Characterization of the microRNAome in porcine reproductive and respiratory syndrome virus infected macrophages.
ABSTRACT: Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), a member of the arterivirus family, is the causative agent of Porcine Reproductive and Respiratory Syndrome (PRRS). PRRS is characterized by late term abortions and respiratory disease, particularly in young pigs. Small regulatory RNAs termed microRNA (miRNA) are associated with gene regulation at the post-transcriptional level. MiRNAs are known to play many diverse and complex roles in viral infections. To discover the impact of PRRSV infections on the cellular miRNAome, Illumina deep sequencing was used to construct small RNA expression profiles from in vitro cultured PRRSV-infected porcine alveolar macrophages (PAMs). A total of forty cellular miRNAs were significantly differentially expressed within the first 48 hours post infection (hpi). The expression of six miRNAs, miR-30a-3p, miR-132, miR-27b*, miR-29b, miR-146a and miR-9-2, were altered at more than one time point. Target gene identification suggests that these miRNAs are involved in regulating immune signaling pathways, cytokine, and transcription factor production. The most highly repressed miRNA at 24 hpi was miR-147. A miR-147 mimic was utilized to maintain miR-147 levels in PRRSV-infected PAMs. PRRSV replication was negatively impacted by high levels of miR-147. Whether down-regulation of miR-147 is directly induced by PRRSV or if it is part of the cellular response and PRRSV indirectly benefits remains to be determined. No evidence could be found of PRRSV-encoded miRNAs. Overall, the present study has revealed that a large and diverse group of miRNAs are expressed in swine alveolar macrophages and that the expression of a subset of these miRNAs is altered in PRRSV infected macrophages.
Project description:Porcine reproductive and respiratory syndrome (PRRS) caused by a single-stranded RNA virus (PRRSV) is a highly infectious respiratory disease and leads to huge economic losses to the swine industry worldwide. To investigate the role of miRNAs in the infection and lung injury induced by PRRSV, the differentially expressed miRNAs (DE-miRs) were isolated from PRRSV-2 infected/mock-infected PAMs of Meishan, Landrace, Pietrain, and Qingping pigs at 9, 36, and 60 hpi. Mir-331-3p was the only common DE-miR in each set of miRNA expression profile at 36 hpi. Mir-210 was one of 7 common DE-miRs between PRRSV infected and mock-infected PAMs of Meishan, Pietrain, and Qingping pigs at 60 hpi. Mir-331-3p/mir-210 could target PRRSV-2 ORF1b, bind and downregulate porcine TNF-?/STAT1 expression, and inhibit PRRSV-2 replication, respectively. Furthermore, STAT1 and TNF-? could mediate the transcriptional activation of MCP-1, VCAM-1, and ICAM-1. STAT1 could also upregulate the expression of TNF-? by binding to its promoter region. In vivo, pEGFP-N1-mir-331-3p could significantly reduce viral replication and pathological changes in PRRSV-2 infected piglets. Taken together, Mir-331-3p/mir-210 have significant roles in the infection and lung injury caused by PRRSV-2, and they may be promising therapeutic targets for PRRS and lung injury/inflammation.
Project description:Porcine reproductive and respiratory syndrome virus (PRRSV) has been recognized to inhibit the response of type I interferon (IFN) both in vivo and in vitro. However, the post-transcriptional mechanism by which PRRSV suppresses type I IFN induction in virus-infected host cells remains unclear. The present study first demonstrated that PRRSV inhibited post-transcriptionally the protein induction of IFN-β in primary porcine alveolar macrophages (PAMs) during early infection, and the inhibition effect mediated by the Chinese highly pathogenic (HP)-PRRSV was stronger. Next, we analyzed the cellular microRNA (miRNA)-modulated protein expression of porcine IFN-β by dual firefly/Renilla luciferase reporter assay, transfection of miRNA mimics and inhibitor assay and polyinosinic-polycytidylic acid (poly I:C) treatment of PAMs, showing that porcine miRNAs including let-7b, miR-26a, miR-34a and miR-145 are able to inhibit IFN-β protein expression in primary PAMs by directly targeting sequences within the porcine IFN-β 3'UTR locating at 160-181, 9-31, 27-47 and 12-32 bp, respectively. Finally, we confirmed that let-7b, miR-26a, miR-34a and miR-145, were upregulated in PRRSV-infected PAMs early in vitro, and the expression level of these miRNAs in HP-PRRSV JXwn06-infected PAMs were higher than those in low pathogenic PRRSV HB-1/3.9-infected PAMs. The endogenous cellular miRNA-mediated inhibition of IFN-β induction in PRRSV-infected PAMs early could be relieved by miRNA antagonists. Taken together, our findings suggest for the first time that PRRSV can suppress post-transcriptionally protein expression of IFN-β by upregulating cellular miRNAs in PAMs in vitro, providing novel insight into mechanisms in relation to the PRRSV-mediated immunomodulation of porcine innate immunity.
