Coronavirus Endoribonuclease Activity in Porcine Epidemic Diarrhea Virus Suppresses Type I and Type III Interferon Responses

ABSTRACTIdentifying 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 pathogenesisin vivo. We conclude that PEDV EndoU activity is a key virulence factor that suppresses both type I and type III IFN responses.IMPORTANCECoronaviruses (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.

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Type-III interferon, not type-I, is the predominant interferon induced by respiratory viruses in nasal epithelial cells

Virus Research ◽

10.1016/j.virusres.2011.07.011 ◽

2011 ◽

Vol 160 (1-2) ◽

pp. 360-366 ◽

Cited By ~ 82

Author(s):

Tamaki Okabayashi ◽

Takashi Kojima ◽

Tomoyuki Masaki ◽

Shin-ichi Yokota ◽

Tadaatsu Imaizumi ◽

...

Keyword(s):

Epithelial Cells ◽

Respiratory Viruses ◽

Type I ◽

Nasal Epithelial Cells ◽

Type Iii ◽

Type Iii Interferon

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Type III Interferon Restriction by Porcine Epidemic Diarrhea Virus and the Role of Viral Protein nsp1 in IRF1 Signaling

Journal of Virology ◽

10.1128/jvi.01677-17 ◽

2017 ◽

pp. JVI.01677-17 ◽

Cited By ~ 24

Author(s):

Qingzhan Zhang ◽

Hanzhong Ke ◽

Anthony Blikslager ◽

Takashi Fujita ◽

Dongwan Yoo

Keyword(s):

Epithelial Cells ◽

Porcine Epidemic Diarrhea Virus ◽

Nuclear Translocation ◽

Intestinal Epithelial Cells ◽

Cell Model ◽

Intestinal Epithelial ◽

Diarrhea Virus ◽

Type Iii ◽

Porcine Epidemic Diarrhea ◽

Type Iii Ifn

Type III interferons (IFN-λs) play a vital role to maintain 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 of the possible immune evasion of porcine epidemic diarrhea virus (PEDV) from type III IFN response, a line of porcine intestinal epithelial cells was developed as a cell model for PEDV replication. IFN-λ1 and IFN-λ3 inhibited the PEDV replication, indicating the anti-PEDV activity of type III IFNs. Of the 21 PEDV proteins, nsp1, nsp3, nsp5, nsp8, nsp14, nsp15, nsp16, ORF3, E, M, and N were found to suppress the type III IFN activities, and the IRF1 signaling mediated the suppression. PEDV specifically inhibited IRF1 nuclear translocation. Peroxisome is the innate antiviral signaling platform for activation of IRF1-mediated IFN-λ production, and peroxisomes were found to decrease in number 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 the conserved residues of nsp1 were crucial for IRF1-mediated IFN-λ suppression. Our study for the first time provides the evidence that the porcine enteric virus PEDV downregulates and evades the IRF1-mediated type III IFN responses by reducing the peroxisomes.IMPORTANCEPorcine epidemic diarrhea virus (PEDV) is a highly contagious enteric coronavirus emerged in swine in the US and has caused severe economic losses. PEDV targets the 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 modulation of type III IFN response by PEDV in the intestinal epithelial cells. In this study, we established a porcine intestinal epithelial cell model for PEDV replication. We found that PEDV inhibited the IRF1-mediated type III IFN production by decreasing the peroxisomes in number in the 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 showed the PEDV evasion of type III IFN response in the intestinal epithelial cells. It provides valuable information on the host cell-virus interactions not only for PEDV but also other enteric viral infections in swine.

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Critical role of type III interferon in controlling SARS-CoV-2 infection, replication and spread in primary human intestinal epithelial cells

10.1101/2020.04.24.059667 ◽

2020 ◽

Cited By ~ 13

Author(s):

Megan L. Stanifer ◽

Carmon Kee ◽

Mirko Cortese ◽

Sergio Triana ◽

Markus Mukenhirn ◽

...

