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The Nucleocapsid Protein and Nonstructural Protein 10 of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Enhance CD83 Production via NF-?B and Sp1 Signaling Pathways.

ABSTRACT: Porcine reproductive and respiratory syndrome, caused by porcine reproductive and respiratory syndrome virus (PRRSV), is a panzootic disease that is one of the most economically costly diseases to the swine industry. A key aspect of PRRSV virulence is that the virus suppresses the innate immune response and induces persistent infection, although the underlying mechanisms are not well understood. The dendritic cell (DC) marker CD83 belongs to the immunoglobulin superfamily and is associated with DC activation and immunosuppression of T cell proliferation when expressed as soluble CD83 (sCD83). In this study, we show that PRRSV infection strongly stimulates CD83 expression in porcine monocyte-derived DCs (MoDCs) and that the nucleocapsid (N) protein and nonstructural protein 10 (nsp10) of PRRSV enhance CD83 promoter activity via the NF-?B and Sp1 signaling pathways. R43A and K44A amino acid substitution mutants of the N protein suppress the N protein-mediated increase of CD83 promoter activity. Similarly, P192-5A and G214-3A mutants of nsp10 (with 5 and 3 alanine substitutions beginning at residues P192 and G214, respectively) abolish the nsp10-mediated induction of the CD83 promoter. Using reverse genetics, four mutant viruses (rR43A, rK44A, rP192-5A, and rG214-3A) and four revertants [rR43A(R), rK44A(R), rP192-5A(R), and rG214-3A(R)] were generated. Decreased induction of CD83 in MoDCs was observed after infection by mutants rR43A, rK44A, rP192-5A, and rG214-3A, in contrast to the results obtained using rR43A(R), rK44A(R), rP192-5A(R), and rG214-3A(R). These findings suggest that PRRSV N and nsp10 play important roles in modulating CD83 signaling and shed light on the mechanism by which PRRSV modulates host immunity.IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically costly pathogens affecting the swine industry. It is unclear how PRRSV inhibits the host's immune response and induces persistent infection. The dendritic cell (DC) marker CD83 belongs to the immunoglobulin superfamily and has previously been associated with DC activation and immunosuppression of T cell proliferation and differentiation when expressed as soluble CD83 (sCD83). In this study, we found that PRRSV infection induces sCD83 expression in porcine MoDCs via the NF-?B and Sp1 signaling pathways. The viral nucleocapsid protein, nonstructural protein 1 (nsp1), and nsp10 were shown to enhance CD83 promoter activity. Amino acids R43 and K44 of the N protein, as well as residues 192 to 196 (P192-5) and 214 to 216 (G214-3) of nsp10, play important roles in CD83 promoter activation. These findings provide new insights into the molecular mechanism of immune suppression by PRRSV.

SUBMITTER: Chen X

PROVIDER: S-EPMC5571251 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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The Nucleocapsid Protein and Nonstructural Protein 10 of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Enhance CD83 Production via NF-κB and Sp1 Signaling Pathways.

Chen Xi X Zhang Qiaoya Q Bai Juan J Zhao Yongxiang Y Wang Xianwei X Wang Haiyan H Jiang Ping P

Journal of virology 20170824 18

Porcine reproductive and respiratory syndrome, caused by porcine reproductive and respiratory syndrome virus (PRRSV), is a panzootic disease that is one of the most economically costly diseases to the swine industry. A key aspect of PRRSV virulence is that the virus suppresses the innate immune response and induces persistent infection, although the underlying mechanisms are not well understood. The dendritic cell (DC) marker CD83 belongs to the immunoglobulin superfamily and is associated with ...[more]

PMID: 28659471

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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:Transmissible gastroenteritis virus (TGEV) infection causes acute enteritis in swine of all ages, and especially in suckling piglets. Small intestinal inflammation is considered a central event in the pathogenesis of TGEV infections, and nuclear factor-kappa B (NF-κB) is a key transcription factor in the inflammatory response. However, it is unclear whether NF-κB is crucial for inducing inflammation during a TGEV infection. Our results show that NF-κB was activated in swine testicular (ST) cells and intestinal epithelial cell lines J2 (IPEC-J2) cells infected with TGEV, which is consistent with the up-regulation of pro-inflammatory cytokines. Treatment of TGEV-infected ST cells and IPEC-J2 cells with the NF-κB-specific inhibitor caused the down-regulation of pro-inflammatory cytokine expression, but did not significantly affect TGEV replication. Individual TGEV protein screening results demonstrated that Nsp2 exhibited a high potential for activating NF-κB and enhancing the expression of pro-inflammatory cytokines. Functional domain analyzes indicated that the first 120 amino acid residues of Nsp2 were essential for NF-κB activation. Taken together, these data suggested that NF-κB activation was a major contributor to TGEV infection-induced inflammation, and that Nsp2 was the key viral protein involved in the regulation of inflammation, with amino acids 1-120 playing a critical role in activating NF-κB. Abbreviations: TCID50: 50% tissue culture infectious dose; DMEM: Dulbecco's Modified Eagle Medium; eNOS: Endothelial nitric oxide synthase; FBS: fetal bovine serum; IFA: Indirect immunofluorescence; IκB: inhibitor of nuclear factor kappa-B; IL: interleukin; IPEC-J2: intestinal epithelial cell lines J2; IKK: IκB kinase; Luc: luciferase reporter gene; mAbs: monoclonal antibodies; MOI: multiple of infection; Nsp: nonstructural protein; NF-κB: nuclear factor-kappa ; ORFs: open reading frames; PBS: phosphate-buffered saline; p65 p-p65: phosphorylated; RT-PCR: reverse transcription PC; SeV: Sendai virus; ST: swine testicular; TGEV: Transmissible gastroenteritis virus; TNF-α: tumor necrosis factor α; UV-TGEV: Ultraviolet light-inactivated TGEV; ZnF: zinc finger.

