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

Abstract

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.

Keywords: Dengue virus; RNA affinity chromatography; Ribonucleoprotein complex.

Figure 1. Schematic of affinity purification technique

Cells are infected with dengue virus at an MOI of 1. Mock infected cells serve as a negative control. RNA-protein cross-links are induced 30 hours post-infection by exposing the cells to 254 nm UV. Cells are lysed under denaturing conditions and incubated with biotinylated antisense DNA oligonucleotides. RNA-protein complexes are captured on streptavidin-coated magnetic beads. Protein can be digested with proteinase K for subsequent RNA analysis by RT-qPCR. Alternatively, proteins can be liberated from the RNA by RNase treatment and analyzed by Western blot or en masse by mass spectrometry.

Figure 2. RNase H mapping antisense oligonucleotide positions

A) Schematic of the RNase H mapping assay. Target RNA is incubated with complementary antisense DNA oligonucleotides and RNase H. If hybridization occurs, RNase H will cleave the RNA involved in the RNA:DNA hybrid. Cleavage is measured using RT-qPCR. B) Infected cells were cross-linked, lysed, and incubated with candidate antisense DNA oligonucleotides and RNase H. Purified RNA from mock cross-linked cells was used as a positive control for RNase H cleavage. RNA was purified from RNase H reactions and cleavage assessed by RT-qPCR using qPCR primers that amplify across the oligonucleotide hybridization site. Data is represented as the amount of RNA remaining relative to the no oligo control.

Figure 3. Optimization of cell lysis conditions

Infected Huh7 cells were lysed in buffer containing 200 mM KCl, 20 mM HEPES pH 7.2, and the indicated detergents. Insoluble material was cleared from the lysate by centrifugation and total RNA was purified from both soluble and pellet fractions. DENV and nuclear U6 RNAs were quantitated by RT-qPCR. DDM = n-dodecyl β-D-maltoside, NP40 = igepal CA-360, DOC = deoxycholic acid

Figure 4. Validation of RNA affinity purification procedure

A) RNA was purified from affinity purified eluates (N=14) and the yield of DENV and actin RNA was determined by RT-qPCR. B) The enrichment of viral RNA per actin after affinity purification was determined by RT-qPCR. C) Affinity purified protein prepared from Mock and DENV infected samples was separated by SDS-PAGE and visualized by silver stain.