Phosphatase Cdc25A Negatively Regulates the Antiviral Immune Response by Inhibiting TBK1 Activity

ABSTRACT The phosphatase Cdc25A plays an important role in cell cycle regulation by dephosphorylating its substrates, such as cyclin-dependent kinases. In this study, we demonstrate that Cdc25A negatively regulates RIG-I-mediated antiviral signaling. We found that ectopic expression of Cdc25A in 293T cells inhibits the activation of beta interferon (IFN-β) induced by Sendai virus and poly(I·C), while knockdown of Cdc25A enhances the transcription of IFN-β stimulated by RNA virus infection. The inhibitory effect of Cdc25A on the antiviral immune response is mainly dependent on its phosphatase activity. Data from a luciferase assay indicated that Cdc25A can inhibit TBK1-mediated activation of IFN-β. Further analysis indicated that Cdc25A can interact with TBK1 and reduce the phosphorylation of TBK1 at S172, which in turn decreases the phosphorylation of its downstream substrate IRF3. Consistently, knockdown of Cdc25A upregulates the phosphorylation of both TBK1-S172 and IRF3 in Sendai virus-infected or TBK1-transfected 293T cells. In addition, we confirmed that Cdc25A can directly dephosphorylate TBK1-S172-p. These results demonstrate that Cdc25A inhibits the antiviral immune response by reducing the active form of TBK1. Using herpes simplex virus 1 (HSV-1) infection, an IFN-β reporter assay, and reverse transcription-quantitative PCR (RT-qPCR), we demonstrated that Cdc25A can also inhibit DNA virus-induced activation of IFN-β. Using a vesicular stomatitis virus (VSV) infection assay, we confirmed that Cdc25A can repress the RIG-I-like receptor (RLR)-mediated antiviral immune response and influence the antiviral status of cells. In conclusion, we demonstrate that Cdc25A negatively regulates the antiviral immune response by inhibiting TBK1 activity. IMPORTANCE The RLR-mediated antiviral immune response is critical for host defense against RNA virus infection. However, the detailed mechanism for balancing the RLR signaling pathway in host cells is not well understood. We found that the phosphatase Cdc25A negatively regulates the RNA virus-induced innate immune response. Our studies indicate that Cdc25A inhibits the RLR signaling pathway via its phosphatase activity. We demonstrated that Cdc25A reduces TBK1 activity and consequently restrains the activation of IFN-β transcription as well as the antiviral status of nearby cells. We showed that Cdc25A can also inhibit DNA virus-induced activation of IFN-β. Taken together, our findings uncover a novel function and mechanism for Cdc25A in regulating antiviral immune signaling. These findings reveal Cdc25A as an important negative regulator of antiviral immunity and demonstrate its role in maintaining host cell homeostasis following viral infection.

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Long Noncoding RNA Lnc-MxA Inhibits Beta Interferon Transcription by Forming RNA-DNA Triplexes at Its Promoter

Journal of Virology ◽

10.1128/jvi.00786-19 ◽

2019 ◽

Vol 93 (21) ◽

Cited By ~ 8

Author(s):

Xinda Li ◽

Guijie Guo ◽

Min Lu ◽

Wenjia Chai ◽

Yucen Li ◽

...

Keyword(s):

