SARS-CoV-2 Nucleocapsid Protein Targets RIG-I-Like Receptor Pathways to Inhibit the Induction of Interferon Response

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19) that has resulted in the current pandemic. The lack of highly efficacious antiviral drugs that can manage this ongoing global emergency gives urgency to establishing a comprehensive understanding of the molecular pathogenesis of SARS-CoV-2. We characterized the role of the nucleocapsid protein (N) of SARS-CoV-2 in modulating antiviral immunity. Overexpression of SARS-CoV-2 N resulted in the attenuation of retinoic acid inducible gene-I (RIG-I)-like receptor-mediated interferon (IFN) production and IFN-induced gene expression. Similar to the SARS-CoV-1 N protein, SARS-CoV-2 N suppressed the interaction between tripartate motif protein 25 (TRIM25) and RIG-I. Furthermore, SARS-CoV-2 N inhibited polyinosinic: polycytidylic acid [poly(I:C)]-mediated IFN signaling at the level of Tank-binding kinase 1 (TBK1) and interfered with the association between TBK1 and interferon regulatory factor 3 (IRF3), subsequently preventing the nuclear translocation of IRF3. We further found that both type I and III IFN production induced by either the influenza virus lacking the nonstructural protein 1 or the Zika virus were suppressed by the SARS-CoV-2 N protein. Our findings provide insights into the molecular function of the SARS-CoV-2 N protein with respect to counteracting the host antiviral immune response.

Download Full-text

PRV UL13 inhibits cGAS–STING-mediated IFN-β production by phosphorylating IRF3

Veterinary Research ◽

10.1186/s13567-020-00843-4 ◽

2020 ◽

Vol 51 (1) ◽

Author(s):

Zongyi Bo ◽

Yurun Miao ◽

Rui Xi ◽

Qiuping Zhong ◽

Chenyi Bao ◽

...

Keyword(s):

Transcriptional Activation ◽

Pseudorabies Virus ◽

Nuclear Translocation ◽

Economic Loss ◽

Inhibitory Effect ◽

Poly I:C ◽

Interferon Response ◽

Type I ◽

Creb Binding Protein ◽

Swine Industry

Abstract Cyclic GMP-AMP (cGAMP) synthase (cGAS) is an intracellular sensor of cytoplasmic viral DNA created during virus infection, which subsequently activates the stimulator of interferon gene (STING)-dependent type I interferon response to eliminate pathogens. In contrast, viruses have developed different strategies to modulate this signalling pathway. Pseudorabies virus (PRV), an alphaherpesvirus, is the causative agent of Aujeszky’s disease (AD), a notable disease that causes substantial economic loss to the swine industry globally. Previous reports have shown that PRV infection induces cGAS-dependent IFN-β production, conversely hydrolysing cGAMP, a second messenger synthesized by cGAS, and attenuates PRV-induced IRF3 activation and IFN-β secretion. However, it is not clear whether PRV open reading frames (ORFs) modulate the cGAS–STING-IRF3 pathway. Here, 50 PRV ORFs were screened, showing that PRV UL13 serine/threonine kinase blocks the cGAS–STING-IRF3-, poly(I:C)- or VSV-mediated transcriptional activation of the IFN-β gene. Importantly, it was discovered that UL13 phosphorylates IRF3, and its kinase activity is indispensable for such an inhibitory effect. Moreover, UL13 does not affect IRF3 dimerization, nuclear translocation or association with CREB-binding protein (CBP) but attenuates the binding of IRF3 to the IRF3-responsive promoter. Consistent with this, it was discovered that UL13 inhibits the expression of multiple interferon-stimulated genes (ISGs) induced by cGAS–STING or poly(I:C). Finally, it was determined that PRV infection can activate IRF3 by recruiting it to the nucleus, and PRVΔUL13 mutants enhance the transactivation level of the IFN-β gene. Taken together, the data from the present study demonstrated that PRV UL13 inhibits cGAS–STING-mediated IFN-β production by phosphorylating IRF3.

