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Porcine reproductive and respiratory syndrome virus induces interleukin-15 through the NF-?B signaling pathway.

ABSTRACT: Porcine reproductive and respiratory syndrome virus (PRRSV) mainly infects macrophages/dendritic cells and modulates cytokine expression in these cells. Interleukin-15 (IL-15) is a pleiotropic cytokine involved in wide range of biological activities. It has been shown to be essential for the generation, activation, and proliferation of NK and NKT cells and for the survival and activation of CD8(+) effector and memory T cells. In this study, we discovered that PRRSV infection upregulated IL-15 production at both the mRNA and protein levels in porcine alveolar macrophages (PAMs), blood monocyte-derived macrophages (BMo), and monocyte-derived dendritic cells (DCs). We subsequently demonstrated that the NF-?B signaling pathway was essential for PRRSV infection-induced IL-15 production. First, addition of an NF-?B inhibitor drastically reduced PRRSV infection-induced IL-15 production. We then found that NF-?B was indeed activated upon PRRSV infection, as evidenced by I?B phosphorylation and degradation. Moreover, we revealed an NF-?B binding motif in the cloned porcine IL-15 (pIL-15) promoter, deletion of which abrogated the pIL-15 promoter activity in PRRSV-infected alveolar macrophages. In addition, we demonstrated that PRRSV nucleocapsid (N) protein had the ability to induce IL-15 production in porcine alveolar macrophage cell line CRL2843 by transient transfection, which was mediated by its multiple motifs, and it also activated NF-?B. These data indicated that PRRSV infection-induced IL-15 production was likely through PRRSV N protein-mediated NF-?B activation. Our findings provide new insights into the molecular mechanisms underling the IL-15 production induced by PRRSV infection.

SUBMITTER: Fu Y

PROVIDER: S-EPMC3416278 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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Porcine reproductive and respiratory syndrome virus induces interleukin-15 through the NF-κB signaling pathway.

Fu Yi Y Quan Rong R Zhang Hexiao H Hou Jun J Tang Jun J Feng Wen-hai WH

Journal of virology 20120509 14

Porcine reproductive and respiratory syndrome virus (PRRSV) mainly infects macrophages/dendritic cells and modulates cytokine expression in these cells. Interleukin-15 (IL-15) is a pleiotropic cytokine involved in wide range of biological activities. It has been shown to be essential for the generation, activation, and proliferation of NK and NKT cells and for the survival and activation of CD8(+) effector and memory T cells. In this study, we discovered that PRRSV infection upregulated IL-15 pr ...[more]

PMID: 22573868

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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-?B 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-?B 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. IMPORTANCE: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-?B 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:Signal transducer and activator of transcription 3 (STAT3) is a pleiotropic signaling mediator of many cytokines, including interleukin-6 (IL-6) and IL-10. STAT3 is known to play critical roles in cell growth, proliferation, differentiation, immunity and inflammatory responses. The objective of this study was to determine the effect of porcine reproductive and respiratory syndrome virus (PRRSV) infection on the STAT3 signaling since PRRSV induces a weak protective immune response in host animals. We report here that PRRSV infection of MARC-145 cells and primary porcine pulmonary alveolar macrophages led to significant reduction of STAT3 protein level. Several strains of both PRRSV type 1 and type 2 led to a similar reduction of STAT3 protein level but had a minimal effect on its transcripts. The PRRSV-mediated STAT3 reduction was in a dose-dependent manner as the STAT3 level decreased, along with incremental amounts of PRRSV inocula. Further study showed that nonstructural protein 5 (nsp5) of PRRSV induced the STAT3 degradation by increasing its polyubiquitination level and shortening its half-life from 24 h to ?3.5 h. The C-terminal domain of nsp5 was shown to be required for the STAT3 degradation. Moreover, the STAT3 signaling in the cells transfected with nsp5 plasmid was significantly inhibited. These results indicate that PRRSV antagonizes the STAT3 signaling by accelerating STAT3 degradation via the ubiquitin-proteasomal pathway. This study provides insight into the PRRSV interference with the JAK/STAT3 signaling, leading to perturbation of the host innate and adaptive immune responses. IMPORTANCE:The typical features of immune responses in PRRSV-infected pigs are delayed onset and low levels of virus neutralizing antibodies, as well as weak cell-mediated immunity. Lymphocyte development and differentiation rely on cytokines, many of which signal through the JAK/STAT signaling pathway to exert their biological effects. Here, we discovered that PRRSV antagonizes the JAK/STAT3 signaling by inducing degradation of STAT3, a master transcription activator involved in multiple cellular processes and the host immune responses. The nsp5 protein of PRRSV is responsible for the accelerated STAT3 degradation. The PRRSV-mediated antagonizing STAT3 could lead to suppression of a broad spectrum of cytokines and growth factors to allow virus replication and spread in host animals. This may be one of the reasons for the PRRSV interference with the innate immunity and its poor elicitation of protective immunity. This finding provides insight into PRRSV pathogenesis and its interference with the host immune responses.

