Virus-triggered Signaling 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.

Kinetics of interferon-λ and receptor expression in response to in vitro respiratory viral infection.

The major protective immune response against viruses is the production of type Iand III interferons (IFNs). IFNs induce the expression of hundreds of IFN-stimulated genes (ISGs) that block viral replication and further viral spread. In this report, we analyzed the expression of IFNs and some ISGs (MxA, PKR, OAS-1, IFIT-1, RIG-1, MDA5, SOCS-1) in alveolar epithelial cells (A549) in response to infection with influenza Aviruses (A/California/07/09 (H1N1pdm); A/Texas/50/12 (H3N2)); influenza Bvirus (B/Phuket/3073/13); adenovirus type 5 and 6; or respiratory syncytial virus (strain A2). Influenza Bvirus had the ability to most rapidly induce IFNs and ISGs as well as to stimulate excessive IFN-α, IFN-β and IFN-λ secretion. It seems curious that IAV H1N1pdm did not induce IFN-λ secretion, but enhanced type IIFN and interleukin (IL)-6 production. We emphasized the importance of the negative regulation of virus-triggered signaling and cellular IFN response. We showed adecrease in IFNLR1 mRNA in the case of IBV infection. The attenuation of SOCS-1 expression in IAV H1N1pdm can be considered as the inability of the system to restore the immune status. Presumably, the lack of negative feedback loop regulation of proinflammatory immune response may be afactor contributing to the particular pathogenicity of several strains of influenza. Keywords: lambda interferons; MxA; influenza; respiratory syncytial virus; A549 cells.

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.

Inducible microRNA-590-5p inhibits host antiviral response by targeting the soluble interleukin-6 (IL6) receptor

MicroRNA (miR)-590-5p has been identified as an important regulator of some signaling pathways such as cell proliferation and tumorigenesis. However, little is known about its role during viral infection. Here, we report that miR-590-5p was significantly induced by various viruses and effectively potentiated virus replication in different viral infection systems. Furthermore, miR-590-5p substantially attenuated the virus-induced expression of type I and type III interferons (IFNs) and inflammatory cytokines, resulting in impaired downstream antiviral signaling. Interleukin-6 receptor (IL6R) was identified as a target of miR-590-5p. Interestingly, the role of miR-590-5p in virus-triggered signaling was abolished in IL6R knockout cells, and this could be rescued by restoring the expression of the soluble IL6R (sIL6R) but not the membrane-bound IL6R (mIL6R), suggesting that sIL6R is indispensable for miR-590-5p in modulating the host antiviral response. Furthermore, miR-590-5p down-regulated endogenous sIL6R and mIL6R expression through a translational repression mechanism. These findings thus uncover a previously uncharacterized role and the underlying mechanism of miR-590-5p in the innate immune response to viral infection.

ZCCHC3 is a co-sensor of cGAS for dsDNA recognition in innate immune response

Cyclic GMP-AMP synthase (cGAS) senses double-strand (ds) DNA in the cytosol and then catalyzes synthesis of the second messenger cGAMP, which activates the adaptor MITA/STING to initiate innate antiviral response. How cGAS activity is regulated remains enigmatic. Here, we identify ZCCHC3, a CCHC-type zinc-finger protein, as a positive regulator of cytosolic dsDNA- and DNA virus-triggered signaling. We show that ZCCHC3-deficiency inhibits dsDNA- and DNA virus-triggered induction of downstream effector genes, and that ZCCHC3-deficient mice are more susceptible to lethal herpes simplex virus type 1 or vaccinia virus infection. ZCCHC3 directly binds to dsDNA, enhances the binding of cGAS to dsDNA, and is important for cGAS activation following viral infection. Our results suggest that ZCCHC3 is a co-sensor for recognition of dsDNA by cGAS, which is important for efficient innate immune response to cytosolic dsDNA and DNA virus.

ZDHHC11 modulates innate immune response to DNA virus by mediating MITA-IRF3 association.