Download Full-text

Clinically observed deletions in SARS-CoV-2 Nsp1 affect protein stability and its ability to inhibit translation

10.1101/2021.11.03.467065 ◽

2021 ◽

Author(s):

Pravin Kumar ◽

Erin Schexnaydre ◽

Karim Rafie ◽

Ilya Terenin ◽

Vasili Hauryliuk ◽

...

Keyword(s):

Nonstructural Protein ◽

Ribosomal Subunit ◽

Fine Tuning ◽

In Vitro Translation ◽

Interferon Response ◽

Type I ◽

Biochemical Basis ◽

Translation Inhibition ◽

Terminal Domain ◽

Nonstructural Protein 1

Nonstructural protein 1 (Nsp1) is a major pathogenicity factor of SARS-CoV-2. It inhibits host-cell translation, primarily through a direct interaction between its C-terminal domain and the mRNA entry channel of the 40S small ribosomal subunit, with an N-terminal β-barrel domain fine-tuning the inhibition and promoting selective translation of viral mRNA. SARS-CoV-2 nsp1 is a target of recurring deletions, some of which are associated with altered COVID-19 disease progression. To provide the biochemical basis for this, it is essential to characterize the efficiency of translational inhibition by the said protein variants. Here, we use an in vitro translation system to investigate the translation inhibition capacity of a series of clinically observed Nsp1 deletion variants. We find that a frequently observed deletion of residues 79-89 destabilized the N-terminal domain (NTD) and severely reduced the capacity of Nsp1 to inhibit translation. Interestingly, shorter deletions in the same region have been reported to effect the type I interferon response but did not affect translation inhibition, indicating a possible translation-independent role of the Nsp1 NTD in interferon response modulation. Taken together, our data provide a mechanistic basis for understanding how deletions in Nsp1 influence SARS-CoV-2 induction of interferon response and COVID-19 progression.

Download Full-text

Peste des Petits Ruminants Virus Nucleocapsid Protein Inhibits Beta Interferon Production by Interacting with IRF3 To Block Its Activation

Journal of Virology ◽

10.1128/jvi.00362-19 ◽

2019 ◽

Vol 93 (16) ◽

Cited By ~ 9

Author(s):

Zixiang Zhu ◽

Pengfei Li ◽

Fan Yang ◽

Weijun Cao ◽

Xiangle Zhang ◽

...

Keyword(s):

Nuclear Translocation ◽

Type I Interferon ◽

Interferon Regulatory Factor ◽

Type I ◽

Peste Des Petits Ruminants ◽

Type I Ifn ◽

Regulatory Factor ◽

N Protein ◽

Beta Interferon ◽

Natural Hosts

ABSTRACTPeste des petits ruminants virus (PPRV) is the etiological agent of peste des petits ruminants, causing acute immunosuppression in its natural hosts. However, the molecular mechanisms by which PPRV antagonizes the host immune responses have not been fully characterized. In particular, how PPRV suppresses the activation of the host RIG-I-like receptor (RLR) pathway has yet to be clarified. In this study, we demonstrated that PPRV infection significantly suppresses RLR pathway activation and type I interferon (IFN) production and identified PPRV N protein as an extremely important antagonistic viral factor that suppresses beta interferon (IFN-β) and IFN-stimulated gene (ISG) expression. A detailed analysis showed that PPRV N protein inhibited type I IFN production by targeting interferon regulatory factor 3 (IRF3), a key molecule in the RLR pathway required for type I IFN induction. PPRV N protein interacted with IRF3 (but not with other components of the RLR pathway, including MDA5, RIG-I, VISA, TBK1, and MITA) and abrogated the phosphorylation of IRF3. As expected, PPRV N protein also considerably impaired the nuclear translocation of IRF3. The TBK1-IRF3 interaction was involved significantly in IRF3 phosphorylation, and we showed that PPRV N protein inhibits the association between TBK1 and IRF3, which in turn inhibits IRF3 phosphorylation. The amino acid region 106 to 210 of PPRV N protein was determined to be essential for suppressing the nuclear translocation of IRF3 and IFN-β production, and the 140 to 400 region of IRF3 was identified as the crucial region for the N-IRF3 interaction. Together, our findings demonstrate a new mechanism evolved by PPRV to inhibit type I IFN production and provide structural insights into the immunosuppression caused by PPRV.IMPORTANCEPeste des petits ruminants is a highly contagious animal disease affecting small ruminants, which threatens both small livestock and endangered susceptible wildlife populations in many countries. The causative agent, peste des petits ruminants virus (PPRV), often causes acute immunosuppression in its natural hosts during infection. Here, for the first time, we demonstrate that N protein, the most abundant protein of PPRV, plays an extremely important role in suppression of interferon regulatory factor 3 (IRF3) function and type I interferon (IFN) production by interfering with the formation of the TBK1-IRF3 complex. This study explored a novel antagonistic mechanism of PPRV.

