Cytoskeletal Prestress Regulates RIG-I-Mediated Innate Immunity

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.

The coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), warranting urgent study of the molecular mechanisms of SARS-CoV-2 infection and host immune response. Type I interferon (IFN-I) is a key component of host innate immune system responsible for eliminating the virus at the early stage of infection. In contrast, SARS-CoV-2 has evolved multiple strategies to evade innate immune response to facilitate viral replication, transmission, and pathogenesis. This review summarizes the recent progresses on SARS-CoV-2 proteins that antagonize host IFN-I production and/or signaling. These progresses have provided knowledge for new vaccine and antiviral development to prevent and control COVID-19.

Beyond sensing: Retinoic acid inducible gene-I (RIG-I) continues to expand its antiviral effector functions.

LL-37 transports immunoreactive cGAMP to activate STING signaling and enhance interferon-mediated host antiviral immunity.

Interferon-stimulated genes and their role in controlling hepatitis C virus

TANK-binding kinase 1 (TBK1) is an important serine/threonine-protein kinase that mediates phosphorylation and nuclear translocation of IRF3, which contributes to induction of type I interferons (IFNs) in the innate antiviral response. In mammals, TBK1 spliced isoform negatively regulates the virus-triggered IFN-β signaling pathway by disrupting the interaction between retinoic acid-inducible gene I (RIG-I) and mitochondria antiviral-signaling protein (MAVS). However, it is still unclear whether alternative splicing patterns and the function of TBK1 isoform(s) exist in teleost fish. In this study, we identify two alternatively spliced isoforms of TBK1 from zebrafish, termed TBK1_tv1 and TBK1_tv2. Both TBK1_tv1 and TBK1_tv2 contain an incomplete STKc_TBK1 domain. Moreover, the UBL_TBK1_like domain is also missing for TBK1_tv2. TBK1_tv1 and TBK1_tv2 are expressed in zebrafish larvae. Overexpression of TBK1_tv1 and TBK1_tv2 inhibits RIG-I-, MAVS-, TBK1-, and IRF3-mediated activation of IFN promoters in response to spring viremia of carp virus infection. Also, TBK1_tv1 and TBK1_tv2 inhibit expression of IFNs and IFN-stimulated genes induced by MAVS and TBK1. Mechanistically, TBK1_tv1 and TBK1_tv2 competitively associate with TBK1 and IRF3 to disrupt the formation of a functional TBK1-IRF3 complex, impeding the phosphorylation of IRF3 mediated by TBK1. Collectively, these results demonstrate that TBK1 spliced isoforms are dominant negative regulators in the RIG-I/MAVS/TBK1/IRF3 antiviral pathway by targeting the functional TBK1-IRF3 complex formation. Identification and functional characterization of piscine TBK1 spliced isoforms may contribute to understanding the role of TBK1 expression in innate antiviral response.