Project description:BackgroundTransmissible gastroenteritis virus (TGEV), a member of the family Coronaviridae, causes lethal watery diarrhea in piglets. Previous studies have revealed that the coronaviruses develop various strategies to evade the host innate immunity through the inhibition of nuclear factor kappa B (NF-κB) signaling pathway. However, the ability of TGEV to inhibit the host innate immune response by modulating the NF-κB signaling pathway is not clear.MethodsIn this study, a dual luciferase reporter assay was used to confirm the inhibition of NF-κB by TGEV infection and to identify the major viral proteins involved in the inhibition of NF-κB signaling. Real-time quantitative PCR was used to quantify the mRNA expression of inflammatory factors. The deubiquitination of Nsp3 domains and its effect on IκBα and p65 were analyzed by western blotting. The ubiquitination level of IκBα was analyzed by immunoprecipitation.ResultsIn ST and IPEC-J2 cells, TGEV exhibited a dose-dependent inhibition of NF-κB activity. Individual TGEV protein screening revealed the high potential of non-structural protein 3 (Nsp3) to inhibit NF-κB signaling, and leading to the downregulation of the NF-κB-induced cytokine production. We demonstrated that the inhibitory effect of Nsp3 was mainly mediated through the suppression of IκBα degradation as well as the inhibition of p65 phosphorylation and nuclear translocation. Furthermore, the amino acid residues at positions 590-1,215 in Nsp3 were demonstrated to inhibit the degradation of IκBα by inhibiting the IκBα ubiquitination.ConclusionTGEV infection can inhibit the activation of the NF-κB signaling pathway, which is mainly mediated by Nsp3 through the canonical pathway. The amino acid residues at positions 590-1,215 in Nsp3 compose the critical domain that mediates NF-κB inhibition. We speculate that this inhibitory effect is likely to be related to the structure of PLP2 with deubiquitinating enzyme activity of the amino acid residues at positions 590-1,215 in Nsp3. Our study provides a better understanding of the TGEV-mediated innate immune modulation and lays the basis for studies on the pathogenesis of coronavirus.

Project description:Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive-stranded RNA virus belonging to the family Arteriviridae Synthesis of the viral RNA is directed by replication/transcription complexes (RTC) that are mainly composed of a network of PRRSV nonstructural proteins (nsps) and likely cellular proteins. Here, we mapped the interaction network among PRRSV nsps by using yeast two-hybrid screening in conjunction with coimmunoprecipitation (co-IP) and cotransfection assays. We identified a total of 24 novel interactions and found that the interactions were centered on open reading frame 1b (ORF1b)-encoded nsps that were mainly connected by the transmembrane proteins nsp2, nsp3, and nsp5. Interestingly, the interactions of the core enzymes nsp9 and nsp10 with transmembrane proteins did not occur in a straightforward manner, as they worked in the co-IP assay but were poorly capable of finding each other within intact mammalian cells. Further proof that they can interact within cells required the engineering of N-terminal truncations of both nsp9 and nsp10. However, despite the poor colocalization relationship in cotransfected cells, both nsp9 and nsp10 came together with membrane proteins (e.g., nsp2) at the viral replication and transcription complexes (RTC) in PRRSV-infected cells. Thus, our results indicate the existence of a complex interaction network among PRRSV nsps and raise the possibility that the recruitment of key replicase proteins to membrane-associated nsps may involve some regulatory mechanisms during infection.IMPORTANCE Synthesis of PRRSV RNAs within host cells depends on the efficient and correct assembly of RTC that takes places on modified intracellular membranes. As an important step toward dissecting this poorly understood event, we investigated the interaction network among PRRSV nsps. Our studies established a comprehensive interaction map for PRRSV nsps and revealed important players within the network. The results also highlight the likely existence of a regulated recruitment of the PRRSV core enzymes nsp9 and nsp10 to viral membrane nsps during PRRSV RTC assembly.

Dataset Information

The Nucleocapsid Protein and Nonstructural Protein 10 of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Enhance CD83 Production via NF-?B and Sp1 Signaling Pathways.

Publications

The Nucleocapsid Protein and Nonstructural Protein 10 of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Enhance CD83 Production via NF-κB and Sp1 Signaling Pathways.

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