Antisense-mediated affinity purification of dengue virus ribonucleoprotein complexes from infected cells.
The identification of RNA-binding proteins that physically associate with viral RNA molecules during infection can provide insight into the molecular mechanisms of RNA virus replication. Until recently, such RNA-protein interactions have been identified predominantly with the use of in vitro assays that may not accurately reflect associations that occur in the context of a living cell. Here we describe a method for the specific affinity purification of dengue virus RNA and associated proteins using in vivo cross-linking followed by antisense-mediated affinity purification. RNA-binding proteins that specifically co-purify with viral RNA using this method can be identified en masse by mass spectrometry. This strategy can potentially be adapted to the purification of any viral RNA species of interest.
Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1055, United States; Department of Microbiology and Molecular Genetics, Center for RNA Biology and Department of Medicine, Duke University, 213 Research Drive, Durham, NC 27710, United States.
Stacia L
SL
Phillips
Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1055, United States; Department of Microbiology and Molecular Genetics, Center for RNA Biology and Department of Medicine, Duke University, 213 Research Drive, Durham, NC 27710, United States.
Mariano A
MA
Garcia-Blanco
Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1055, United States; Department of Microbiology and Molecular Genetics, Center for RNA Biology and Department of Medicine, Duke University, 213 Research Drive, Durham, NC 27710, United States. Electronic address: ssbradri@utmb.edu.
Shelton S
SS
Bradrick
D002846:Chromatography, Affinity / Q000379:methods*; D003716:Dengue Virus / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D020319:Oligodeoxyribonucleotides, Antisense; D012367:RNA, Viral / Q000302:isolation & purification* / Q000378:metabolism; D012261:Ribonucleoproteins / Q000302:isolation & purification* / Q000378:metabolism
D020319:Oligodeoxyribonucleotides, Antisense; D012367:RNA, Viral; D012261:Ribonucleoproteins
Dengue virus; RNA affinity chromatography; Ribonucleoprotein complex