Porcine Reproductive and Respiratory Syndrome Virus Antagonizes JAK/STAT3 Signaling via nsp5, Which Induces STAT3 Degradation.
ABSTRACT: 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:The porcine reproductive and respiratory syndrome virus (PRRSV) is the cause of one of the most economically important diseases affecting swine worldwide. Efforts to develop a next-generation vaccine have largely focused on envelope glycoproteins to target virus-neutralizing antibody responses. However, these approaches have failed to demonstrate the necessary efficacy to progress toward market. T cells are crucial to the control of many viruses through cytolysis and cytokine secretion. Since control of PRRSV infection is not dependent on the development of neutralizing antibodies, it has been proposed that T cell-mediated immunity plays a key role. Therefore, we hypothesized that conserved T cell antigens represent prime candidates for the development a novel PRRS vaccine. Antigens were identified by screening a proteome-wide synthetic peptide library with T cells from cohorts of pigs rendered immune by experimental infections with a closely related (subtype 1) or divergent (subtype 3) PRRSV-1 strain. Dominant T cell IFN-? responses were directed against the non-structural protein 5 (NSP5), and to a lesser extent, the matrix (M) protein. The majority of NSP5-specific CD8 T cells and M-specific CD4 T cells expressed a putative effector memory phenotype and were polyfunctional as assessed by coexpression of TNF-? and mobilization of the cytotoxic degranulation marker CD107a. Both antigens were generally well conserved among strains of both PRRSV genotypes. Thus, M and NSP5 represent attractive vaccine candidate T cell antigens, which should be evaluated further in the context of PRRSV vaccine development.
Project description:BACKGROUND:Autophagy is an essential process in eukaryotic cells in which autophagosomes form to deliver cellular organelles and long-lived proteins to lysosomes for degradation. Many studies have recently identified the regulatory mechanisms involved in the interaction between viral infection and autophagy. METHODS:LC3 turnover and the proteins in the endoplasmic reticulum (ER) stress pathway were investigated using western blot analysis. The formation and degradation of autophagosomes were detected using immunofluorescence staining. RESULTS:Autophagy was activated by porcine reproductive and respiratory syndrome virus (PRRSV) NSP3, NSP5 and NSP9, which are two transmembrane proteins and an RNA-dependent RNA polymerase, respectively. The formation of autophagosomes was induced by NSP3 and NSP5 and developed from the ER; the fusion of these autophagosomes with lysosomes was limited. Although NSP3 and NSP5 are ER transmembrane proteins, these proteins did not activate the ER stress signaling pathways. In addition, the cytoplasmic domain of NSP3 plays a pivotal role in activating autophagy. CONCLUSIONS:The data presented in this study reveal an important relationship between PRRSV NSPs and autophagy and provide new insights that improve our understanding of the involvement of PRRSV NSPs in the autophagy process.
Project description:Porcine reproductive and respiratory syndrome virus (PRRSV) has caused tremendous economic losses in the swine industry since its emergence in the late 1980s. PRRSV exploits various strategies to evade immune responses and establish chronic persistent infections. Suppressor of cytokine signaling (SOCS) 1, a member of the SOCS family, is a crucial intracellular negative regulator of innate immunity. In this study, it was shown that SOCS1 can be co-opted by PRRSV to evade host immune responses, facilitating viral replication. It was observed that PRRSV induced SOCS1 production in porcine alveolar macrophages, monkey-derived Marc-145 cells, and porcine-derived CRL2843-CD163 cells. SOCS1 inhibited the expression of IFN-? and IFN-stimulated genes, thereby markedly enhancing PRRSV replication. It was observed that the PRRSV N protein has the ability to upregulate SOCS1 production and that nuclear localization signal-2 (NLS-2) is essential for SOCS1 induction. Moreover, SOCS1 upregulation was dependent on p38/AP-1 and JNK/AP-1 signaling pathways rather than classical type I IFN signaling pathways. In summary, to our knowledge, the findings of this study uncovered the molecular mechanism that underlay SOCS1 induction during PRRSV infection, providing new insights into viral immune evasion and persistent infection.