Immune Response ◽

Immune Responses ◽

Noncoding Rna ◽

Influenza A ◽

Sendai Virus ◽

Negative Regulator ◽

Host Cells ◽

Luciferase Assay ◽

Antiviral Immune Response ◽

Beta Interferon

ABSTRACT Previously, we identified a set of long noncoding RNAs (lncRNAs) that were differentially expressed in influenza A virus (IAV)-infected cells. In this study, we focused on lnc-MxA, which is upregulated during IAV infection. We found that the overexpression of lnc-MxA facilitates the replication of IAV, while the knockdown of lnc-MxA inhibits viral replication. Further studies demonstrated that lnc-MxA is an interferon-stimulated gene. However, lnc-MxA inhibits the Sendai virus (SeV)- and IAV-induced activation of beta interferon (IFN-β). A luciferase assay indicated that lnc-MxA inhibits the activation of the IFN-β reporter upon stimulation with RIG-I, MAVS, TBK1, or active IRF3 (IRF3-5D). These data indicated that lnc-MxA negatively regulates the RIG-I-mediated antiviral immune response. A chromatin immunoprecipitation (ChIP) assay showed that the enrichment of IRF3 and p65 at the IFN-β promoter in lnc-MxA-overexpressing cells was significantly lower than that in control cells, indicating that lnc-MxA interfered with the binding of IRF3 and p65 to the IFN-β promoter. Chromatin isolation by RNA purification (ChIRP), triplex pulldown, and biolayer interferometry assays indicated that lnc-MxA can bind to the IFN-β promoter. Furthermore, an electrophoretic mobility shift assay (EMSA) showed that lnc-MxA can form complexes with the IFN-β promoter fragment. These results demonstrated that lnc-MxA can form a triplex with the IFN-β promoter to interfere with the activation of IFN-β transcription. Using a vesicular stomatitis virus (VSV) infection assay, we confirmed that lnc-MxA can repress the RIG-I-like receptor (RLR)-mediated antiviral immune response and influence the antiviral status of cells. In conclusion, we revealed that lnc-MxA is an interferon-stimulated gene (ISG) that negatively regulates the transcription of IFN-β by forming an RNA-DNA triplex. IMPORTANCE IAV can be recognized as a nonself molecular pattern by host immune systems and can cause immune responses. However, the intense immune response induced by influenza virus, known as a “cytokine storm,” can also cause widespread tissue damage (X. Z. J. Guo and P. G. Thomas, Semin Immunopathol 39:541–550, 2017, https://doi.org/10.1007/s00281-017-0636-y; S. Yokota, Nihon Rinsho 61:1953–1958, 2003; I. A. Clark, Immunol Cell Biol 85:271–273, 2007). Meanwhile, the detailed mechanisms involved in the balancing of immune responses in host cells are not well understood. Our studies reveal that, as an IFN-inducible gene, lnc-MxA functions as a negative regulator of the antiviral immune response. We uncovered the mechanism by which lnc-MxA inhibits the activation of IFN-β transcription. Our findings demonstrate that, as an ISG, lnc-MxA plays an important role in the negative-feedback loop involved in maintaining immune homeostasis.

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Inhibition of Antiviral Innate Immunity by Avibirnavirus VP3 via Blocking TBK1-TRAF3 Complex Formation and IRF3 Activation

mSystems ◽

10.1128/msystems.00016-21 ◽

2021 ◽

Vol 6 (3) ◽

Author(s):

Tingjuan Deng ◽

Boli Hu ◽

Xingbo Wang ◽

Lulu Lin ◽

Jianwei Zhou ◽

...

Keyword(s):

Immune Response ◽

Innate Immunity ◽

Virus Infection ◽

Complex Formation ◽

Rna Virus ◽

Critical Role ◽

Type I ◽

Antiviral Immune Response ◽

Critical Residue ◽

Irf3 Activation

ABSTRACT The host innate immune system develops various strategies to antagonize virus infection, and the pathogen subverts or evades host innate immunity for self-replication. In the present study, we discovered that Avibirnavirus infectious bursal disease virus (IBDV) VP3 protein significantly inhibits MDA5-induced beta interferon (IFN-β) expression by blocking IRF3 activation. Binding domain mapping showed that the CC1 domain of VP3 and the residue lysine-155 of tumor necrosis factor receptor-associated factor 3 (TRAF3) are essential for the interaction. Furthermore, we found that the CC1 domain was required for VP3 to downregulate MDA5-mediated IFN-β production. A ubiquitination assay showed that lysine-155 of TRAF3 was the critical residue for K33-linked polyubiquitination, which contributes to the formation of a TRAF3-TBK1 complex. Subsequently, we revealed that VP3 blocked TRAF3-TBK1 complex formation through reducing K33-linked polyubiquitination of lysine-155 on TRAF3. Taken together, our data reveal that VP3 inhibits MDA5-dependent IRF3-mediated signaling via blocking TRAF3-TBK1 complex formation, which improves our understanding of the interplay between RNA virus infection and the innate host antiviral immune response. IMPORTANCE Type I interferon plays a critical role in the host response against virus infection, including Avibirnavirus. However, many viruses have developed multiple strategies to antagonize the innate host antiviral immune response during coevolution with the host. In this study, we first identified that K33-linked polyubiquitination of lysine-155 of TRAF3 enhances the interaction with TBK1, which positively regulates the host IFN immune response. Meanwhile, we discovered that the interaction of the CC1 domain of the Avibirnavirus VP3 protein and the residue lysine-155 of TRAF3 reduced the K33-linked polyubiquitination of TRAF3 and blocked the formation of the TRAF3-TBK1 complex, which contributed to the downregulation of host IFN signaling, supporting viral replication.