Download Full-text

Synergistic benefit in inflammatory arthritis by targeting IκB kinase ϵ and interferon β

Annals of the Rheumatic Diseases ◽

10.1136/ard.2008.095356 ◽

2008 ◽

Vol 68 (2) ◽

pp. 257-263 ◽

Cited By ~ 28

Author(s):

M Corr ◽

D L Boyle ◽

L Ronacher ◽

N Flores ◽

G S Firestein

Keyword(s):

Gene Expression ◽

Low Dose ◽

Serum Levels ◽

Poly I:C ◽

Type I ◽

Wild Type ◽

Polycytidylic Acid ◽

Iκb Kinase ◽

Novel Approach ◽

Clinical Arthritis

Objectives:The IκB kinase (IKK)-related kinase IKKϵ regulates type I interferon expression and responses as well as proinflammatory mediator production. We examined the role of IKKϵ in arthritis and its ability to enhance the therapeutic response to systemic interferon (IFN) β therapy in passive murine K/BxN arthritis.Methods:IKKϵ–/–, IFNα∼βR–/– and wild type mice were given K/BxN serum and treated with polyinosinic polycytidylic acid (poly(I:C)), IFNβ, or normal saline. Clinical response and histological scores were assessed. Gene expression in the paws was measured by quantitative PCR. Serum interleukin 1a receptor agonist (IL1Ra) and IL10 were measured by ELISA and multiplex bead array.Results:Arthritis was almost completely blocked in wild type mice if arthritogenic K/BxN serum and the Toll-like receptor (TLR)3 ligand, poly(I:C), were coadministered at the onset of the model, but not in established disease. Mice deficient in IFNα∼βR had an accelerated course of arthritis, and did not respond to poly(I:C). IKKϵ null mice had a modest decrease in clinical arthritis compared with heterozygous mice. Low doses of IFNβ that were ineffective in wild type mice significantly decreased clinical arthritis in IKKϵ null mice. Articular chemokine gene expression was reduced in the IKKϵ–/– mice with arthritis and secreted IL1Ra (sIL1Ra) mRNA was significantly increased. Serum levels of IL1Ra were increased in low dose IFNβ-treated IKKϵ–/– mice.Conclusions:Subtherapeutic doses of IFNβ enhance the anti-inflammatory effects of IKKϵ deficiency, possibly by increasing production of IL1Ra and unmasking the antichemokine effects. Combination therapy with low dose IFNβ and an IKKϵ inhibitor might improve efficacy of either agent alone and offers a novel approach to RA.

Download Full-text

Nonstructural Proteins 1 and 2 of Respiratory Syncytial Virus Suppress Maturation of Human Dendritic Cells

Journal of Virology ◽

10.1128/jvi.00630-08 ◽

2008 ◽

Vol 82 (17) ◽

pp. 8780-8796 ◽

Cited By ~ 76

Author(s):

Shirin Munir ◽

Cyril Le Nouen ◽

Cindy Luongo ◽

Ursula J. Buchholz ◽

Peter L. Collins ◽

...