Project description:The porcine reproductive and respiratory syndrome (PRRS) is a devastating viral disease affecting swine production, health and welfare throughout the world. A synergistic action of the innate and the adaptive immune system of the host is essential for mounting a durable protective immunity through vaccination. Therefore, the current study aimed to investigate the transcriptome profiles of peripheral blood mononuclear cells (PBMCs) to characterize the innate and the adaptive immune response to PRRS Virus (PRRSV) vaccination in Pietrain pigs. The Affymetrix gene chip porcine gene 1.0 ST array was used for the transcriptome profiling of PBMCs collected at immediately before (D0), at one (D1) and 28 days (D28) post PRRSV vaccination with three biological replications. With FDR <0.05 and log2 fold change ±1.5 as cutoff criteria, 295 and 115 transcripts were found to be differentially expressed in PBMCs during the stage of innate and adaptive response, respectively. The microarray expression results were technically validated by qRT-PCR. The gene ontology terms such as viral life cycle, regulation of lymphocyte activation, cytokine activity and inflammatory response were enriched during the innate immunity; cytolysis, T cell mediated cytotoxicity, immunoglobulin production were enriched during adaptive immunity to PRRSV vaccination. Significant enrichment of cytokine-cytokine receptor interaction, signaling by interleukins, signaling by the B cell receptor (BCR), viral mRNA translation, IFN-gamma pathway and AP-1 transcription factor network pathways were indicating the involvement of altered genes in the antiviral defense. Network analysis revealed that four network modules were functionally involved with the transcriptional network of innate immunity, and five modules were linked to adaptive immunity in PBMCs. The innate immune transcriptional network was found to be regulated by LCK, STAT3, ATP5B, UBB and RSP17. While TGFß1, IL7R, RAD21, SP1 and GZMB are likely to be predictive for the adaptive immune transcriptional response to PRRSV vaccine in PBMCs. Results of the current immunogenomics study advances our understanding of PRRS in term of host-vaccine interaction, and thereby contribute to design a rationale for disease control strategy.
Project description:Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling mediates almost all immune regulatory processes, including those that are involved in tumor cell recognition and tumor-driven immune escape. Antitumor immune responses are largely driven by STAT1 and STAT2 induction of type I and II interferons (IFNs) and the downstream programs IFNs potentiate. Conversely, STAT3 has been widely linked to cancer cell survival, immunosuppression, and sustained inflammation in the tumor microenvironment. The discovery of JAK-STAT cross-regulatory mechanisms, post-translational control, and non-canonical signal transduction has added a new level of complexity to JAK-STAT governance over tumor initiation and progression. Endeavors to better understand the vast effects of JAK-STAT signaling on antitumor immunity have unearthed a wide range of targets, including oncogenes, miRNAs, and other co-regulatory factors, which direct specific phenotypical outcomes subsequent to JAK-STAT stimulation. Yet, the rapidly expanding field of therapeutic developments aimed to resolve JAK-STAT aberrations commonly reported in a multitude of cancers has been marred by off-target effects. Here, we discuss JAK-STAT biology in the context of immunity and cancer, the consequences of pathway perturbations and current therapeutic interventions, to provide insight and consideration for future targeting innovations.
Project description:Interferon (IFN)-induced Janus kinase (Jak)/signal transducer and activator of transcription (Stat) pathway is important in controlling immune responses and is negatively response-regulated by the suppressor of cytokine signaling (SOCS) proteins. However, several viruses have developed various strategies to inhibit this pathway to circumvent the anti-viral immunity of the host. The infectious spleen and kidney necrosis virus (ISKNV) is the type species of the genus Megalocytivirus in the family Iridoviridae and a causative agent of epizootics in fish. ISKNV ORF103R encodes a predicted viral SOCS (vSOCS) with high homology to the vertebrate SOCS1, but lacks a SOCS-box domain. Interestingly, vSOCS only exists in the genus Megalocytivirus. ISKNV-vSOCS can block the IFN-?-induced Jak/Stat pathway in HepG2 cells. Over-expression of ISKNV-vSOCS inhibited the activities of IFN-stimulated response element (ISRE) promoter; however, the inhibitions by ISKNV-vSOCS were dose-dependent. ISKNV-vSOCS interacted with Jak1 protein and inhibited its tyrosine kinase activity in vitro. ISKNV-vSOCS also impaired the phosphorylation of Stat1 and Stat3 proteins and suppressed their activations. The point mutations (F18D, S66A, S85A, and R64K) of ISKNV-vSOCS significantly impaired the inhibition of IFN-?-induced ISRE-promoter activation. In conclusion, vSOCS inhibits IFN-?-induced Stat1/Stat3 signaling, suggesting that Megalocytivirus has developed a novel strategy to evade IFN anti-viral immunity via vSOCS protein.