Project description:Porcine reproductive and respiratory syndrome (PRRS) is one of the most ruinous diseases in pig production. Our previous work showed that Tongcheng pigs (TC) were less susceptible to PRRS virus (PRRSV) than Large White (LW) pigs. To elucidate the difference in PRRSV resistance between the two breeds, small RNA-seq and ribo-zero RNA-seq were used to identify differentially expressed non-coding RNAs (including miRNAs and lincRNAs) responded to PRRSV in porcine alveolar macrophages (PAMs) from TC and LW pigs. Totally, 250 known mature miRNAs were detected. For LW pigs, there were 44 down-regulated and 67 up-regulated miRNAs in infection group; while for TC pigs, 12 down-regulated and 23 up-regulated miRNAs in TC infection group were identified. The target genes of the common differentially expressed miRNAs (DEmiRNAs) in these two breeds were enriched in immune-related processes, including apoptosis process, inflammatory response, T cell receptor signaling pathway and so on. In addition, 5 shared DEmiRNAs (miR-181, miR-1343, miR-296-3p, miR-199a-3p and miR-34c) were predicted to target PRRSV receptors, of which miR-199a-3p was validated to inhibit the expression of CD151. Interestingly, miR-378 and miR-10a-5p, which could inhibit PRRSV replication, displayed higher expression level in TC control group than that in LW control group. Contrarily, miR-145-5p and miR-328, which were specifically down-regulated in LW pigs, could target inhibitory immunoreceptors and may involve in immunosuppression caused by PRRSV. This indicates that DEmiRNAs are involved in the regulation of the immunosuppression and immune escape of the two breeds. Furthermore, we identified 616 lincRNA transcripts, of which 48 and 30 lincRNAs were differentially expressed in LW and TC pigs, respectively. LincRNA TCONS_00125566 may play an important role in the entire regulatory network, and was predicted to regulate the expression of immune-related genes through binding with miR-1343 competitively. In conclusion, this study provides an important resource for further revealing the interaction between host and virus, which will specify a new direction for anti-PRRSV research.
Project description:Porcine reproductive and respiratory syndrome virus (PRRSV) infects mainly the porcine alveolar macrophages (PAMs) and causes porcine reproductive and respiratory syndrome (PRRS). Previous studies have analyzed the global gene expression profiles of lung tissue in vivo and PAMs in vitro following infection with PRRSV, however, transcriptome-wide understanding of the interaction between highly pathogenic PRRSV (HP-PRRSV) and PAMs in vivo has not yet been established. In this study, we employed Affymetrix microarrays to investigate the gene expression patterns of PAMs isolated from Tongcheng piglets (a Chinese indigenous breed) after infection with HP-PRRSV. During the infection, Tongcheng piglets exhibited typical clinical signs, e.g. fever, asthma, coughing, anorexia, lethargy and convulsion, but displayed mild regional lung damage at 5 and 7 dpi. Microarray analysis revealed that HP-PRRSV infection has affected PAMs in expression of the important genes involved in cytoskeleton and exocytosis organization, protein degradation and folding, intracellular calcium and zinc homeostasis. Several potential antiviral strategies might be employed in PAMs, including upregulating IFN-induced genes and increasing intracellular zinc ion concentration. And inhibition of the complement system likely attenuated the lung damage during HP-PRRSV infection. Transcriptomic analysis of PAMs in vivo could lead to a better understanding of the HP-PRRSV-host interaction, and to the identification of novel antiviral therapies and genetic components of swine tolerance/susceptibility to HP-PRRS.
Project description:Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically devastating infectious diseases in pigs worldwide. The causative agent is the PRRS virus (PRRSV). In this study, we explored polyethylenimine (PEI), a cationic polymer with different forms (linear or branched), to inhibit the replication of PRRSV. Our results demonstrate that the linear but not the 40 kDa branched PEI, or the 25 kDa linear PEI, were well tolerated in cultured cells and exhibited a broad-spectrum inhibition of heterogeneous PRRSV-2 isolates in both MARC-145 cells and primary porcine pulmonary alveolar macrophages (PAMs). Further analysis suggests that PEI could prevent the attachment of PRRSV virions to the susceptible cells. Notably, PEI had a minimal effect on PRRSV internalization in MARC-145 cells, whereas PEI promoted the internalization of PRRSV virions in PAMs, which suggests that these two types of cells might have different internalization processes of PRRSV virions. In conclusion, our data demonstrate that PEI could be used as a novel inhibitor against PRRSV.