MITA is a central adaptor in innate immune responses to DNA viruses. The mechanisms responsible for recruitment of downstream kinase TBK1 and the transcription factor IRF3 to MITA remains enigmatic. Here we identified ZDHHC11, a member of DHHC palmitoyl transferase family, as a positive regulator of DNA virus-triggered signaling. Overexpression of ZDHHC11 activated the IFN-β promoter, while ZDHHC11-deficiency specifically impaired DNA virus HSV-1-induced transcription of downstream antiviral genes. Zdhhc11-/- mice exhibited lower serum cytokine levels and higher lethality after HSV-1 infection. Mechanistically, ZDHHC11 facilitated the optimal recruitment of IRF3 to MITA. Our findings support an important role for ZDHHC11 in mediating MITA-dependent innate immune responses against DNA viruses.

PKACs attenuate innate antiviral response by phosphorylating VISA and priming it for MARCH5-mediated degradation.

Sensing of viral RNA by RIG-I-like receptors initiates innate antiviral response, which is mediated by the central adaptor VISA. How the RIG-I-VISA-mediated antiviral response is terminated at the late phase of infection is enigmatic. Here we identified the protein kinase A catalytic (PKAC) subunits α and β as negative regulators of RNA virus-triggered signaling in a redundant manner. Viral infection up-regulated cellular cAMP levels and activated PKACs, which then phosphorylated VISA at T54. This phosphorylation abrogated virus-induced aggregation of VISA and primed it for K48-linked polyubiquitination and degradation by the E3 ligase MARCH5, leading to attenuation of virus-triggered induction of downstream antiviral genes. PKACs-deficiency or inactivation by the inhibitor H89 potentiated innate immunity to RNA viruses in cells and mice. Our findings reveal a critical mechanism of attenuating innate immune response to avoid host damage at the late phase of viral infection by the house-keeping PKA kinase.

TRAF6 is a critical factor in fish immune response to virus infection

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is one of the key adaptor molecule in Toll-like receptor signal transduction that triggers downstream cascades involved in innate immunity. In our previous study, the molecular characteristics of EtTRAF6 (TRAF6 from Epinephelus tauvina), the tissue distributions, expression patterns after challenging with bacterial and viral pathogens were investigated. Here we identified EtTRAF6 as an important regulator of virus-triggered signaling pathway. Overexpression of EtTRAF6-ORF and truncated forms of EtTRAF6, including EtTRAF6-C (delete the MATH domain), EtTRAF6-N (delete the RING domain) and EtTRAF6-MATH, inhibited IFN-β activity strongly in grouper spleen (GS) cells. Overexpression of EtTRAF6 repressed virus-induced production of type I IFNs. When EtTRAF6 cotransfected with EcIRF3 or EcIRF7, EtTRAF6 inhibited IRF-induced activation of IFN-β. Over-expressed EtTRAF6 inhibited the transcription of SGIV genes significantly in GS cells. Although TRAF6 has a role in apoptosis regulation, it is not known if EtTRAF6 has any role in apoptosis regulation. Strikingly, when over-expressed in fathead minnow (FHM) cells, EtTRAF6 protected them from cell death induced by SGIV. Therefore, these results suggest that TRAF6 may play a critical role in their response to SGIV infection, through regulation of a cell death pathway that is common to fish and humans.

USP18 recruits USP20 to promote innate antiviral response through deubiquitinating STING/MITA.

STING (also known as MITA) mediates the innate antiviral signaling and ubiquitination of STING is key to its function. However, the deubiquitination process of STING is unclear. Here we report that USP18 recruits USP20 to deconjugate K48-linked ubiquitination chains from STING and promotes the stability of STING and the expression of type I IFNs and proinflammatory cytokines after DNA virus infection. USP18 deficiency or knockdown of USP20 resulted in enhanced K48-linked ubiquitination and accelerated degradation of STING, and impaired activation of IRF3 and NF-κB as well as induction of downstream genes after infection with DNA virus HSV-1 or transfection of various DNA ligands. In addition, Usp18-/- mice were more susceptible to HSV-1 infection compared with the wild-type littermates. USP18 did not deubiquitinate STING in vitro but facilitated USP20 to catalyze deubiquitination of STING in a manner independent of the enzymatic activity of USP18. In addition, reconstitution of STING into Usp18-/- MEFs restored HSV-1-induced expression of downstream genes and cellular antiviral responses. Our findings thus uncover previously uncharacterized roles of USP18 and USP20 in mediating virus-triggered signaling and contribute to the understanding of the complicated regulatory system of the innate antiviral responses.

