Porcine Epidemic Diarrhea Virus nsp15 Antagonizes Interferon Signaling by RNA Degradation of TBK1 and IRF3.
ABSTRACT: Porcine epidemic diarrhea virus (PEDV) causes a porcine disease associated with swine epidemic diarrhea. The type I interferon (IFN-I or IFN ?/?) is a key mediator of innate antiviral response during virus infection. Different antagonistic strategies have been identified and determined as to how PEDV infection inhibits the host's IFN responses to escape the host innate immune pathway, but the pathogenic mechanisms of PEDV infection are not fully elucidated. Our preliminary results revealed that endogenous TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3), the key components in the IFN signaling pathway were downregulated in PEDV infected IPEC-J2 cells by iTRAQ analysis. In this study, we screened nsp15 as the most important viral encoded protein involved in TBK1 and IRF3 reduction. Endoribonuclease (EndoU) activity has been well determined for coronavirus nsp15. Three residues (H226, H241, and K282) of PEDV nsp15 were identified as critical amino acids for PEDV EndoU but not D265, which was not well correlated with published results of other coronaviruses, such as severe acute respiratory syndrome virus (SARS-CoV). Moreover, PEDV nsp15 can directly degrade the RNA levels of TBK1 and IRF3 dependent on its EndoU activity to suppress IFN production and constrain the induction of IFN stimulated genes (ISGs), by which PEDV antagonizes the host innate response to facilitate its replication. Collectively, these results have confirmed that PEDV nsp15 was capable of subverting the IFN response by the RNA degradation of TBK1 and IRF3.
Project description:Porcine epidemic diarrhea virus (PEDV), a porcine enteropathogenic coronavirus, causes lethal watery diarrhea in piglets and results in large economic losses in many Asian and European countries. A large-scale outbreak of porcine epidemic diarrhea occurred in China in 2010, and the virus emerged in the United States in 2013 and spread rapidly, posing significant economic and public health concerns. Previous studies have shown that PEDV infection inhibits the synthesis of type I interferon (IFN), and viral papain-like protease 2 has been identified as an IFN antagonist. In this study, we found that the PEDV-encoded nucleocapsid (N) protein also inhibits Sendai virus-induced IFN-? production, IFN-stimulated gene expression, and activation of the transcription factors IFN regulatory factor 3 (IRF3) and NF-?B. We also found that N protein significantly impedes the activation of the IFN-? promoter stimulated by TBK1 or its upstream molecules (RIG-I, MDA5, IPS-1, and TRAF3) but does not counteract its activation by IRF3. A detailed analysis revealed that the PEDV N protein targets TBK1 by direct interaction and that this binding sequesters the association between TBK1 and IRF3, which in turn inhibits both IRF3 activation and type I IFN production. Together, our findings demonstrate a new mechanism evolved by PEDV to circumvent the host's antiviral immunity.PEDV has received increasing attention since the emergence of a PEDV variant in China and the United States. Here, we identify nucleocapsid (N) protein as a novel PEDV-encoded interferon (IFN) antagonist and demonstrate that N protein antagonizes IFN production by sequestering the interaction between IRF3 and TBK1, a critical step in type I IFN signaling. This adds another layer of complexity to the immune evasion strategies evolved by this economically important viral pathogen. An understanding of its immune evasion mechanism may direct us to novel therapeutic targets and more effective vaccines against PEDV infection.
