology
Volume8
Issue number6
DOIs
StatePublished - Nov 2011
Externally publishedYes

Fingerprint

DEAD-box RNA Helicases
Dengue Virus
3' Untranslated Regions
Mass Spectrometry
RNA
Proteins
Severe Dengue
Virus Replication
5' Untranslated Regions
Heat-Shock Proteins
Untranslated Regions
Flavivirus
Active Immunotherapy
Dengue
Viral RNA
Virus Diseases
Indirect Fluorescent Antibody Technique
Fluorescence Microscopy
Chromatography

Keywords

  • DDX6
  • Dengue
  • Host factors
  • RNA
  • Stress granules

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

Cite this

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. / Ward, Alex Michael; Bidet, Katell; Yinglin, Ang; Ler, Siok Ghee; Hogue, Kelly; Blackstock, Walter; Gunaratne, Jayantha; Garcia-Blanco, Mariano.

In: RNA Biology, Vol. 8, No. 6, 11.2011.

Research output: Contribution to journalArticle

Ward, Alex Michael ; Bidet, Katell ; Yinglin, Ang ; Ler, Siok Ghee ; Hogue, Kelly ; Blackstock, Walter ; Gunaratne, Jayantha ; Garcia-Blanco, Mariano. / 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. In: RNA Biology. 2011 ; Vol. 8, No. 6.
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abstract = "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 positivestrand 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 USP 10, 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 USP 10 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.",
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AU - Blackstock, Walter

AU - Gunaratne, Jayantha

AU - Garcia-Blanco, Mariano

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