Antisense-mediated affinity purification of dengue virus ribonucleoprotein complexes from infected cells

• A method for purification of endogenous dengue virus ribonucleoprotein complexes is described.
• The method incorporates cross-linking of RNA and protein in living cells using ultraviolet light prior to cell lysis.
• Dengue virus RNA and cross-linked proteins are purified using complementary biotinylated oligonucleotides.
• This method can be used in principle to study interactions between RNA-binding proteins and any viral RNA.

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.