Project description:Identifying viral antagonists of innate immunity and determining if they contribute to pathogenesis are critical for developing effective strategies to control emerging viruses. Previously, we reported that an endoribonuclease (EndoU) encoded by murine coronavirus plays a pivotal role in evasion of host innate immune defenses in macrophages. Here, we asked if the EndoU activity of porcine epidemic diarrhea coronavirus (PEDV), which causes acute diarrhea in swine, plays a role in antagonizing the innate response in porcine epithelial cells and macrophages, the sites of viral replication. We constructed an infectious clone of PEDV-Colorado strain (icPEDV-wt) and an EndoU-mutant PEDV (icPEDV-EnUmt) by changing the codon for a catalytic histidine residue of EndoU to alanine (His226Ala). We found that both icPEDV-wt and icPEDV-EnUmt propagated efficiently in interferon (IFN)-deficient Vero cells. In contrast, the propagation of icPEDV-EnUmt was impaired in porcine epithelial cells (LLC-PK1), where we detected an early and robust transcriptional activation of type I and type III IFNs. Infection of piglets with the parental Colorado strain, icPEDV-wt, or icPEDV-EnUmt revealed that all viruses replicated in the gut and induced diarrhea; however, there was reduced viral shedding and mortality in the icPEDV-EnUmt-infected animals. These results demonstrate that EndoU activity is not required for PEDV replication in immortalized, IFN-deficient Vero cells, but is important for suppressing the IFN response in epithelial cells and macrophages, which facilitates replication, shedding, and pathogenesis in vivo We conclude that PEDV EndoU activity is a key virulence factor that suppresses both type I and type III IFN responses.IMPORTANCE Coronaviruses (CoVs) can emerge from an animal reservoir into a naive host species to cause pandemic respiratory or gastrointestinal diseases with significant mortality in humans or domestic animals. Porcine epidemic diarrhea virus (PEDV), an alphacoronavirus (alpha-CoV), infects gut epithelial cells and macrophages, inducing diarrhea and resulting in high mortality in piglets. How PEDV suppresses the innate immune response was unknown. We found that mutating a viral endoribonuclease, EndoU, results in a virus that activates both the type I interferon response and the type III interferon response in macrophages and epithelial cells. This activation of interferon resulted in limited viral replication in epithelial cell cultures and was associated with reduced virus shedding and mortality in piglets. This study reveals a role for EndoU activity as a virulence factor in PEDV infection and provides an approach for generating live-attenuated vaccine candidates for emerging coronaviruses.
Project description:Type III interferons (IFNs) play a vital role in maintaining the antiviral state of the mucosal epithelial surface in the gut, and in turn, enteric viruses may have evolved to evade the type III IFN responses during infection. To study the possible immune evasion of the type III IFN response by porcine epidemic diarrhea virus (PEDV), a line of porcine intestinal epithelial cells was developed as a cell model for PEDV replication. IFN-?1 and IFN-?3 inhibited PEDV replication, indicating the anti-PEDV activity of type III IFNs. Of the 21 PEDV proteins, nsp1, nsp3, nsp5, nsp8, nsp14, nsp15, nsp16, open reading frame 3 (ORF3), E, M, and N were found to suppress type III IFN activities, and IRF1 (interferon regulatory factor 1) signaling mediated the suppression. PEDV specifically inhibited IRF1 nuclear translocation. The peroxisome is the innate antiviral signaling platform for the activation of IRF1-mediated IFN-? production, and the numbers of peroxisomes were found to be decreased in PEDV-infected cells. PEDV nsp1 blocked the nuclear translocation of IRF1 and reduced the number of peroxisomes to suppress IRF1-mediated type III IFNs. Mutational studies showed that the conserved residues of nsp1 were crucial for IRF1-mediated IFN-? suppression. Our study for the first time provides evidence that the porcine enteric virus PEDV downregulates and evades IRF1-mediated type III IFN responses by reducing the number of peroxisomes.IMPORTANCE Porcine epidemic diarrhea virus (PEDV) is a highly contagious enteric coronavirus that emerged in swine in the United States and has caused severe economic losses. PEDV targets intestinal epithelial cells in the gut, and intestinal epithelial cells selectively induce and respond to the production of type III interferons (IFNs). However, little is known about the modulation of the type III IFN response by PEDV in intestinal epithelial cells. In this study, we established a porcine intestinal epithelial cell model for PEDV replication. We found that PEDV inhibited IRF1-mediated type III IFN production by decreasing the number of peroxisomes in porcine intestinal epithelial cells. We also demonstrated that the conserved residues in the PEDV nsp1 protein were crucial for IFN suppression. This study for the first time shows PEDV evasion of the type III IFN response in intestinal epithelial cells, and it provides valuable information on host cell-virus interactions not only for PEDV but also for other enteric viral infections in swine.
Project description:Porcine epidemic diarrhea virus (PEDV), a swine enteropathogenic coronavirus (CoV), is the causative agent of porcine epidemic diarrhea (PED). PED causes lethal watery diarrhea in piglets, which has led to substantial economic losses in many countries and is a great threat to the global swine industry. Interferons (IFNs) are major cytokines involved in host innate immune defense, which induce the expression of a broad range of antiviral effectors that help host to control and antagonize viral infections. PEDV infection does not elicit a robust IFN response, and some of the mechanisms used by the virus to counteract the host innate immune response have been unraveled. PEDV evades the host innate immune response by two main strategies including: (1) encoding IFN antagonists to disrupt innate immune pathway, and (2) hiding its viral RNA to avoid the exposure of viral RNA to immune sensors. This review highlights the immune evasion mechanisms employed by PEDV, which provides insights for the better understanding of PEDV-host interactions and developing effective vaccines and antivirals against CoVs.
