Cao-Qi Lei

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.

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.

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.

Krüppel-like Factor 6 Is a Co-activator of NF-κB That Mediates p65-dependent Transcription of Selected Downstream Genes

Background: Transcription factor NF-κB is involved in various biological processes and regulated at multiple levels. Results: KLF6 modulates NF-κB-mediated transcription by promoting binding of p65 to promoters of downstream genes. Conclusion: KLF6 is a co-activator of NF-κB-mediated transcription of selected downstream genes. Significance: Our study reveals a new regulatory mechanism for NF-κB-mediated transcriptional activation in the nucleus. The transcription factor NF-κB plays a pivotal role in a broad range of physiological and pathological processes, including development, inflammation, and immunity. How NF-κB integrates activating signals to expression of specific sets of target genes is of great interest. Here, we identified Krüppel-like factor 6 (KLF6) as a co-activator of NF-κB after TNFα and IL-1β stimulation. Overexpression of KLF6 enhanced TNFα- and IL-1β-induced activation of NF-κB and transcription of a subset of downstream genes, whereas knockdown of KLF6 had opposite effects. KLF6 interacted with p65 in the nucleus and bound to the promoters of target genes. Upon IL-1β stimulation, KLF6 was recruited to promoters of a subset of NF-κB target genes in a p65-dependent manner, which was in turn required for the optimal binding of p65 to the target gene promoters. Our findings thus identified KLF6 as a previously unknown but essential co-activator of NF-κB and provided new insight into the molecular regulation of p65-dependent gene expression.

FoxO1 negatively regulates cellular antiviral response by promoting degradation of IRF3

Background: The transcription factor IRF3 is critical for type I interferon induction and innate antiviral immune response. Results: FoxO1 mediates IRF3 ubiquitination and degradation. Conclusion: FoxO1 is a negative regulator of cellular antiviral response. Significance: Our study reveals a new mechanism for control of type I interferon induction and cellular antiviral response. Viral infection causes activation of the transcription factor IRF3, which is critical for production of type I interferons (IFNs) and innate antiviral immune response. How virus-induced type I IFN signaling is controlled is not fully understood. Here we identified the transcription factor FoxO1 as a negative regulator for virus-triggered IFN-β induction. Overexpression of FoxO1 inhibited virus-triggered ISRE activation, IFN-β induction as well as cellular antiviral response, whereas knockdown of FoxO1 had opposite effects. FoxO1 interacted with IRF3 in a viral infection-dependent manner and promoted K48-linked polyubiquitination and degradation of IRF3 in the cytosol. Furthermore, FoxO1-mediated degradation of IRF3 was independent of the known E3 ubiquitin ligases for IRF3, including RBCK1 and RAUL. Our findings thus suggest that FoxO1 negatively regulates cellular antiviral response by promoting IRF3 ubiquitination and degradation, providing a previously unknown mechanism for control of type I IFN induction and cellular antiviral response.

WDFY1 mediates TLR3/4 signaling by recruiting TRIF

Toll‐like receptors (TLRs) are pattern recognition receptors that sense a variety of pathogens, initiate innate immune responses, and direct adaptive immunity. All TLRs except TLR3 recruit the adaptor MyD88 to ultimately elicit inflammatory gene expression, whereas TLR3 and internalized TLR4 use TIR‐domain‐containing adaptor TRIF for the induction of type I interferon and inflammatory cytokines. Here, we identify the WD repeat and FYVE‐domain‐containing protein WDFY1 as a crucial adaptor protein in the TLR3/4 signaling pathway. Overexpression of WDFY1 potentiates TLR3‐ and TLR4‐mediated activation of NF‐κB, interferon regulatory factor 3 (IRF3), and production of type I interferons and inflammatory cytokines. WDFY1 depletion has the opposite effect. WDFY1 interacts with TLR3 and TLR4 and mediates the recruitment of TRIF to these receptors. Our findings suggest a crucial role for WDFY1 in bridging the TLR–TRIF interaction, which is necessary for TLR signaling.

The VP3 structural protein of foot-and-mouth disease virus inhibits the IFN-β signaling pathway

Foot‐and‐mouth disease is a frequently occurring disease of cloven‐hoofed animals that is caused by infection with the foot‐and‐mouth virus (FMDV). FMDV circumvents the type‐I IFN response by expressing proteins that antagonize cellular innate immunity, such as leader protease and 3C protease. We identified the FMDV structural protein VP3 as a negative regulator of the virus‐triggered IFN‐β signaling pathway. Expression of FMDV VP3 inhibited the Sendai virus‐triggered activation of IFN regulatory factor‐3 and the expression of retinoic acid‐inducible gene‐I/melanoma differentiation‐associated protein‐5. Transient transfection and coimmunoprecipitation confirmed that the structural protein VP3 interacts with virus‐induced signaling adapter (VISA), which is dependent on the C‐terminal aa 111–220 of VP3. In addition, we found that FMDV VP3 inhibits the expression of VISA by disrupting its mRNA. Taken together, our findings reveal a novel strategy used by the structural VP3 protein of FMDV to evade host innate immunity.—Li, D., Yang, W., Yang, F., Liu, H., Zhu, Z., Lian, K., Lei, C., Li, S., Liu, X., Zheng, H., Shu, H. The VP3 structural protein of foot‐and‐mouth disease virus inhibits the IFN‐β signaling pathway. FASEB J. 30, 1757–1766 (2016). www.fasebj.org

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.

ECSIT Bridges RIG-I-Like Receptors to VISA in Signaling Events of Innate Antiviral Responses

Upon binding to RNA structures from invading viruses, RIG-I and MDA5 are recruited to mitochondria to interact with VISA and initiate antiviral type I interferon (IFN) responses. How this process is mediated is less understood. In this report, we demonstrate that ECSIT is an essential scaffolding protein that mediates the association of VISA and RIG-I or MDA5. Overexpression of ECSIT potentiated virus-triggered activation of IFN-regulatory factor 3 (IRF3) and expression of IFNB1, whereas knockdown of ECSIT impaired viral infection-induced activation of IRF3 and expression of IFNB1 as well as cellular antiviral responses. Mechanistically, ECSIT was associated with VISA on mitochondria and important for bridging RIG-I and MDA5 to VISA. Our findings suggest that ECSIT mediates virus-triggered type I IFN induction by bridging RIG-I and MDA5 to the VISA complex, and provide new insights into the molecular events of innate antiviral immune responses.

Duck Tembusu Virus Nonstructural Protein 1 Antagonizes IFN-β Signaling Pathways by Targeting VISA

Duck Tembusu virus (DTMUV) is an emergent infectious pathogen that has caused severe disease in ducks and huge economic losses to the poultry industry in China since 2009. Previously, we showed that DTMUV inhibits IFN-β induction early in infection; however, the mechanisms of the inhibition of innate immune responses remain poorly understood. In this study, we screened DTMUV-encoded structural and nonstructural proteins using reporter assays and found that DTMUV NS1 markedly suppressed virus-triggered IFN-β expression by inhibiting retinoic acid–inducible gene I–like receptor signaling. Moreover, we found that DTMUV NS1 specifically interacted with the C-terminal domain of virus-induced signaling adaptor and impaired the association of retinoic acid–inducible gene I or melanoma differentiation-associated gene 5 and virus-induced signaling adaptor, thereby downregulating the retinoic acid–inducible gene I–like receptor–mediated signal transduction and cellular antiviral responses, leading to evasion of the innate immune response. Together, our findings reveal a novel mechanism manipulated by DTMUV to circumvent the host antiviral immune response.