The role of Interferon Stimulated Gene 56 (ISG56) in Virus-Triggered Signaling and Cellular Antiviral Response

The role of Interferon Stimulated Gene 56 (ISG56) in Virus-Triggered Signaling and Cellular Antiviral Response

DYRK2 Negatively Regulates Type I Interferon Induction by Promoting TBK1 Degradation via Ser527 Phosphorylation.

Viral infection activates the transcription factors NF-κB and IRF3, which contribute to the induction of type I interferons (IFNs) and cellular antiviral responses. Protein kinases play a critical role in various signaling pathways by phosphorylating their substrates. Here, we identified dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 2 (DYRK2) as a negative regulator of virus-triggered type I IFN induction. DYRK2 inhibited the virus-triggered induction of type I IFNs and promoted the K48-linked ubiquitination and degradation of TANK-binding kinase 1 (TBK1) in a kinase-activity-dependent manner. We further found that DYRK2 phosphorylated Ser527 of TBK1, which is essential for the recruitment of NLRP4 and for the E3 ubiquitin ligase DTX4 to degrade TBK1. These findings suggest that DYRK2 negatively regulates virus-triggered signaling by targeting TBK1 for phosphorylation and priming it for degradation, and these data provide new insights into the molecular mechanisms that dictate the cellular antiviral response.

Regulation of cellular innate antiviral signaling by ubiquitin modification.

Host pattern-recognition receptors (PRRs) recognize pathogen-associated molecular patterns generated by invading viruses and initiate a series of signaling cascades that lead to the activation of interferon-regulatory factor 3 (IRF3) and nuclear factor-κB (NF-κB) and subsequent induction of type I interferons (IFNs). Posttranslational modification of proteins by ubiquitin plays an essential role in mediating or regulating the virus-triggered PRRs-mediated signaling. Deubiquitination is the reversible process of ubiquitination and its role in regulating PRRs-mediated signaling has recently been explored. In this review, we first summarize the ubiquitination events in PRRs-mediated signaling that is triggered by viral nucleic acid and then focus on host and viral deubiquitinating enzymes-mediated regulation of virus-triggered signaling that modulates the activation of IRF3 and NF-κB and subsequent induction of type I IFNs.

Krüppel-like factor 4 negatively regulates cellular antiviral immune response.

Viral infection triggers activation of the transcription factors NF-κB and IRF3, which collaborate to induce the expression of type I interferons (IFNs) and elicit innate antiviral response. In this report, we identified Krüppel-like factor 4 (KLF4) as a negative regulator of virus-triggered signaling. Overexpression of KLF4 inhibited virus-induced activation of ISRE and IFN-β promoter in various types of cells, while knockdown of KLF4 potentiated viral infection-triggered induction of IFNB1 and downstream genes and attenuated viral replication. In addition, KLF4 was found to be localized in the cytosol and nucleus, and viral infection promoted the translocation of KLF4 from cytosol to nucleus. Upon virus infection, KLF4 was bound to the promoter of IFNB gene and inhibited the recruitment of IRF3 to the IFNB promoter. Our study thus suggests that KLF4 negatively regulates cellular antiviral response.

Systematic Analysis of the Mechanisms of Virus-Triggered Type I IFN Signaling Pathways through Mathematical Modeling

Based on biological experimental data, we developed a mathematical model of the virus-triggered signaling pathways that lead to induction of type I IFNs and systematically analyzed the mechanisms of the cellular antiviral innate immune responses, including the negative feedback regulation of ISG56 and the positive feedback regulation of IFNs. We found that the time between 5 and 48 hours after viral infection is vital for the control and/or elimination of the virus from the host cells and demonstrated that the ISG56-induced inhibition of MITA activation is stronger than the ISG56-induced inhibition of TBK1 activation. The global parameter sensitivity analysis suggests that the positive feedback regulation of IFNs is very important in the innate antiviral system. Furthermore, the robustness of the innate immune signaling network was demonstrated using a new robustness index. These results can help us understand the mechanisms of the virus-induced innate immune response at a system level and provide instruction for further biological experiments.

