Viral RNA-Protein Interaction Analysis

Immunoprecipitation was performed using total protein extracts from respective cells in various conditions (~300 mg), together with 1 mg of respective antibodies (as indicated in the figures). After overnight incubation at 4 °C, immune complexes were precipitated together with protein A-Sepharose beads (Amersham Biosciences) and analyzed by immunoblotting as described above. RIP assay was performed using total protein extracts from mock- or viral-infected cells with indicated antibody by RiboCluster Profiler RIP-Assay Kit (MBL, Japan, RN1001) according to the manufacturer’s recommendations. In brief, RNA–protein complex was pulled down either with 5 mg of mouse normal IgG (#sc-2025, Santa Cruz Biotechnology) or antibodies of interest. The bound RNAs were then recovered from the RNA–protein complex and used as a template for cDNA synthesis. Reverse-transcription and quantitative-PCR (RT-qPCR) was further performed to evaluate the RNA level bound to respective pulled-down proteins with the specific primer sets targeting the respective viral-associated gene. As an internal control, the glyceraldehyde 3-phosphatedehydrogenase (GAPDH) gene was used. Northern blotting was performed as previously described [13 (link)]. RNA probes to detect NDV F-mRNA was prepared from NDV cDNA using primer sets: Forward 5′-GCACAGATAACAGCAGCCTC-3′ and reverse 5′-CATCTTCCCAACTGCCACTG-3′.