Project description:Interferons (IFNs) play a crucial role in host antiviral response by activating the JAK/STAT (Janus kinase/signal transducer and activator of transcription) signaling pathway to induce the expression of myriad genes. STAT2 is a key player in the IFN-activated JAK/STAT signaling. Porcine reproductive and respiratory syndrome virus (PRRSV) is an important viral pathogen, causing huge losses to the swine industry. PRRSV infection elicits a meager protective immune response in pigs. The objective of this study was to investigate the effect of PRRSV on STAT2 signaling. Here, we demonstrated that PRRSV downregulated STAT2 to inhibit IFN-activated signaling. PRRSV strains of both PRRSV-1 and PRRSV-2 species reduced the STAT2 protein level, whereas the STAT2 transcript level had minimal change. PRRSV reduced the STAT2 level in a dose-dependent manner and shortened STAT2 half-life significantly from approximately 30 to 5?h. PRRSV-induced STAT2 degradation could be restored by treatment with the proteasome inhibitor MG132 and lactacystin. In addition, PRRSV nonstructural protein 11 (nsp11) was identified to interact with and reduce STAT2. The N-terminal domain (NTD) of nsp11 was responsible for STAT2 degradation and interacted with STAT2 NTD and the coiled-coil domain. Mutagenesis analysis showed that the amino acid residue K59 of nsp11 was indispensable for inducing STAT2 reduction. Mutant PRRSV with the K59A mutation generated by reverse genetics almost lost the ability to reduce STAT2. Together, these results demonstrate that PRRSV nsp11 antagonizes IFN signaling via mediating STAT2 degradation and provide further insights into the PRRSV interference of the innate immunity.IMPORTANCE PRRSV infection elicits a meager protective immune response in pigs. One of the possible reasons is that PRRSV antagonizes interferon induction and its downstream signaling. Interferons are key components in the innate immunity and play crucial roles against viral infection and in the activation of adaptive immune response via JAK/STAT signaling. STAT2 is indispensable in the JAK/STAT signaling since it is also involved in activation of antiviral activity in the absence of STAT1. Here, we discovered that PRRSV nsp11 downregulates STAT2. Interestingly, the N-terminal domain of nsp11 is responsible for inducing STAT2 degradation and directly interacts with STAT2 N-terminal domain. We also identified a crucial amino acid residue K59 in nsp11 since a mutation of it led to loss of the ability to downregulate STAT2. A mutant PRRSV with mutation of K59 had minimal effect on STAT2 reduction. Our data provide further insights into PRRSV interference with interferon signaling.

Project description:Porcine reproductive and respiratory syndrome virus (PRRSV) is widely prevalent in pigs, resulting in significant economic losses worldwide. A compelling impact of PRRSV infection is severe pneumonia. In the present study, we found that interleukin-17 (IL-17) was upregulated by PRRSV infection. Subsequently, we demonstrated that PI3K and p38MAPK signaling pathways were essential for PRRSV-induced IL-17 production as addition of phosphatidylinositol 3-kinase (PI3K) and p38MAPK inhibitors dramatically reduced IL-17 production. Furthermore, we show here that deleting the C/EBP? and CREB binding motif in porcine IL-17 promoter abrogated its activation and that knockdown of C/EBP? and CREB remarkably impaired PRRSV-induced IL-17 production, suggesting that IL-17 expression was dependent on C/EBP? and CREB. More specifically, we demonstrate that PRRSV nonstructural protein 11 (nsp11) induced IL-17 production, which was also dependent on PI3K-p38MAPK-C/EBP?/CREB pathways. We then show that Ser74 and Phe76 amino acids were essential for nsp11 to induce IL-17 production and viral rescue. In addition, IRAK1 was required for nsp11 to activate PI3K and enhance IL-17 expression by interacting with each other. Importantly, we demonstrate that PI3K inhibitor significantly suppressed IL-17 production and lung inflammation caused by HP-PRRSV in vivo, implicating that higher IL-17 level induced by HP-PRRSV might be associated with severe lung inflammation. These findings provide new insights onto the molecular mechanisms of the PRRSV-induced IL-17 production and help us further understand the pathogenesis of PRRSV infection.IMPORTANCE Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) associated with severe pneumonia has been one of the most important viral pathogens in pigs. IL-17 is a proinflammatory cytokine that might be associated with the strong inflammation caused by PRRSV. Therefore, we sought to determine whether PRRSV infection affects IL-17 expression, and if so, determine this might partially explain the underlying mechanisms for the strong inflammation in HP-PRRSV-infected pigs, especially in lungs. Here, we show that PRRSV significantly induced IL-17 expression, and we subsequently dissected the molecular mechanisms about how PRRSV regulated IL-17 production. Furthermore, we show that Ser74 and Phe76 in nsp11 were indispensable for IL-17 production and viral replication. Importantly, we demonstrated that PI3K inhibitor impaired IL-17 production and alleviated lung inflammation caused by HP-PRRSV infection. Our findings will help us for a better understanding of PRRSV pathogenesis.

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Porcine reproductive and respiratory syndrome virus induces interleukin-15 through the NF-?B signaling pathway.

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Porcine reproductive and respiratory syndrome virus induces interleukin-15 through the NF-κB signaling pathway.

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