Innate immune responses to DNA viruses.

The innate immune system acts as the first line of defense against pathogens, which is also essential for initiation of adaptive immunity. Innate immune responses are initiated by pattern-recognition receptors (PRRs), which recognize conserved molecular structures of pathogens called pathogen-associated molecular patterns (PAMPs). The infection of virus triggers a series of signaling events leading to transcriptional induction of type I interferons (IFNs) and proinflammatory cytokines. In recent years, the mechanisms of viral RNA recognition and RNA virus-triggered signaling pathways have been well studied. However, it remains unclear on how DNA virus infection is sensed by host cells and triggers the host antiviral defense. Although ten potential viral DNA sensors have been reported, none of them is validated as a generally used sensor for distinct DNA viruses in divergent cell types and animals. Here, we provide a summary and perspective on recent advances in innate immune responses to DNA viruses.

Modeling and Dynamical Analysis of Virus-Triggered Innate Immune Signaling Pathways

The investigation of the dynamics and regulation of virus-triggered innate immune signaling pathways at a system level will enable comprehensive analysis of the complex interactions that maintain the delicate balance between resistance to infection and viral disease. In this study, we developed a delayed mathematical model to describe the virus-induced interferon (IFN) signaling process by considering several key players in the innate immune response. Using dynamic analysis and numerical simulation, we evaluated the following predictions regarding the antiviral responses: (1) When the replication ratio of virus is less than 1, the infectious virus will be eliminated by the immune system’s defenses regardless of how the time delays are changed. (2) The IFN positive feedback regulation enhances the stability of the innate immune response and causes the immune system to present the bistability phenomenon. (3) The appropriate duration of viral replication and IFN feedback processes stabilizes the innate immune response. The predictions from the model were confirmed by monitoring the virus titer and IFN expression in infected cells. The results suggest that the balance between viral replication and IFN-induced feedback regulation coordinates the dynamical behavior of virus-triggered signaling and antiviral responses. This work will help clarify the mechanisms of the virus-induced innate immune response at a system level and provide instruction for further biological experiments.

Regulation of Virus-triggered Signaling by OTUB1- and OTUB2-mediated Deubiquitination of TRAF3 and TRAF6

Ubiquitination and deubiquitination have emerged as critical post-translational regulatory mechanisms for activation or attenuation of the virus-triggered type I interferon (IFN)(2) induction pathways. In this study, we identified two deubiquitinating enzymes, OTUB1 and OTUB2, as negative regulators of virus-triggered type I IFN induction. Overexpression of OTUB1 and OTUB2 inhibited virus-induced activation of IRF3 and NF-kappaB, transcription of the IFNB1 gene as well as cellular antiviral response, whereas knockdown of OTUB1 and OTUB2 had opposite effects. Coimmunoprecipitations indicated OTUB1 and -2 interacted with TRAF3 and TRAF6, two E3 ubiquitin ligases required for virus-triggered IRF3 and NF-kappaB activation, respectively. Furthermore, we found that OTUB1 and OTUB2 mediated virus-triggered deubiquitination of TRAF3 and -6. These findings suggest that OTUB1 and OTUB2 negatively regulate virus-triggered type I IFN induction and cellular antiviral response by deubiquitinating TRAF3 and -6.

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.

Negative Regulation of Virus-triggered IFN-β Signaling Pathway by Alternative Splicing of TBK1

Induction of Type I IFNs is a central event in antiviral responses and must be tightly controlled. The protein kinase TBK1 is critically involved in virus-triggered type I IFN signaling. In this study, we identify an alternatively spliced isoform of TBK1, termed TBK1s, which lacks exons 3-6. Upon Sendai virus (SeV) infection, TBK1s is induced in both human and mouse cells and binds to RIG-1, disrupting the interaction between RIG-I and VISA. Consistent with that result, overexpression of TBK1s inhibits IRF3 nuclear translocation and leads to a shutdown of SeV-triggered IFN-beta production. Taken together, our data indicate that TBK1s plays an inhibitory role in virus-triggered IFN-beta signaling pathways.