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Dephosphorylation of cGAS by PPP6C impairs its substrate binding activity and innate antiviral response

Protein & Cell ◽

10.1007/s13238-020-00729-3 ◽

2020 ◽

Vol 11 (8) ◽

pp. 584-599 ◽

Cited By ~ 3

Author(s):

Mi Li ◽

Hong-Bing Shu

Keyword(s):

Immune Response ◽

Virus Infection ◽

Innate Immune Response ◽

Substrate Binding ◽

Binding Activity ◽

Innate Immune ◽

Negative Regulator ◽

Autoimmune Response ◽

Serine Residue ◽

Dna Virus

Abstract The cyclic GMP-AMP (cGAMP) synthase (cGAS) plays a critical role in host defense by sensing cytosolic DNA derived from microbial pathogens or mis-located cellular DNA. Upon DNA binding, cGAS utilizes GTP and ATP as substrates to synthesize cGAMP, leading to MITA-mediated innate immune response. In this study, we identified the phosphatase PPP6C as a negative regulator of cGAS-mediated innate immune response. PPP6C is constitutively associated with cGAS in un-stimulated cells. DNA virus infection causes rapid disassociation of PPP6C from cGAS, resulting in phosphorylation of human cGAS S435 or mouse cGAS S420 in its catalytic pocket. Mutation of this serine residue of cGAS impairs its ability to synthesize cGAMP upon DNA virus infection. In vitro experiments indicate that S420-phosphorylated mcGAS has higher affinity to GTP and enzymatic activity. PPP6C-deficiency promotes innate immune response to DNA virus in various cells. Our findings suggest that PPP6C-mediated dephosphorylation of a catalytic pocket serine residue of cGAS impairs its substrate binding activity and innate immune response, which provides a mechanism for keeping the DNA sensor cGAS inactive in the absence of infection to avoid autoimmune response.

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Activation and Evasion of RLR Signaling by DNA Virus Infection

Frontiers in Microbiology ◽

10.3389/fmicb.2021.804511 ◽

2021 ◽

Vol 12 ◽

Author(s):

Junli Jia ◽

Jiangan Fu ◽

Huamin Tang

Keyword(s):

Immune Response ◽

Virus Infection ◽

Viral Infections ◽

Infection Process ◽

Research Progress ◽

Ligand Recognition ◽

Dna Virus ◽

Antiviral Immune Response ◽

Host Immune Responses ◽

Inducible Gene

Antiviral innate immune response triggered by nucleic acid recognition plays an extremely important role in controlling viral infections. The initiation of antiviral immune response against RNA viruses through ligand recognition of retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) was extensively studied. RLR’s role in DNA virus infection, which is less known, is increasing attention. Here, we review the research progress of the ligand recognition of RLRs during the DNA virus infection process and the viral evasion mechanism from host immune responses.

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TAX1BP1 Restrains Virus-Induced Apoptosis by Facilitating Itch-Mediated Degradation of the Mitochondrial Adaptor MAVS

Molecular and Cellular Biology ◽

10.1128/mcb.00422-16 ◽

2016 ◽

Vol 37 (1) ◽

Cited By ~ 14

Author(s):

Young Bong Choi ◽

Noula Shembade ◽

Kislay Parvatiyar ◽

Siddharth Balachandran ◽

Edward William Harhaj

Keyword(s):