Project description:Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important viral diseases affecting swine industry worldwide. Despite routine farm vaccination, effective control strategies for PRRS remained elusive which underscores the need for in-depth studies to gain insight into the host immune response to vaccines. The current study aimed to investigate transcriptional responses to PRRS Virus (PRRSV) vaccine in the peripheral blood mononuclear cells (PBMCs) within 3 days following vaccination in German Landrace pigs.Transcriptome profiling of PBMCs from PRRSV vaccinated and age-matched unvaccinated pigs at right before (0 h), and at 6, 24 and 72 h after PRRSV vaccination was performed using the Affymetrix gene chip porcine gene 1.0 st array. Comparison of PBMCs transcriptome profiles between vaccinated and unvaccinated pigs revealed a distinct host innate immune transcriptional response to PRRSV vaccine. There was a significant temporal variation in transcriptional responses of PRRSV vaccine in PBMCs accounting 542, 2,263 and 357 differentially expressed genes (DEGs) at 6, 24 and 72 h post vaccination, respectively compared to the time point before vaccination (controls). Gene ontology analysis revealed the involvement of these DEGs in various biological process including innate immune response, signal transduction, positive regulation of MAP kinase activity, TRIF-dependent toll-like receptor signaling pathway, T cell differentiation and apoptosis. Immune response specific pathways such as cytokine-cytokine receptor interaction, chemokine signaling pathway, signal transduction, JAK-STAT pathway and regulation, TRAF6 mediated induction of NF-kB and MAPK, the NLRP3 inflammasome, endocytosis and interferon signaling were under regulation during the early stage of PRRSV vaccination. Network enrichment analysis revealed APP, TRAF6, PIN1, FOS, CTNNB1, TNFAIP3, TIP1, CDKN1, SIRT1, ESR1 and HDAC5 as the highly interconnected hubs of the functional network of PRRSV vaccine induced transcriptome changes in PBMCs.This study showed that a massive gene expression change occurred in PBMCs following PRRSV vaccination in German Landrace pigs. Within first 3 days of vaccine exposure, the highest transcript abundance was observed at 24 h after vaccination compared to that of control. Results of this study suggest that APP, TRAF6, PIN1, FOS, CDKN1A and TNFAIP3 could be considered as potential candidate genes for PRRSV vaccine responsiveness.
Project description:In this study, we show that porcine reproductive and respiratory syndrome virus (PRRSV) non-structural protein 1? (nsp1?) facilitates PRRSV escape from innate immune by modulating nuclear to cytoplasmic translocation and distribution ratio of TRAIP to promote virus proliferation. Mechanistically, TRAIP interacts with PRRSV nsp1? via its K205 site, while NSP1? decreases the SUMOylation and K48 ubiquitination independent of the TRAIP interaction K205 site. Modulation of the dual modification of TRAIP by PRRSV nsp1? results in over-enrichment of TRAIP in the cytoplasm. Enrichment of nsp1?-induced cytoplasmic TRAIP in turn leads to excessive K48 ubiquitination and degradation of serine/threonine-protein kinase (TBK1), thereby antagonizing TBK1-IRF3-IFN signaling. This study proposes a novel mechanism by which PRRSV utilizes host proteins to regulate innate immunity. Findings from this study provides novel perspective to advance our understanding in the pathogenesis of PRRSV.
Project description:This work describes peptides from non-structural proteins (nsp) of porcine reproductive and respiratory syndrome virus (PRRSV) predicted as potential T cell epitopes by bioinfornatics and tested for their ability to induce IFN-? and IL-10 responses. Pigs immunized with either genotype 1 or genotype 2 PRRSV attenuated vaccines (n=5/group) and unvaccinated pigs (n = 4) were used to test the peptides. Swine leukocyte antigen haplotype of each pig was also determined. Pigs were initially screened for IFN-? responses (ELISPOT) and three peptides were identified; two of them in non-conserved segments of nsp2 and nsp5 and the other in a conserved region of nsp5 peptide. Then, peptides were screened for IL-10 inducing properties. Six peptides were found to induce IL-10 release in PBMC and some of them were also able to inhibit IFN-? responses on PHA-stimulated cells. Interestingly, the IFN-? low responder pigs against PRRSV were mostly homozygous for their SLA haplotypes. In conclusion, these results indicate that nsp of PRRSV contain T-cell epitopes inducing IFN-? responses as well as IL-10 inducing segments with inhibitory capabilities.