Quantitative affinity purification coupled to mass spectrometry identifies OASL as an ORF20 interaction partner.

<p>(A) Scheme of the quantitative affinity purification coupled to mass spectrometry (q-AP-MS) workflow used to identify cellular interaction partners of ORF20. In the forward experiment, HeLa S3 cells were labeled with heavy or light amino acids, then transfected with ORF20WT-myc or LacZ-myc, respectively. In the crossover experiment, ORF20WT-myc was labeled with light amino acids and LacZ-myc with heavy amino acids. Lysates were immunoprecipitated with anti-myc magnetic beads, combined for affinity purification, processed, and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). ORF20 interaction partners were identified based on the increased abundance of heavy labeled proteins compared to light labeled proteins. In the parallel crossover experiment, specific interaction partners were identified based on increased abundance of light labeled proteins. Proteins with a 1:1 ratio of light and heavy amino acids are non-specific binding partners or contaminants. (B) Quantitative proteomics results displayed graphically. The abundances of proteins identified in the forward and crossover experiments are graphed, with ORF20 interacting partners located in the lower right quadrant. Each point represents one protein. 40S and 60S ribosomal proteins are shown in blue and OASL is indicated in red. (C) 293T, 293T OASL^-/-, and HeLa S3 cells were transfected with ORF20WT-myc (+) or empty vector (-) and/or RIG-I N (+) or empty vector (-). An anti-myc immunoprecipitation of NP-40 lysates was performed and input lysates and immunoprecipitates were immunoblotted with anti-OASL and anti-myc antibodies. *: Nonspecific background band. Immunoblots are representative of two independent experiments. (D) 293T cells were transfected with myc-tagged ORF20 isoforms, LacZ-myc, OASL-V5, and/or EV as indicated. An anti-myc immunoprecipitation of RIPA lysates was performed and input lysates and immunoprecipitates were immunoblotted with anti-V5 and anti-myc antibodies. Data are representative of three independent experiments. (E) HeLa S3 cells were transfected individually with ORF20WT-myc or OASL-V5, then labeled with antibodies against myc or V5 as appropriate (green) and fibrillarin (red). (F) HeLa S3 cells were co-transfected with ORF20WT-myc and OASL-V5, and then labeled with anti-V5 (green) and anti-myc (red) antibodies. Nuclei were counterstained with Hoechst (blue). Images are representative of three independent experiments. Scale bar = 10 μm (E) and 20 μm (F).