Keyword(s):

Epithelial Cells ◽

De Novo ◽

Intestinal Epithelial Cells ◽

Critical Role ◽

Type I ◽

Intestinal Epithelial ◽

Virus Particles ◽

Type Iii ◽

Human Intestinal Epithelial Cells ◽

Type Iii Interferon

SummarySARS-CoV-2 is an unprecedented worldwide health problem that requires concerted and global approaches to better understand the virus in order to develop novel therapeutic approaches to stop the COVID-19 pandemic and to better prepare against potential future emergence of novel pandemic viruses. Although SARS-CoV-2 primarily targets cells of the lung epithelium causing respiratory infection and pathologies, there is growing evidence that the intestinal epithelium is also infected. However, the importance of the enteric phase of SARS-CoV-2 for virus-induced pathologies, spreading and prognosis remains unknown. Here, using both colon-derived cell lines and primary non-transformed colon organoids, we engage in the first comprehensive analysis of SARS-CoV-2 lifecycle in human intestinal epithelial cells. Our results demonstrate that human intestinal epithelial cells fully support SARS-CoV-2 infection, replication and production of infectious de-novo virus particles. Importantly, we identified intestinal epithelial cells as the best culture model to propagate SARS-CoV-2. We found that viral infection elicited an extremely robust intrinsic immune response where, interestingly, type III interferon mediated response was significantly more efficient at controlling SARS-CoV-2 replication and spread compared to type I interferon. Taken together, our data demonstrate that human intestinal epithelial cells are a productive site of SARS-CoV-2 replication and suggest that the enteric phase of SARS-CoV-2 may participate in the pathologies observed in COVID-19 patients by contributing in increasing patient viremia and by fueling an exacerbated cytokine response.

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The papain-like protease of porcine epidemic diarrhea virus negatively regulates type I interferon pathway by acting as a viral deubiquitinase

Journal of General Virology ◽

10.1099/vir.0.051169-0 ◽

2013 ◽

Vol 94 (7) ◽

pp. 1554-1567 ◽

Cited By ~ 76

Author(s):

Yaling Xing ◽

Jianfei Chen ◽

Jian Tu ◽

Bailing Zhang ◽

Xiaojuan Chen ◽

...

Keyword(s):

Porcine Epidemic Diarrhea Virus ◽

Innate Immune ◽

Signalling Pathway ◽

Type I ◽

Polyubiquitin Chains ◽

Diarrhea Virus ◽

Ubiquitinated Proteins ◽

Interferon Pathway ◽

Porcine Epidemic Diarrhea ◽

Swine Disease

Porcine epidemic diarrhea virus (PEDV) is the cause of an economically important swine disease. Previous studies suggested that PEDV does not elicit a robust IFN response, but the mechanism(s) used to evade or block this innate immune response was not known. In this study, we found that PEDV infection blocked synthetic dsRNA-induced IFN-β production by interfering with the activation of interferon regulatory factor 3 (IRF3). We identified PEDV replicase encoded papain-like protease 2 (PLP2) as an IFN antagonist that depends on catalytic activity for its function. We show that levels of ubiquitinated proteins are reduced during PEDV infection and that PEDV PLP2 has deubiquitinase (DUB) activity that recognizes and processes both K-48 and K-63 linked polyubiquitin chains. Furthermore, we found that PEDV PLP2 strongly inhibits RIG-I- and STING-activated IFN expression and that PEDV PLP2 can be co-immunoprecipitated with and deubiquitinates RIG-I and STING, the key components of the signalling pathway for IFN expression. These results show that PEDV infection suppresses production of IFN-β and provides evidence indicating that the PEDV papain-like protease 2 acts as a viral DUB to interfere with the RIG-I- and STING-mediated signalling pathway.

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Efficient Replication of the Novel Human Betacoronavirus EMC on Primary Human Epithelium Highlights Its Zoonotic Potential

mBio ◽

10.1128/mbio.00611-12 ◽

2013 ◽

Vol 4 (1) ◽

Cited By ~ 120

Author(s):

Eveline Kindler ◽

Hulda R. Jónsdóttir ◽

Doreen Muth ◽

Ole J. Hamming ◽

Rune Hartmann ◽

...