Project description:The lack of optimal porcine cell lines has severely impeded the study and progress in elucidation of porcine epidemic diarrhea virus (PEDV) pathogenesis. Vero cell, an African green monkey kidney cell line, was often used to isolate and propagate PEDV. Nonetheless, the target cells of PEDV in vivo are intestinal epithelial cells, during infection, intestinal epithelia would be damaged and resulted in digestive disorders. The immune functions of porcine epithelial cells and interactions with other immune cell populations display a number of differences compared to other species. Type I interferon (IFN) plays an important role in antiviral immune response. Limited reports showed that PEDV could inhibit type I interferon production. In this study, porcine small intestinal epithelial cells (IECs), the target cells of PEDV, were used as the infection model in vitro to identify the possible molecular mechanisms of PEDV-inhibition IFN-? production.PEDV not only failed to induce IFN-? expression, but also inhibited dsRNA-mediated IFN-? production in IECs. As the key IFN-? transcription factors, we found that dsRNA-induced activation of IFN regulatory factor 3 (IRF-3) was inhibited after PEDV infection, but not nuclear factor-kappaB (NF-?B). To identify the mechanism of PEDV intervention with dsRNA-mediated IFN-? expression more accurately, the role of individual molecules of RIG-I signaling pathway were investigated. In the upstream of IRF-3, TANK-binding kinase 1 (TBK1)-or inhibitor of ?B kinase-? (IKK?)-mediated IFN-? production was not blocked by PEDV, while RIG-I-and its adapter molecule IFN-? promoter stimulator 1 (IPS-1)-mediated IFN-? production were completely inhibited after PEDV infection.Taken together, our data demonstrated for the first time that PEDV infection of its target cell line, IECs, inhibited dsRNA-mediated IFN-? production by blocking the activation of IPS-1 in RIG-I-mediated pathway. Our studies offered new visions in understanding of the interaction between PEDV and host innate immune system.
Project description:Type I interferons (IFN-?/?) are the major components of the innate immune response of hosts, and in turn many viruses have evolved to modulate the host response during infection. We found that the IFN-? production was significantly suppressed during PEDV infection in cells. To identify viral IFN antagonists and to study their suppressive function, viral coding sequences for the entire structural and nonstructural proteins were cloned and expressed. Of 16 PEDV nonstructural proteins (nsps), nsp1, nsp3, nsp7, nsp14, nsp15 and nsp16 were found to inhibit the IFN-? and IRF3 promoter activities. The sole accessory protein ORF3, structure protein envelope (E), membrane (M), and nucleocapsid (N) protein were also shown to inhibit such activities. PEDV nsp1 did not interfere the IRF3 phosphorylation and nuclear translocation but interrupted the enhanceosome assembly of IRF3 and CREB-binding protein (CBP) by degrading CBP. A further study showed that the CBP degradation by nsp1 was proteasome-dependent. Our data demonstrate that PEDV modulates the host innate immune responses by degrading CBP and suppressing ISGs expression.
Project description:Type-I IFNs (IFN-I) provide a key mediator of innate antiviral response during virus proliferation. Porcine epidemic diarrhea virus (PEDV), which causes diarrhea in swine of all ages, is a worldwide-distributed alphacoronavirus with economic importance. Here, we screened PEDV RNA modification enzymes involved in regulating antiviral response. Whereas the PEDV nsp13 barely regulates type I IFN, inflammatory cytokines (IL-6, TNF-a) and MHCII, nsp16 and nsp14 (to a lesser extent) down-regulate these antiviral effectors. Importantly, we found nsp16 KDKE tetrad appears to play a key role in interferon inhibition by mutating the D129 catalytic residue. Mechanistically, nsp16 down-regulates the activities of RIG-I and MDA5 mediated IFN-? and ISRE. In turn, the mRNA levels of IFIT family members (IFIT1, IFIT2, IFIT3) was inhibited in cells overexpressing nsp16. In addition, nsp10 enhanced the inhibitory effect of nsp16 on IFN-?. Altogether these results indicate PEDV nsp16 negatively regulates innate immunity to promote viral proliferation. Findings from this study provides novel perspective to advance the understanding in the pathogenesis of PEDV.
