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MiR-125b reduces porcine reproductive and respiratory syndrome virus replication by negatively regulating the NF-?B pathway.

ABSTRACT: 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.

SUBMITTER: Wang D

PROVIDER: S-EPMC3566999 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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MiR-125b reduces porcine reproductive and respiratory syndrome virus replication by negatively regulating the NF-κB pathway.

Wang Dang D Cao Lu L Xu Zheng Z Fang Liurong L Zhong Yao Y Chen Quangang Q Luo Rui R Chen Huanchun H Li Kui K Xiao Shaobo S

PloS one 20130207 2

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 ex ...[more]

PMID: 23409058

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Project description:Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses to the pork industry worldwide each year. Our previous research demonstrated that heme oxygenase-1 (HO-1) can suppress PRRSV replication via an unknown molecular mechanism. In this study, inhibition of PRRSV replication was demonstrated to be mediated by carbon monoxide (CO), a downstream metabolite of HO-1. Using several approaches, we demonstrate that CO significantly inhibited PRRSV replication in both a PRRSV permissive cell line, MARC-145, and the predominant cell type targeted during in vivo PRRSV infection, porcine alveolar macrophages (PAMs). Our results showed that CO inhibited intercellular spread of PRRSV; however, it did not affect PRRSV entry into host cells. Furthermore, CO was found to suppress PRRSV replication via the activation of the cyclic GMP/protein kinase G (cGMP/PKG) signaling pathway. CO significantly inhibits PRRSV-induced NF-κB activation, a required step for PRRSV replication. Moreover, CO significantly reduced PRRSV-induced proinflammatory cytokine mRNA levels. In conclusion, the present study demonstrates that CO exerts its anti-PRRSV effect by activating the cellular cGMP/PKG signaling pathway and by negatively regulating cellular NF-κB signaling. These findings not only provide new insights into the molecular mechanism of HO-1 inhibition of PRRSV replication but also suggest potential new control measures for future PRRSV outbreaks. PRRSV causes great economic losses each year to the swine industry worldwide. Carbon monoxide (CO), a metabolite of HO-1, has been shown to have antimicrobial and antiviral activities in infected cells. Our previous research demonstrated that HO-1 can suppress PRRSV replication. Here we show that endogenous CO produced through HO-1 catalysis mediates the antiviral effect of HO-1. CO inhibits PRRSV replication by activating the cellular cGMP/PKG signaling pathway and by negatively regulating cellular NF-κB signaling. These findings not only provide new insights into the molecular mechanism of HO-1 inhibition of PRRSV replication but also suggest potential new control measures for future PRRSV outbreaks.

Project description:UnlabelledPorcine reproductive and respiratory syndrome virus (PRRSV) is a highly infectious pathogen that causes severe diseases in pigs and great economic losses to the swine industry worldwide. Type I interferons (IFNs) play a crucial role in antiviral immunity. In the present study, we demonstrated that infection with the highly pathogenic PRRSV strain JXwn06 antagonized type I IFN expression induced by poly(I·C) in both porcine alveolar macrophages (PAMs) and blood monocyte-derived macrophages (BMo). Subsequently, we showed that the inhibition of poly(I·C)-induced IFN-β production by PRRSV was dependent on the blocking of NF-κB signaling pathways. By screening PRRSV nonstructural and structural proteins, we demonstrated that nonstructural protein 4 (nsp4), a viral 3C-like serine protease, significantly suppressed IFN-β expression. Moreover, we verified that nsp4 inhibited NF-κB activation induced by signaling molecules, including RIG-I, VISA, TRIF, and IKKβ. nsp4 was shown to target the NF-κB essential modulator (NEMO) at the E349-S350 site to mediate its cleavage. Importantly, nsp4 mutants with defective protease activity abolished its ability to cleave NEMO and inhibit IFN-β production. These findings might have implications for our understanding of PRRSV pathogenesis and its mechanisms for evading the host immune response.ImportancePorcine reproductive and respiratory syndrome virus (PRRSV) is a major agent of respiratory diseases in pigs. Like many other viruses, PRRSV has evolved a variety of strategies to evade host antiviral innate immunity for survival and propagation. In this study, we show that PRRSV nsp4 is a novel antagonist of the NF-κB signaling pathway, which is responsible for regulating the expression of type I interferons and other crucial cytokines. We then investigated the underlying mechanism used by nsp4 to suppress NF-κB-mediated IFN-β production. We found that nsp4 interfered with the NF-κB signaling pathway through the cleavage of NEMO (a key regulator of NF-κB signaling) at the E349-S350 site, leading to the downregulation of IFN-β production induced by poly(I·C). The data presented here may help us to better understand PRRSV pathogenesis.

Project description:MicroRNAs (miRNAs) contribute to gene regulation at the post-transcriptional level and are capable of mRNA silencing by binding to target sites exhibiting high degrees of complementarity. Therefore, cloning host miRNA-recognition sequences into the genome of RNA viruses represents a rational strategy for manipulating viral replication. Here, we performed deep sequencing to obtain small-RNA (sRNA)-expression profiles from in vitro-cultured MARC-145 cells post infection with porcine reproductive and respiratory syndrome virus (PRRSV) and chose six candidate miRNAs of different abundance (miR-21, miR-140-3p, miR-185, miR-26a, miR-505, and miR-199a) for further study. Based on the full-length cDNA clone p7USC, we constructed a number of PRRSV mutants that provided complementary base-pairing target sites for the miRNAs in 3' untranslated regions. Our results showed that all low- and moderate- abundant miRNA-target mutants showed similar growth properties, whereas the highest-abundant miRNA-target mutant blocked both viral transcription and replication. Discontinuous mutations in high-abundant miRNA-target sites subsequently recovered viral viability and propagation. These results demonstrated the copy number of endogenous miRNAs and the extent of sRNA complementarity were key factors to silence potential mRNA expression/translation, thereby determining PRRSV viability. Interestingly, the mutant containing miR-140-target sites (v140-t) showed strong suppression of viral replication from P1 to P3 in vitro, as shown by virus titer, plaque morphology, and qRT-PCR assays. To assess genetic stability, sequencing of v140-t (P1, P3, P5 and P10) revealed spontaneous mutations preferentially located among several nucleotides near the 3' end of the insertion region and corresponding to the "seed region" of miR-140-3p, explaining the induced viral repression and the direction of virus evolution. This approach provided a general silencing strategy for limiting PRRSV replication by endogenous miRNAs in MARC-145 cells.

Dataset Information

MiR-125b reduces porcine reproductive and respiratory syndrome virus replication by negatively regulating the NF-?B pathway.

Publications

MiR-125b reduces porcine reproductive and respiratory syndrome virus replication by negatively regulating the NF-κB pathway.

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