Keyword(s):

Airway Epithelium ◽

Respiratory Viruses ◽

Innate Immune ◽

Type I ◽

Target Tissue ◽

Primary Target ◽

Human Coronavirus ◽

Type Iii ◽

Human Airway Epithelium ◽

Type Iii Interferon

ABSTRACT The recent emergence of a novel human coronavirus (HCoV-EMC) in the Middle East raised considerable concerns, as it is associated with severe acute pneumonia, renal failure, and fatal outcome and thus resembles the clinical presentation of severe acute respiratory syndrome (SARS) observed in 2002 and 2003. Like SARS-CoV, HCoV-EMC is of zoonotic origin and closely related to bat coronaviruses. The human airway epithelium (HAE) represents the entry point and primary target tissue for respiratory viruses and is highly relevant for assessing the zoonotic potential of emerging respiratory viruses, such as HCoV-EMC. Here, we show that pseudostratified HAE cultures derived from different donors are highly permissive to HCoV-EMC infection, and by using reverse transcription (RT)-PCR and RNAseq data, we experimentally determined the identity of seven HCoV-EMC subgenomic mRNAs. Although the HAE cells were readily responsive to type I and type III interferon (IFN), we observed neither a pronounced inflammatory cytokine nor any detectable IFN responses following HCoV-EMC, SARS-CoV, or HCoV-229E infection, suggesting that innate immune evasion mechanisms and putative IFN antagonists of HCoV-EMC are operational in the new host. Importantly, however, we demonstrate that both type I and type III IFN can efficiently reduce HCoV-EMC replication in HAE cultures, providing a possible treatment option in cases of suspected HCoV-EMC infection. IMPORTANCE A novel human coronavirus, HCoV-EMC, has recently been described to be associated with severe respiratory tract infection and fatalities, similar to severe acute respiratory syndrome (SARS) observed during the 2002-2003 epidemic. Closely related coronaviruses replicate in bats, suggesting that, like SARS-CoV, HCoV-EMC is of zoonotic origin. Since the animal reservoir and circumstances of zoonotic transmission are yet elusive, it is critically important to assess potential species barriers of HCoV-EMC infection. An important first barrier against invading respiratory pathogens is the epithelium, representing the entry point and primary target tissue of respiratory viruses. We show that human bronchial epithelia are highly susceptible to HCoV-EMC infection. Furthermore, HCoV-EMC, like other coronaviruses, evades innate immune recognition, reflected by the lack of interferon and minimal inflammatory cytokine expression following infection. Importantly, type I and type III interferon treatment can efficiently reduce HCoV-EMC replication in the human airway epithelium, providing a possible avenue for treatment of emerging virus infections.

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Impaired type I and type III interferon induction and rhinovirus control in human cystic fibrosis airway epithelial cells

Thorax ◽

10.1136/thoraxjnl-2011-200405 ◽

2012 ◽

Vol 67 (6) ◽

pp. 517-525 ◽

Cited By ~ 25

Author(s):

Marjolaine Vareille ◽

Elisabeth Kieninger ◽

Marco P Alves ◽

Brigitte S Kopf ◽

Alexander Möller ◽

...

Keyword(s):

Cystic Fibrosis ◽

Epithelial Cells ◽

Airway Epithelial Cells ◽

Type I ◽

Airway Epithelial ◽

Type Iii ◽

Interferon Induction ◽

Cystic Fibrosis Airway ◽

Type Iii Interferon

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Parainfluenza Virus 3 Blocks Antiviral Mediators Downstream of the Interferon Lambda Receptor by Modulating Stat1 Phosphorylation

Journal of Virology ◽

10.1128/jvi.02502-15 ◽

2015 ◽

Vol 90 (6) ◽

pp. 2948-2958 ◽

Cited By ~ 12

Author(s):

Kirsten C. Eberle ◽

Jodi L. McGill ◽

Timothy A. Reinhardt ◽

Randy E. Sacco

Keyword(s):