Project description:Coronaviruses (CoVs), such as human coronavirus NL63 (HCoV-NL63), severe acute respiratory syndrome CoV (SARS-CoV), murine hepatitis virus (MHV), porcine epidemic diarrhea virus (PEDV), and Middle East Respiratory Syndrome Coronavirus (MERS-CoV), encode papain-like (PL) proteases that inhibit Sendai virus- (SeV-) induced interferon (IFN-?) production. Recently, the crystal structure of transmissible gastroenteritis virus (TGEV) PL1 has been solved, which was similar to that of SARS-CoV PL2pro, which may antagonize host innate immunity. However, very little is known about whether TGEV PL1 can antagonize host innate immune response. Here, we presented evidence that TGEV PL1 encoded by the replicase gene could suppress the IFN-? expression and inhibit the nuclear translocation of interferon regulatory factor 3 (IRF3). The ability to antagonize IFN-? production was dependent on the intact catalytic activity of PL1. Furthermore, TGEV PL1 exerted deubiquitinase (DUB) activity which strongly inhibited the retinoic acid-induced gene I- (RIG-1-) and stimulator of interferon gene- (STING-) dependent IFN expression. Our data collectively suggest that TGEV PL1 can inhibit the IFN-? expression and interfere with RIG-1- and STING-mediated signaling through a viral DUB activity. Our study has yielded strong evidence for the TGEV PL1 mechanisms that counteract the host innate immunity.
Project description:Porcine deltacoronavirus (PDCoV) is an emerging swine coronavirus causing diarrhea and intestinal damage in nursing piglets. Previous work showed that PDCoV infection inhibits type I interferon (IFN) production. To further identify and characterize the PDCoV-encoded IFN antagonists will broaden our understanding of its pathogenesis. Nonstructural protein 15 (nsp15) encodes an endoribonuclease that is highly conserved among vertebrate nidoviruses (coronaviruses and arteriviruses) and plays a critical role in viral replication and transcription. Here, we found that PDCoV nsp15 significantly inhibits Sendai virus (SEV)-induced IFN-? production. PDCoV nsp15 disrupts the phosphorylation and nuclear translocation of NF-?B p65 subunit, but not antagonizes the activation of transcription factor IRF3. Interestingly, site-directed mutagenesis found that PDCoV nsp15 mutants (H129A, H234A, K269A) lacking endoribonuclease activity also suppress SEV-induced IFN-? production and NF-?B activation, suggesting that the endoribonuclease activity is not required for its ability to antagonize IFN-? production. Taken together, our results demonstrate that PDCoV nsp15 is an IFN antagonist and it inhibits interferon-? production via an endoribonuclease activity-independent mechanism.
Project description:Porcine diarrhea disease in newborn and suckling piglets due to infection with porcine epidemic diarrhea virus (PEDV) is a leading cause of economic loss in the pig industry globally. In this study, we investigated the molecular mechanism of the host innate immune response to PEDV infection. The expression dynamics of antiviral genes (e.g., RIG-1, PKR, OAS1, Mx1, and Mx2) and inflammatory cytokines (e.g., IFN-?, IFN-?, TNF-?, IL-6, IL-8, and IL-12) in porcine small intestinal epithelial (IPEC-J2) cells were analyzed following PEDV stimulation. The results showed that the expression of antiviral genes (e.g., PKR, OAS1, and Mx2) and inflammatory cytokines (e.g., IFN-? and TNF-?) were significantly reduced within 0-4 h post-infection (P < 0.05). However, all antiviral genes and inflammatory cytokines were up-regulated from 12 to 24 h (P < 0.05), and cytopathic changes were observed during this time. The expression of RIG-1, PKR, OAS1, Mx1, and Mx2 were significantly and positively correlated to each other during the entire infection (P < 0.01). The results suggested that the RIG-1, PKR, OAS1, Mx1, and Mx2 genes may play an important role in PEDV infection in piglets. Initially, PEDV displayed cellular invasion by inhibiting IFN-? transcription and interfering with the antiviral function of PKR, OAS1, and Mx2, ultimately induced an intense inflammatory response. The relationship between antiviral genes and inflammatory cytokines with PEDV infection at the cellular level provides a reference for studying the mechanism of resistance to PEDV infection in piglets.