Abstract
Host miRNAs are known to modulate the cell response to virus infections. We characterized the miRNA-targeted transcriptome of porcine alveolar macrophages (PAMs) at early times after infection with a subtype 1.1 strain of PRRSV (Porcine Reproductive and Respiratory Syndrome Virus). We performed the immunoprecipitation of RISC (RNA-induced Silencing Complex) followed by microarray analysis of the RISC-bound miRNA targets (RIP-Chip) to evaluate the relative enrichment or depletion of expressed genes in RISC. The miRNA-mediated regulation occurred early after PRRSV infection and decreased fast (1,241 and 141 RISC-bound genes at 7 h and 10 h post-infection, respectively); it affected several cell functions with evidence of miRNA buffering of upregulated interferon-related genes. Eight miRNAs were highly enriched in RISC of both control and infected cells with no evidence of differential expression. Although miR-335-5p was the miRNA with most predicted targets among enriched RISC-bound genes, no effects on surface markers, cytokine expression and PRRSV replication were detected upon miR-335-5p mimics of primary PAMs. Our results do not point to specific miRNA-driven mechanisms regulating the early response to infection with this PRRSV 1.1 strain and indicate that the miRNome expressed by steady-state PAMs reacts promptly to counterbalance PRRSV infection by a pervasive modulation of host functions.
Highlights
The Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) emerged in the early 1990s and since represents a major problem to the swine industry worldwide[1,2]
(“Finistère”)[29] was performed on porcine alveolar macrophages (PAMs) isolated by bronchoalveolar lavages from four specific-pathogen-free piglets, with multiplicity of infection (MOI) of 2
108 PAMs were used for each experimental condition to ensure equal RNA input amounts for each microarray hybridization (50 ng and 10 ng of whole cell and RNA-induced silencing complex (RISC)-bound RNA, respectively)
Summary
The Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) emerged in the early 1990s and since represents a major problem to the swine industry worldwide[1,2]. PRRSV has been extensively investigated on these aspects, with several individual miRNAs reported to play important roles in PRRSV infection and replication, and in modulating host antiviral responses (reviewed by)[26] These studies have made use of different cell systems, PRRSV strains, infection conditions and choice of time points for in vitro kinetics, making it difficult to infer unique or common patterns of miRNA host response between studies. We aimed at identifying the whole set of host genes undergoing miRNA-mediated post-transcriptional regulation (i.e. the miRNA-targeted transcriptome) in the main target cells of PRRSV (porcine alveolar macrophages) during the early infection phase, when miRNAs may determine phenotypes not yet overruled by the cell immune responses or by cell death and other indirect effects To this issue we carried out the immunoprecipitation of RISC followed by microarray analysis of the RISC-bound miRNA targets (RIP-Chip), as this high-throughput biochemical assay allows the genome-wide identification of genes targeted by cellular miRNAs in an unbiased and physiologically relevant manner[27,28]
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