Keyword(s):

Dendritic Cells ◽

Respiratory Syncytial Virus ◽

Fluorescent Protein ◽

Nonstructural Protein ◽

Type I ◽

Ns1 Protein ◽

Myeloid Dendritic Cells ◽

Nonstructural Protein 1 ◽

Syncytial Virus ◽

Dc Maturation

ABSTRACT Human respiratory syncytial virus (RSV) is the most important agent of serious pediatric respiratory tract disease worldwide. One of the main characteristics of RSV is that it readily reinfects and causes disease throughout life without the need for significant antigenic change. The virus encodes nonstructural protein 1 (NS1) and NS2, which are known to suppress type I interferon (IFN) production and signaling. In the present study, we monitored the maturation of human monocyte-derived myeloid dendritic cells (DC) following inoculation with recombinant RSVs bearing deletions of the NS1 and/or NS2 proteins and expressing enhanced green fluorescent protein. Deletion of the NS1 protein resulted in increased expression of cell surface markers of DC maturation and an increase in the expression of multiple cytokines and chemokines. This effect was enhanced somewhat by further deletion of the NS2 protein, although deletion of NS2 alone did not have a significant effect. The upregulation was largely inhibited by pretreatment with a blocking antibody against the type I IFN receptor, suggesting that suppression of DC maturation by NS1/2 is, at least in part, a result of IFN antagonism mediated by these proteins. Therefore, this study identified another effect of the NS1 and NS2 proteins. The observed suppression of DC maturation may result in decreased antigen presentation and T-lymphocyte activation, leading to incomplete and/or weak immune responses that might contribute to RSV reinfection.

Download Full-text

HCFC2 is needed for IRF1- and IRF2-dependent Tlr3 transcription and for survival during viral infections

Journal of Experimental Medicine ◽

10.1084/jem.20161630 ◽

2017 ◽

Vol 214 (11) ◽

pp. 3263-3277 ◽

Cited By ~ 12

Author(s):

Lei Sun ◽

Zhengfan Jiang ◽

Victoria A. Acosta-Rodriguez ◽

Michael Berger ◽

Xin Du ◽

...

Keyword(s):

Viral Infections ◽

Poly I:C ◽

Type I ◽

Induced Mutations ◽

Herpes Simplex Virus 1 ◽

Double Stranded Rna ◽

Polycytidylic Acid ◽

Interferon Regulatory Factor 1 ◽

Mouse Macrophages ◽

Simplex Virus

Transcriptional regulation of numerous interferon-regulated genes, including Toll-like receptor 3 (Tlr3), which encodes an innate immune sensor of viral double-stranded RNA, depends on the interferon regulatory factor 1 (IRF1) and IRF2 transcription factors. We detected specific abrogation of macrophage responses to polyinosinic-polycytidylic acid (poly(I:C)) resulting from three independent N-ethyl-N-nitrosourea–induced mutations in host cell factor C2 (Hcfc2). Hcfc2 mutations compromised survival during influenza virus and herpes simplex virus 1 infections. HCFC2 promoted the binding of IRF1 and IRF2 to the Tlr3 promoter, without which inflammatory cytokine and type I IFN responses to the double-stranded RNA analogue poly(I:C) are reduced in mouse macrophages. HCFC2 was also necessary for the transcription of a large subset of other IRF2-dependent interferon-regulated genes. Deleterious mutations of Hcfc2 may therefore increase susceptibility to diverse infectious diseases.

Download Full-text

Statins attenuate antiviral IFN-β and ISG expression via inhibiting IRF3/JAK/STAT signaling in poly(I:C)-treated hyperlipidemic mice and macrophages

10.1101/2020.06.21.163873 ◽

2020 ◽

Author(s):

Atsushi Koike ◽

Kaito Tsujinaka ◽

Ko Fujimori

Keyword(s):

