Virus-triggered Activation Of IRF3 Research Articles

RNF138 Downregulates Antiviral Innate Immunity by Inhibiting IRF3 Activation.

A viral infection activates the transcription factors IRF3 and NF-κB, which synergistically induces type I interferons (IFNs). Here, we identify the E3 ubiquitin ligase RNF138 as an important negative regulator of virus-triggered IRF3 activation and IFN-β induction. The overexpression of RNF138 inhibited the virus-induced activation of IRF3 and the transcription of the IFNB1 gene, whereas the knockout of RNF138 promoted the virus-induced activation of IRF3 and transcription of the IFNB1 gene. We further found that RNF138 promotes the ubiquitination of PTEN and subsequently inhibits PTEN interactions with IRF3, which is essential for the PTEN-mediated nuclear translocation of IRF3, thereby inhibiting IRF3 import into the nucleus. Our findings suggest that RNF138 negatively regulates virus-triggered signaling by inhibiting the interaction of PTEN with IRF3, and these data provide new insights into the molecular mechanisms of cellular antiviral responses.

Induction of OTUD4 by viral infection promotes antiviral responses through deubiquitinating and stabilizing MAVS.

The activity and stability of the adapter protein MAVS (also known as VISA, Cardif and IPS-1), which critically mediates cellular antiviral responses, are extensively regulated by ubiquitination. However, the process whereby MAVS is deubiquitinated is unclear. Here, we report that the ovarian tumor family deubiquitinase 4 (OTUD4) targets MAVS for deubiquitination. Viral infection leads to the IRF3/7-dependent upregulation of OTUD4 which interacts with MAVS to remove K48-linked polyubiquitin chains, thereby maintaining MAVS stability and promoting innate antiviral signaling. Knockout or knockdown of OTUD4 impairs RNA virus-triggered activation of IRF3 and NF-κB, expression of their downstream target genes, and potentiates VSV replication in vitro and in vivo. Consistently, Cre-ER Otud4fl/fl or Lyz2-Cre Otud4fl/fl mice produce decreased levels of type I interferons and proinflammatory cytokines and exhibit increased sensitivity to VSV infection compared to their control littermates. In addition, reconstitution of MAVS into OTUD4-deficient cells restores virus-induced expression of downstream genes and cellular antiviral responses. Together, our findings uncover an essential role of OTUD4 in virus-triggered signaling and contribute to the understanding of deubiquitination-mediated regulation of innate antiviral responses.

Foot-and-mouth disease virus non-structural protein 3A inhibits the interferon-β signaling pathway.

Foot-and-mouth disease virus (FMDV) is the etiological agent of FMD, which affects cloven-hoofed animals. The pathophysiology of FMDV has not been fully understood and the evasion of host innate immune system is still unclear. Here, the FMDV non-structural protein 3A was identified as a negative regulator of virus-triggered IFN-β signaling pathway. Overexpression of the FMDV 3A inhibited Sendai virus-triggered activation of IRF3 and the expressions of RIG-I/MDA5. Transient transfection and co-immunoprecipitation experiments suggested that FMDV 3A interacts with RIG-I, MDA5 and VISA, which is dependent on the N-terminal 51 amino acids of 3A. Furthermore, 3A also inhibited the expressions of RIG-I, MDA5, and VISA by disrupting their mRNA levels. These results demonstrated that 3A inhibits the RLR-mediated IFN-β induction and uncovered a novel mechanism by which the FMDV 3A protein evades the host innate immune system.

Characterization of a MAVS ortholog from the Chinese tree shrew (Tupaia belangeri chinensis)

Human mitochondrial antiviral signaling protein (hMAVS, also known as IPS-1, VISA, or Cardif) is essential for antiviral innate immunity. The Chinese tree shrew (Tupaia belangeri chinenses), a close relative of primates, is emerging as a potential animal model for investigating viral infection. However, there is a lack of biological knowledge about the antiviral innate immunity of the tree shrew. In this study, we identified and characterized the function of the Chinese tree shrew MAVS gene (tMAVS). The cDNA of tMAVS was 2771 bp in length and encoded a polypeptide of 501 amino acids. Phylogenetic analyses based on the amino acid sequences revealed a closer affinity of tMAVS with those of primates. Quantitative real-time PCR analysis indicated that tMAVS mRNA was constitutively expressed in all seven tissues analyzed in this study. The tMAVS mRNA expression was rapidly and significantly increased after RNA virus infections. Ectopic-expression of tMAVS significantly potentiated the virus-triggered activation of IRF3, NF-κB and interferon-β (IFN-β), whereas knockdown of tMAVS displayed the opposite effect. Furthermore, tMAVS mutants lacking the caspase activation and recruitment (CARD) domains or the transmembrane (TM) domain were unable to induce IFN-β. Similar with hMAVS, mitochondrial localization of tMAVS was dependent on its domain. Collectively, this study revealed evolutionary conservation of the MAVS antiviral signaling pathway in the Chinese tree shrew.

