The miRNA-targeted transcriptome of porcine alveolar macrophages upon infection with Porcine Reproductive and Respiratory Syndrome Virus (RISC bound RNA)

Porcine alveolar macrophages were challenged in vitro with a low virulent strain of the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) to identify and characterize the population of host genes subjected to miRNA post-transcriptional regulation durign early viral infection. We employed a high-throughput biochemical assay, i.e. the immunoprecipitation of RISCs followed by microarray analysis of the RISC-bound miRNA targets (RIP-Chip). qPCR analyses were carried out to validate the resolution of the microarray and to determine levels of expression of a panel of eight miRNAs (ssc-miR-142-3p, ssc-miR-142-5p, ssc-let-7f, miR-146a-5p, miR-155-5p, ssc-miR-181a, ssc-miR-21 and ssc-miR-335). Lung alveolar macrophages were obtained by lung lavage from 4 animals. Cells were infected in vitro with the strain Finistère at multiplicity of infection of 2, or mock infected. Infected cells were harvested at 7 and 10 hours post-infection; control (mock infected) cells were collected at the same times. Total RNAs were extracted from total cell contents and from RISC (RNA-induced Silencing Complex) immunoprecipitates. RNAs were hybridized on microarrays and transcripts analyzed for relative enrichment in RISC (RIP-Chip technology).