Project description:Porcine Reproductive and Respiratory Syndrome (PRRS) is a panzootic infectious disease of pigs, causing major economic losses to the world-wide pig industry. PRRS manifests differently in pigs of all ages but primarily causes late-term abortions and stillbirths in sows and respiratory disease in piglets. The causative agent of the disease is the positive-strand RNA PRRS virus (PRRSV). PRRSV has a narrow host cell tropism, limited to cells of the monocyte/macrophage lineage. CD163 has been described as a fusion receptor for PRRSV, whereby the scavenger receptor cysteine-rich domain 5 (SRCR5) region was shown to be an interaction site for the virus in vitro. CD163 is expressed at high levels on the surface of macrophages, particularly in the respiratory system. Here we describe the application of CRISPR/Cas9 to pig zygotes, resulting in the generation of pigs with a deletion of Exon 7 of the CD163 gene, encoding SRCR5. Deletion of SRCR5 showed no adverse effects in pigs maintained under standard husbandry conditions with normal growth rates and complete blood counts observed. Pulmonary alveolar macrophages (PAMs) and peripheral blood monocytes (PBMCs) were isolated from the animals and assessed in vitro. Both PAMs and macrophages obtained from PBMCs by CSF1 stimulation (PMMs) show the characteristic differentiation and cell surface marker expression of macrophages of the respective origin. Expression and correct folding of the SRCR5 deletion CD163 on the surface of macrophages and biological activity of the protein as hemoglobin-haptoglobin scavenger was confirmed. Challenge of both PAMs and PMMs with PRRSV genotype 1, subtypes 1, 2, and 3 and PMMs with PRRSV genotype 2 showed complete resistance to viral infections assessed by replication. Confocal microscopy revealed the absence of replication structures in the SRCR5 CD163 deletion macrophages, indicating an inhibition of infection prior to gene expression, i.e. at entry/fusion or unpacking stages.
Project description:Porcine reproductive and respiratory syndrome (PRRS), which caused by the porcine reproductive and respiratory syndrome virus (PRRSV), is a serious viral disease affecting global swine industry. At present, PRRSV vaccines fail to prevent this disease. Consequently, new antiviral strategies to compensate for the inefficacy of available vaccines are urgently required. Lysine acetylation is an important post-translational modification (PTM) regulating an array of pathological and physiological conditions. In this study, we profiled the global acetylome using acetylation specific antibody based enrichment and Tandem mass tag (TMT) label LC-MS in PRRSV-infected pulmonary alveolar macrophages (PAMs). As a result, 3731 lysine acetylation sites on 1421 cellular proteins were identified and quantified 6 hours post infection (hpi). Bioinformatics analysis of the differentially acetylated proteins revealed their involvement in various biological processes, including the host immune response and energy metabolism.
Project description: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.
Project description:Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus that has been devastating the swine industry worldwide since the late 1980s. To investigate the impact of cellular microRNAs (miRNAs) on the replication of PRRSV, we screened 10 highly conserved miRNAs implicated in innate immunity or antiviral function and identified miR-125b as an inhibitor of PRRSV replication. Virus titer and western blot assays demonstrated that miR-125b reduced PRRSV replication and viral gene expression in a dose-dependent manner in both MARC-145 cell line and primary porcine alveolar macrophages. Mechanistically, miR-125b did not target the PRRSV genome. Rather, it inhibited activation of NF-?B, which we found to be required for PRRSV replication. PRRSV, in turn, down-regulated miR-125b expression post-infection to promote viral replication. Collectively, miR-125b is an antiviral host factor against PRRSV, but it is subject to manipulation by PRRSV. Our study reveals an example of manipulation of a cellular miRNA by an arterivirus to re-orchestrate host gene expression for viral propagation and sheds new light on targeting host factors to develop effective control measures for PRRS.
Project description:Porcine reproductive and respiratory syndrome virus (PRRSV) causes porcine reproductive and respiratory syndrome (PRRS), which is currently insufficiently controlled. From a previous small-scale screen we identified an effective DNA-based short antisense oligonucleotide (AS-ON) targeting viral NSP9, which could inhibit PRRSV replication in both Marc-145 cells and pulmonary alveolar macrophages (PAMs). The objective of this study was to explore the strategy of incorporating locked nucleic acids (LNAs) to achieve better inhibition of PRRSV replication in vitro.The effective DNA-based AS-ON (YN8) was modified with LNAs at both ends as gap-mer (LNA-YN8-A) or as mix-mer (LNA-YN8-B). Marc-145 cells or PAMs were infected with PRRSV and subsequently transfected.Compared with the DNA-based YN8 control, the two AS-ONs modified with LNAs were found to be significantly more effective in decreasing the cytopathic effect (CPE) induced by PRRSV and thus in maintaining cell viability. LNA modifications conferred longer lifetimes to the AS-ON in the cell culture model. Viral ORF7 levels were more significantly reduced at both RNA and protein levels as shown by quantitative PCR, western blot and indirect immunofluorescence staining. Moreover, transfection with LNA modified AS-ON reduced the PRRSV titer by 10-fold compared with the YN8 control.Taken together, incorporation of LNA into AS-ON technology holds higher therapeutic promise for PRRS control.