TRIM4 modulates type I interferon induction and cellular antiviral response by targeting RIG-I for K63-linked ubiquitination.

RIG-I is a pivotal cytoplasmic sensor that recognizes different species of viral RNAs. This recognition leads to activation of the transcription factors NF-κB and IRF3, which collaborate to induce type I interferons (IFNs) and innate antiviral response. In this study, we identified the TRIM family protein TRIM4 as a positive regulator of RIG-I-mediated IFN induction. Overexpression of TRIM4 potentiated virus-triggered activation of IRF3 and NF-κB, as well as IFN-β induction, whereas knockdown of TRIM4 had opposite effects. Mechanistically, TRIM4 associates with RIG-I and targets it for K63-linked polyubiquitination. Our findings demonstrate that TRIM4 is an important regulator of the virus-induced IFN induction pathways by mediating RIG-I for K63-linked ubiquitination.

Glycogen Synthase Kinase 3β Regulates IRF3 Transcription Factor-Mediated Antiviral Response via Activation of the Kinase TBK1

Viral infection activates transcription factors IRF3 and NF-κB, which collaborate to induce type I interferons (IFNs). Here, we identified glycogen synthase kinase 3β (GSK3β) as an important regulator for virus-triggered IRF3 and NF-κB activation, IFN-β induction, and cellular antiviral response. Overexpression of GSK3β potentiated virus-induced activation of IRF3 and transcription of the IFNB1 gene, whereas reduced expression or deletion of GSK3β impaired virus-induced IRF3 and NF-κB activation, transcription of the IFNB1 gene, as well as cellular antiviral response. GSK3β physically associated with the kinase TBK1 in a viral infection-dependent manner. GSK3β promoted TBK1 self-association and autophosphorylation at Ser172, which is critical for virus-induced IRF3 activation and IFN-β induction. The effect of GSK3β on virus-induced signaling is independent of its kinase activity. Our findings suggest that GSK3β plays important roles in virus-triggered IRF3 activation by promoting TBK1 activation and provide new insights to the molecular mechanisms of cellular antiviral response.

Virus-triggered Ubiquitination of TRAF3/6 by cIAP1/2 Is Essential for Induction of Interferon-β (IFN-β) and Cellular Antiviral Response

Viral infection causes activation of transcription factors NF-kappaB and IRF3, which collaborate to induce type I interferons (IFNs) and cellular antiviral response. Here we show that knockdown of the E3 ubiquitin ligases cIAP1 and cIAP2 markedly inhibited virus-triggered activation of IRF3 and NF-kappaB as well as IFN-beta induction. Knockdown of cIAP1 and cIAP2 also inhibited cytoplasmic dsRNA-triggered cellular antiviral response. Endogenous coimmunoprecipitation experiments indicated that viral infection caused recruitment of cIAP1 and cIAP2 to TRAF3, TRAF6, and VISA. Furthermore, we demonstrated that cIAP1- and cIAP2-mediated virus-triggered ubiquitination of TRAF3 and TRAF6. These findings suggest that virus-triggered ubiquitination of TRAF3 and TRAF6 by cIAP1 and cIAP2 is essential for type I IFN induction and cellular antiviral response.

WDR5 is essential for assembly of the VISA-associated signaling complex and virus-triggered IRF3 and NF-κB activation

Viral infection causes activation of the transcription factors NF-kappaB and IRF3, which collaborate to induce type I interferons (IFNs) and cellular antiviral response. The mitochondrial outer membrane protein VISA acts as a critical adapter for assembling a virus-induced complex that signals NF-kappaB and IRF3 activation. Using a biochemical purification approach, we identified the WD repeat protein WDR5 as a VISA-associated protein. WDR5 was recruited to VISA in a viral infection dependent manner. Viral infection also caused translocation of WDR5 from the nucleus to mitochondria. Knockdown of WDR5 impaired the formation of virus-induced VISA-associated complex. Consistently, knockdown of WDR5 inhibited virus-triggered activation of IRF3 and NF-kappaB as well as transcription of the IFNB1 gene. These findings suggest that WDR5 is essential in assembling a virus-induced VISA-associated complex and plays an important role in virus-triggered induction of type I IFNs.