Virus Infection ◽

Sendai Virus ◽

Apoptotic Cell ◽

Rna Virus ◽

Adaptor Protein ◽

Negative Regulator ◽

Type I ◽

Adaptor Molecule ◽

Highly Sensitive ◽

Induced Apoptosis

ABSTRACTThe host response to RNA virus infection consists of an intrinsic innate immune response and the induction of apoptosis as mechanisms to restrict viral replication. The mitochondrial adaptor molecule MAVS plays critical roles in coordinating both virus-induced type I interferon production and apoptosis; however, the regulation of MAVS-mediated apoptosis is poorly understood. Here, we show that the adaptor protein TAX1BP1 functions as a negative regulator of virus-induced apoptosis. TAX1BP1-deficient cells are highly sensitive to apoptosis in response to infection with the RNA viruses vesicular stomatitis virus and Sendai virus and to transfection with poly(I·C). TAX1BP1 undergoes degradation during RNA virus infection, and loss of TAX1BP1 is associated with apoptotic cell death. TAX1BP1 deficiency augments virus-induced activation of proapoptotic c-Jun N-terminal kinase (JNK) signaling. Virus infection promotes the mitochondrial localization of TAX1BP1 and concomitant interaction with the mitochondrial adaptor MAVS. TAX1BP1 recruits the E3 ligase Itch to MAVS to trigger its ubiquitination and degradation, and loss of TAX1BP1 or Itch results in increased MAVS protein expression. Together, these results indicate that TAX1BP1 functions as an adaptor molecule for Itch to target MAVS during RNA virus infection and thus restrict virus-induced apoptosis.

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Dendritic Cell Precursors Are Permissive to Dengue Virus and Human Immunodeficiency Virus Infection

Journal of Virology ◽

10.1128/jvi.79.12.7291-7299.2005 ◽

2005 ◽

Vol 79 (12) ◽

pp. 7291-7299 ◽

Cited By ~ 34

Author(s):

Wing-Hong Kwan ◽

Anna-Marija Helt ◽

Concepción Marañón ◽

Jean-Baptiste Barbaroux ◽

Anne Hosmalin ◽

...

Keyword(s):

Immune Response ◽

Human Immunodeficiency Virus ◽

Virus Infection ◽

Dengue Virus ◽

Blood Monocytes ◽

Dengue Virus Infection ◽

Differentiation Potential ◽

Antiviral Immune Response ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT CD14+ interstitial cells reside beneath the epidermis of skin and mucosal tissue and may therefore play an important role in viral infections and the shaping of an antiviral immune response. However, in contrast to dendritic cells (DC) or blood monocytes, these antigen-presenting cells (APC) have not been well studied. We have previously described long-lived CD14+ cells generated from CD34+ hematopoietic progenitors, which may represent model cells for interstitial CD14+ APC. Here, we show that these cells carry DC-SIGN and differentiate into immature DC in the presence of granulocyte-macrophage colony-stimulating factor. We have compared the CD14+ cells and the DC derived from these cells with respect to dengue virus and human immunodeficiency virus type 1 (HIV-1) infection. Both cell types are permissive to dengue virus infection, but the CD14+ cells secrete the anti-inflammatory cytokine interleukin 10 and no tumor necrosis factor alpha. Regarding HIV, the CD14+ cells are permissive to HIV-1, release higher p24 levels than the derived DC, and more efficiently activate HIV Pol-specific CD8+ memory T cells. The CD14+ DC precursors infected with either virus retain their DC differentiation potential. The results suggest that interstitial CD14+ APC may contribute to HIV-1 and dengue virus infection and the shaping of an antiviral immune response.

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JNK pathway restricts DENV, ZIKV and CHIKV infection by activating complement and apoptosis in mosquito salivary glands

10.1101/2020.03.01.972026 ◽

2020 ◽

Cited By ~ 1

Author(s):

Avisha Chowdhury ◽

Cassandra M. Modahl ◽

Siok Thing Tan ◽

Benjamin Wong Wei Xiang ◽

Dorothée Missé ◽

...

Keyword(s):

Immune Response ◽

Virus Infection ◽

Salivary Glands ◽

Antiviral Effect ◽

Negative Regulator ◽

Chikv Infection ◽

Jnk Pathway ◽

Antiviral Effects ◽

Antiviral Function ◽

Arbovirus Infection

AbstractArbovirus infection of Aedes aegypti salivary glands (SGs) determines transmission. However, there is a dearth of knowledge on SG immunity. Here, we characterized SG immune response to dengue, Zika and chikungunya viruses using high-throughput transcriptomics. The three viruses regulate components of Toll, IMD and JNK pathways. However, silencing of Toll and IMD components showed variable effects on SG infection by each virus. In contrast, regulation of JNK pathway produced consistent responses. Virus infection increased with depletion of component Kayak and decreased with depletion of negative regulator Puckered. Virus-induced JNK pathway regulates complement and apoptosis in SGs via TEP20 and Dronc, respectively. Individual and co-silencing of these genes demonstrate their antiviral effects and that both may function together. Co-silencing either TEP20 or Dronc with Puckered annihilates JNK pathway antiviral effect. We identified and characterized the broad antiviral function of JNK pathway in SGs, expanding the immune arsenal that blocks arbovirus transmission.

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