Epithelial Cell ◽

Epithelial Cells ◽

Signaling Pathways ◽

Vero Cells ◽

Parainfluenza Virus ◽

Epithelial Cell Line ◽

Type I ◽

Type Iii ◽

Parainfluenza Viruses ◽

Type Iii Ifn

ABSTRACTParainfluenza viruses are known to inhibit type I interferon (IFN) production; however, there is a lack of information regarding the type III IFN response during infection. Type III IFNs signal through a unique heterodimeric receptor, IFN-λR1/interleukin-10R2 (IL-10R2), which is primarily expressed by epithelial cells. Parainfluenza virus 3 (PIV-3) infection is highly restricted to the airway epithelium. We therefore sought to examine type III IFN signaling pathways during PIV-3 infection of epithelial cells. We used three strains of PIV-3: human PIV-3 (HPIV-3), bovine PIV-3 (BPIV-3), and dolphin PIV-1 (Tursiops truncatusPIV-1, or TtPIV-1). Here, we show that message levels of IL-29 are significantly increased during PIV-3 infection, yet downstream antiviral signaling molecules are not upregulated to levels similar to those of the positive control. Furthermore, in Vero cells infected with PIV-3, stimulation with recombinant IL-29/-28A/-28B does not cause upregulation of downstream antiviral molecules, suggesting that PIV-3 interferes with the JAK/STAT pathway downstream of the IFN-λR1/IL-10R2 receptor. We used Western blotting to examine the phosphorylation of Stat1 and Stat2 in Vero cells and the bronchial epithelial cell line BEAS-2B. In Vero cells, we observed reduced phosphorylation of the serine 727 (S727) site on Stat1, while in BEAS-2B cells Stat1 phosphorylation was decreased at the tyrosine 701 (Y701) site during PIV-3 infection. PIV-3 therefore interferes with the phosphorylation of Stat1 downstream of the type III IFN receptor. These data provide new evidence regarding strategies employed by parainfluenza viruses to effectively circumvent respiratory epithelial cell-specific antiviral immunity.IMPORTANCEParainfluenza virus (PIV) in humans is associated with bronchiolitis and pneumonia and can be especially problematic in infants and the elderly. Also seen in cattle, bovine PIV-3 causes respiratory infections in young calves. In addition, PIV-3 is one of a number of pathogens that contribute to the bovine respiratory disease complex (BRDC). As their name suggests, interferons (IFNs) are produced by cells to interfere with viral replication. Paramyxoviruses have previously been shown to block production and downstream signaling of type I IFNs. For the first time, it is shown here that PIV-3 can induce protective type III IFNs in epithelial cells, the primary site of PIV-3 infection. However, we found that PIV-3 modulates signaling pathways downstream of the type III IFN receptor to block production of several specific molecules that aid in a productive antiviral response. Importantly, this work expands our understanding of how PIV-3 effectively evades host innate immunity.

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Porcine Epidemic Diarrhea Virus Infection Inhibits Interferon Signaling by Targeted Degradation of STAT1

Journal of Virology ◽

10.1128/jvi.01091-16 ◽

2016 ◽

Vol 90 (18) ◽

pp. 8281-8292 ◽

Cited By ~ 34

Author(s):

Longjun Guo ◽

Xiaolei Luo ◽

Ren Li ◽

Yunfei Xu ◽

Jian Zhang ◽

...

Keyword(s):

Virus Infection ◽

Porcine Epidemic Diarrhea Virus ◽

Molecular Mechanisms ◽

Type I Interferon ◽

Infection Type ◽

Interferon Response ◽

Type I ◽

Dependent Manner ◽

Diarrhea Virus ◽

Porcine Epidemic Diarrhea

ABSTRACTPorcine epidemic diarrhea virus (PEDV) is a worldwide-distributed alphacoronavirus, but the pathogenesis of PEDV infection is not fully characterized. During virus infection, type I interferon (IFN) is a key mediator of innate antiviral responses. Most coronaviruses develop some strategy for at least partially circumventing the IFN response by limiting the production of IFN and by delaying the activation of the IFN response. However, the molecular mechanisms by which PEDV antagonizes the antiviral effects of interferon have not been fully characterized. Especially, how PEDV impacts IFN signaling components has yet to be elucidated. In this study, we observed that PEDV was relatively resistant to treatment with type I IFN. Western blot analysis showed that STAT1 expression was markedly reduced in PEDV-infected cells and that this reduction was not due to inhibition of STAT1 transcription. STAT1 downregulation was blocked by a proteasome inhibitor but not by an autophagy inhibitor, strongly implicating the ubiquitin-proteasome targeting degradation system. Since PEDV infection-induced STAT1 degradation was evident in cells pretreated with the general tyrosine kinase inhibitor, we conclude that STAT1 degradation is independent of the IFN signaling pathway. Furthermore, we report that PEDV-induced STAT1 degradation inhibits IFN-α signal transduction pathways. Pharmacological inhibition of STAT1 degradation rescued the ability of the host to suppress virus replication. Collectively, these data show that PEDV is capable of subverting the type I interferon response by inducing STAT1 degradation.IMPORTANCEIn this study, we show that PEDV is resistant to the antiviral effect of IFN. The molecular mechanism is the degradation of STAT1 by PEDV infection in a proteasome-dependent manner. This PEDV infection-induced STAT1 degradation contributes to PEDV replication. Our findings reveal a new mechanism evolved by PEDV to circumvent the host antiviral response.

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