Bronchoalveolar Lavage ◽

Molecular Mechanisms ◽

Bronchoalveolar Lavage Fluid ◽

Lavage Fluid ◽

Janus Kinase ◽

Poly I:C ◽

Induced Expression ◽

Antiviral Immune Response ◽

Polycytidylic Acid ◽

Stat Signaling

AbstractViral infection is a significant burden to healthcare worldwide. Statins, 3-hydroxy-3-methyl glutaryl coenzyme A reductase inhibitors, are widely used as cholesterol-lowering drugs. Recently, long term statin therapy was shown to reduce the antiviral immune response; however, the underlying molecular mechanisms are unclear. Here, we found that simvastatin decreased polyinosinic-polycytidylic acid [poly(I:C)]-induced expression of antiviral interferon (IFN)-β and IFN-stimulated genes (ISGs) in the bronchoalveolar lavage fluid and lungs of mice with high-fat diet-induced hyperlipidemia. As macrophages were the dominant cell type in the bronchoalveolar lavage fluid of poly(I:C)-treated mice, we examined the molecular mechanisms of statin-mediated inhibition of antiviral gene expression using murine J774.1/JA-4 macrophages. Simvastatin and pitavastatin decreased poly(I:C)-induced expression of IFN-β and ISGs. Moreover, they repressed poly(I:C)-induced phosphorylation of IFN regulatory factor (IRF) 3 and signal transducers and activators of transcription (STAT) 1, which is involved in Janus kinase (JAK)/STAT signaling. Mevalonate and geranylgeranylpyrophosphate (GGPP), but not cholesterol, counteracted the negative effect of statins on IFN-β and ISG expression and phosphorylation of IRF3 and STAT1. These results suggest that statins suppressed the expression of IFN-β and ISGs in poly(I:C)-treated hyperlipidemic mice and murine macrophages, and that these effects occured through the inhibition of IRF3-mediated JAK/STAT signaling in macrophages. Furthermore, GGPP recovered the statin-suppressed IRF3/JAK/STAT signaling pathway in poly(I:C)-treated macrophages.

Download Full-text

Swine Acute Diarrhea Syndrome Coronavirus Nucleocapsid Protein Antagonizes Interferon-β Production via Blocking the Interaction Between TRAF3 and TBK1

Frontiers in Immunology ◽

10.3389/fimmu.2021.573078 ◽

2021 ◽

Vol 12 ◽

Author(s):

Zhihai Zhou ◽

Yuan Sun ◽

Jingya Xu ◽

Xiaoyu Tang ◽

Ling Zhou ◽

...

Keyword(s):

Innate Immunity ◽

Acute Diarrhea ◽

Nuclear Translocation ◽

Sendai Virus ◽

Type I ◽

Innate Immune Responses ◽

N Protein ◽

Antiviral Innate Immunity ◽

Diarrhea Syndrome

Swine acute diarrhea syndrome coronavirus (SADS-CoV), first discovered in 2017, is a porcine enteric coronavirus that can cause acute diarrhea syndrome (SADS) in piglets. Here, we studied the role of SADS-CoV nucleocapsid (N) protein in innate immunity. Our results showed that SADS-CoV N protein could inhibit type I interferon (IFN) production mediated by Sendai virus (Sev) and could block the phosphorylation and nuclear translocation of interferon regulatory factor 3 (IRF3). Simultaneously, the IFN-β promoter activity mediated by TANK binding kinase 1 (TBK1) or its upstream molecules in the RLRs signal pathway was inhibited by SADS-CoV N protein. Further investigations revealed that SADS-CoV N protein could counteract interaction between TNF receptor-associated factor 3 (TRAF3) and TBK1, which led to reduced TBK1 activation and IFN-β production. Our study is the first report of the interaction between SADS-CoV N protein and the host antiviral innate immune responses, and the mechanism utilized by SADS-CoV N protein provides a new insight of coronaviruses evading host antiviral innate immunity.

Download Full-text

Saudi Arabian SARS-CoV-2 genomes implicate a mutant Nucleocapsid protein in modulating host interactions and increased viral load in COVID-19 patients

10.1101/2021.05.06.21256706 ◽

2021 ◽

Author(s):

Tobias Mourier ◽

Muhammad Shuaib ◽

Sharif Hala ◽

Sara Mfarrej ◽

Fadwa Alofi ◽

...