ISG56 is a negative-feedback regulator of virus-triggered signaling and cellular antiviral response

IFN-stimulated gene 56 (ISG56) is one of the first identified proteins induced by viruses and type I IFNs. In this study, we identified ISG56 as a virus-induced protein associated with MITA, an adapter protein involved in virus-triggered induction of type I IFNs. Overexpression of ISG56 inhibited Sendai virus-triggered activation of IRF3, NF-kappaB, and the IFN-beta promoter, whereas knockdown of ISG56 had opposite effects. Consistently, overexpression of ISG56 reversed cytoplasmic poly(I:C)-induced inhibition of vesicular stomatitis virus (VSV) replication, whereas knockdown of ISG56 inhibited VSV replication. Competitive coimmunoprecipitation experiments indicated that ISG56 disrupted the interactions between MITA and VISA or TBK1, two components in the virus-triggered IFN signaling pathways. These results suggest that ISG56 is a mediator of negative-feedback regulation of virus-triggered induction of type I IFNs and cellular antiviral responses.

The Ubiquitin Ligase RNF5 Regulates Antiviral Responses by Mediating Degradation of the Adaptor Protein MITA

Viral infection activates transcription factors NF-kappaB and IRF3, which collaborate to induce type I interferons (IFNs) and elicit innate antiviral response. MITA (also known as STING) has recently been identified as an adaptor that links virus-sensing receptors to IRF3 activation. Here, we showed that the E3 ubiquitin ligase RNF5 interacted with MITA in a viral-infection-dependent manner. Overexpression of RNF5 inhibited virus-triggered IRF3 activation, IFNB1 expression, and cellular antiviral response, whereas knockdown of RNF5 had opposite effects. RNF5 targeted MITA at Lys150 for ubiquitination and degradation after viral infection. Both MITA and RNF5 were located at the mitochondria and endoplasmic reticulum (ER) and viral infection caused their redistribution to the ER and mitochondria, respectively. We further found that virus-induced ubiquitination and degradation of MITA by RNF5 occurred at the mitochondria. These findings suggest that RNF5 negatively regulates virus-triggered signaling by targeting MITA for ubiquitination and degradation at the mitochondria.

The Adaptor Protein MITA Links Virus-Sensing Receptors to IRF3 Transcription Factor Activation

Viral infection triggers activation of transcription factors such as NF-kappaB and IRF3, which collaborate to induce type I interferons (IFNs) and elicit innate antiviral response. Here, we identified MITA as a critical mediator of virus-triggered type I IFN signaling by expression cloning. Overexpression of MITA activated IRF3, whereas knockdown of MITA inhibited virus-triggered activation of IRF3, expression of type I IFNs, and cellular antiviral response. MITA was found to localize to the outer membrane of mitochondria and to be associated with VISA, a mitochondrial protein that acts as an adaptor in virus-triggered signaling. MITA also interacted with IRF3 and recruited the kinase TBK1 to the VISA-associated complex. MITA was phosphorylated by TBK1, which is required for MITA-mediated activation of IRF3. Our results suggest that MITA is a critical mediator of virus-triggered IRF3 activation and IFN expression and further demonstrate the importance of certain mitochondrial proteins in innate antiviral immunity.

SIKE is an IKKε/TBK1-associated suppressor of TLR3- and virus-triggered IRF-3 activation pathways

Viral infection or TLR3 engagement causes activation of the transcription factors IRF-3 and NF-kappaB, which collaborate to induce transcription of type I IFN genes. IKKepsilon and TBK1 are two IKK-related kinases critically involved in virus- and TLR3-triggered activation of IRF-3. We identified a protein termed SIKE (for Suppressor of IKKepsilon) that interacts with IKKepsilon and TBK1. SIKE is associated with TBK1 under physiological condition and dissociated from TBK1 upon viral infection or TLR3 stimulation. Overexpression of SIKE disrupted the interactions of IKKepsilon or TBK1 with TRIF, RIG-I and IRF-3, components in virus- and TLR3-triggered IRF-3 activation pathways, but did not disrupt the interactions of TRIF with TRAF6 and RIP, components in TLR3-triggered NF-kappaB activation pathway. Consistently, overexpression of SIKE inhibited virus- and TLR3-triggered interferon-stimulated response elements (ISRE) but not NF-kappaB activation. Knockdown of SIKE potentiated virus- and TLR3-triggered ISRE but not NF-kappaB activation. Moreover, overexpression of SIKE inhibited IKKepsilon- and TBK1-mediated antiviral response. These findings suggest that SIKE is a physiological suppressor of IKKepsilon and TBK1 and plays an inhibitory role in virus- and TLR3-triggered IRF-3 but not NF-kappaB activation pathways.