Keyword(s):

Saudi Arabia ◽

Viral Load ◽

Nucleocapsid Protein ◽

Rna Binding ◽

Genomic Sequence ◽

Mass Spectrometry Analysis ◽

Interferon Response ◽

Sequence Information ◽

Effective Population ◽

N Protein

Monitoring SARS-CoV-2 spread and evolution through genome sequencing is essential in handling the COVID-19 pandemic. The availability of patient hospital records is crucial for linking the genomic sequence information to virus function during the course of infections. Here, we sequenced 892 SARS-CoV-2 genomes collected from patients in Saudi Arabia from March to August 2020. From the assembled sequences, we estimate the SARS-CoV-2 effective population size and infection rate and outline the epidemiological dynamics of import and transmission events during this period in Saudi Arabia. We show that two consecutive mutations (R203K/G204R) in the SARS-CoV-2 nucleocapsid (N) protein are associated with higher viral loads in COVID-19 patients. Our comparative biochemical analysis reveals that the mutant N protein displays enhanced viral RNA binding and differential interaction with key host proteins. We found hyper-phosphorylation of the adjacent serine site (S206) in the mutant N protein by mass-spectrometry analysis. Furthermore, analysis of the host cell transcriptome suggests that the mutant N protein results in dysregulated interferon response genes. We provide crucial information in linking the R203K/G204R mutations in the N protein as a major modulator of host-virus interactions and increased viral load and underline the potential of the nucleocapsid protein as a drug target during infection.

Download Full-text

Imiquimod Boosts Interferon Response, and Decreases ACE2 and Pro-Inflammatory Response of Human Bronchial Epithelium in Asthma

Frontiers in Immunology ◽

10.3389/fimmu.2021.743890 ◽

2021 ◽

Vol 12 ◽

Author(s):

Juan José Nieto-Fontarigo ◽

Sofia Tillgren ◽

Samuel Cerps ◽

Asger Sverrild ◽

Morten Hvidtfeldt ◽

...

Keyword(s):

Viral Infections ◽

Ex Vivo ◽

Bronchial Epithelium ◽

Poly I:C ◽

Interferon Response ◽

Cytokine Signaling ◽

Type I ◽

Protective Effects ◽

Bronchial Epithelial ◽

Proteomic Analyses

BackgroundBoth anti-viral and anti-inflammatory bronchial effects are warranted to treat viral infections in asthma. We sought to investigate if imiquimod, a TLR7 agonist, exhibits such dual actions in ex vivo cultured human bronchial epithelial cells (HBECs), targets for SARS-CoV-2 infectivity.ObjectiveTo investigate bronchial epithelial effects of imiquimod of potential importance for anti-viral treatment in asthmatic patients.MethodsEffects of imiquimod alone were examined in HBECs from healthy (N=4) and asthmatic (N=18) donors. Mimicking SARS-CoV-2 infection, HBECs were stimulated with poly(I:C), a dsRNA analogue, or SARS-CoV-2 spike-protein 1 (SP1; receptor binding) with and without imiquimod treatment. Expression of SARS-CoV-2 receptor (ACE2), pro-inflammatory and anti-viral cytokines were analyzed by RT-qPCR, multiplex ELISA, western blot, and Nanostring and proteomic analyses.ResultsImiquimod reduced ACE2 expression at baseline and after poly(I:C) stimulation. Imiquimod also reduced poly(I:C)-induced pro-inflammatory cytokines including IL-1β, IL-6, IL-8, and IL-33. Furthermore, imiquimod increased IFN-β expression, an effect potentiated in presence of poly(I:C) or SP1. Multiplex mRNA analysis verified enrichment in type-I IFN signaling concomitant with suppression of cytokine signaling pathways induced by imiquimod in presence of poly(I:C). Exploratory proteomic analyses revealed potentially protective effects of imiquimod on infections.ConclusionImiquimod triggers viral resistance mechanisms in HBECs by decreasing ACE2 and increasing IFN-β expression. Additionally, imiquimod improves viral infection tolerance by reducing viral stimulus-induced epithelial cytokines involved in severe COVID-19 infection. Our imiquimod data highlight feasibility of producing pluripotent drugs potentially suited for anti-viral treatment in asthmatic subjects.

Download Full-text