Innate immunity and HCV

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality in the United States. Lung adenocarcinomas are highly correlated with smoking and are characterized by mutations in KRAS, EGFR, BRAF and other oncogenes. KRAS mutations are widespread in adenocarcinomas among smokers and known to be a key player in various downstream signaling pathways contributing to the tumorigenesis. Recently, a non-canonical IκB kinase, Tank Binding Kinase 1 (TBK1), has been found to contribute in KRAS mutant cancers. TBK1 has well documented functions in immune response, cell survival and in mitosis. While it has been suggested that TBK1-mediated regulation of Akt signaling might facilitate oncogenesis, the molecular mechanisms underlying TBK1 function downstream of KRAS is not fully elucidated. Yes associated protein 1 (YAP1) is an oncogenic component of the Hippo signaling cascade which could promote KRAS mediated oncogenesis and could substitute for the loss of Kras in mouse models of pancreatic cancer. In our present study, we demonstrate a unique and novel interplay between TBK1 and the oncogenic Hippo effector molecule, YAP1. YAP1 and its paralog, TAZ are known transcriptional co-activators that function to maintain organ size during development and is often activated in cancers. We find that TBK1 could physically interact with YAP1 and phosphorylate it at T110, T114, S128 and S131 residues in vitro. Knocking down (KD) or knock out (KO) of TBK1 resulted in a significant elevation of YAP1 expression at the protein level; surprisingly, without any effect at the mRNA level. Interestingly, the upregulation of YAP1 upon depletion of TBK1 was restricted to KRAS mutant NSCLC cell-lines and not in EGFR mutant cell lines. This elevation of YAP1 upon TBK1 KD was mainly observed in the nucleus; notably, there were only minimal changes in the levels of MST and LATS, raising the possibility that these changes occur independent of the classic hippo signaling pathway. Treatment with cycloheximide, an inhibitor of protein-translation, could not diminish the elevated level of YAP1 protein in the TBK1 depleted cells, indicating that the increased YAP1 level is due to enhanced protein stability, probably as a result of post-translational modification(s). Depletion of TBK1 also resulted in the induction of EMT-like features, promoting cell-migration in scratch assays and elevated the proportion of stem-like side-population cells, probably in a YAP1-dependent manner. An unbiased RNA-Seq analysis in A549 and H460 cells indicated that MAP kinase (MAPK) pathway is activated upon TBK1 KD, which might also be involved in the YAP1 protein regulation. Our recent experiments further support this argument. The in-depth molecular mechanism(s) by which TBK1 regulates YAP1 in KRAS mutant cells are under investigation, and we hypothesize that this regulatory event contributes to KRAS mediated oncogenesis in NSCLC. Citation Format: Biswarup Saha, Neha Jaiswal, Namrata Bora-Singhal, Srikumar Chellappan. Molecular interplay between Tank-binding kinase (TBK1) and Yes-associated protein (YAP1) in KRAS mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4394.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality in the United States. Lung adenocarcinomas are highly correlated with smoking and are characterized by mutations in KRAS , EGFR , BRAF and other oncogenes. KRAS mutations are widespread in adenocarcinomas among smokers and known to be a key player in various downstream signaling pathways contributing to the tumorigenesis. Recently, a non-canonical IκB kinase, Tank Binding Kinase 1 (TBK1), has been found to contribute in KRAS mutant cancers. TBK1 has well documented functions in immune response, cell survival and in mitosis. While it has been suggested that TBK1-mediated regulation of Akt signaling might facilitate oncogenesis, the molecular mechanisms underlying TBK1 function downstream of KRAS is not fully elucidated. Yes associated protein 1 (YAP1) is an oncogenic component of the Hippo signaling cascade which could promote KRAS mediated oncogenesis and could substitute for the loss of Kras in mouse models of pancreatic cancer. In our present study, we demonstrate a unique and novel interplay between TBK1 and the oncogenic Hippo effector molecule, YAP1. YAP1 and its paralog, TAZ are known transcriptional co-activators that function to maintain organ size during development and is often activated in cancers. We find that TBK1 could physically interact with YAP1 and phosphorylate it at T110, T114, S128 and S131 residues in vitro . Knocking down (KD) or knock out (KO) of TBK1 resulted in a significant elevation of YAP1 expression at the protein level; surprisingly, without any effect at the mRNA level. Interestingly, the upregulation of YAP1 upon depletion of TBK1 was restricted to KRAS mutant NSCLC cell-lines and not in EGFR mutant cell lines. This elevation of YAP1 upon TBK1 KD was mainly observed in the nucleus; notably, there were only minimal changes in the levels of MST and LATS, raising the possibility that these changes occur independent of the classic hippo signaling pathway. Treatment with cycloheximide, an inhibitor of protein-translation, could not diminish the elevated level of YAP1 protein in the TBK1 depleted cells, indicating that the increased YAP1 level is due to enhanced protein stability, probably as a result of post-translational modification(s). Depletion of TBK1 also resulted in the induction of EMT-like features, promoting cell-migration in scratch assays and elevated the proportion of stem-like side-population cells, probably in a YAP1-dependent manner. An unbiased RNA-Seq analysis in A549 and H460 cells indicated that MAP kinase (MAPK) pathway is activated upon TBK1 KD, which might also be involved in the YAP1 protein regulation. Our recent experiments further support this argument. The in-depth molecular mechanism(s) by which TBK1 regulates YAP1 in KRAS mutant cells are under investigation, and we hypothesize that this regulatory event contributes to KRAS mediated oncogenesis in NSCLC. Citation Format: Biswarup Saha, Neha Jaiswal, Namrata Bora-Singhal, Srikumar Chellappan. Molecular interplay between Tank-binding kinase (TBK1) and Yes-associated protein (YAP1) in KRAS mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4394.

Structural Basis for the Activation of Innate Immune Pattern-Recognition Receptor RIG-I by Viral RNA

Cytoplasmic RNA Sensor Pathways and Nitazoxanide Broadly Inhibit Intracellular Mycobacterium tuberculosis Growth.

Nucleic acids from bacteria or viruses induce potent immune responses in infected cells1–4. The detection of pathogen-derived nucleic acids is a central strategy by which the host senses infection and initiates protective immune responses5,6. Cyclic GMP-AMP synthase (cGAS) is a double-stranded DNA sensor7,8. It catalyzes the synthesis of cyclic GMP-AMP (cGAMP)9–12, which stimulates the induction of type I interferons (IFN-Is) through the STING-TBK1-IRF-3 signaling axis13–15. Stimulator of interferon genes (STING) oligomerizes upon cGAMP binding, leading to the recruitment and activation of tank-binding kinase 1 (TBK1)8,16. Interferon regulatory factor 3 (IRF-3) is then recruited to the signaling complex and activated by TBK18,17–20. Phosphorylated IRF-3 translocates to the nucleus and initiates the expression of IFN-Is21. However, the precise mechanisms governing STING activation by cGAMP and subsequent TBK1 activation by STING remained poorly understood. Here we show that a conserved PLPLRT/SD motif within the C-terminal tail of STING mediates the recruitment and activation of TBK1. Crystal structures of TBK1 bound to STING reveal that the PLPLRT/SD motif binds to the dimer interface of TBK1. Cell-based studies confirm that the direct interaction between TBK1 and STING is essential for IFN-β induction upon cGAMP stimulation. Moreover, we show that full-length STING oligomerizes upon cGAMP binding and highlight this as an essential step in the activation of STING-mediated signaling.

In addition to silencing specific genes, small interfering RNA (siRNA) transfection is also associated with the non-specific induction of inflammatory cytokines and type I interferon. Those so-called “off-target” effects have considerable implications for the interpretation of in vitro studies and clinical application of siRNA. The present study attempted to develop a better understanding of the mechanism involved in these off target effects. Synthesized siRNA significantly enhances DNA-mediated interferon lambda-1 response (IFN-λ1/IL-29), a newly characterized antiviral interferon in non-immune or primary immune cells. This enhancement was most pronounced by double-stranded siRNA with at least a 2-nucleotide overhang at one 3′ terminus in a dose-dependent manner, while the presence of DNA was indispensable. A pull-down assay using biotinylated siRNA- or DNA-conjugated beads indicated that retinoic acid-inducible gene I (RIG-I) and interferon gamma-inducible protein 16 (IFI16) were involved in the sensing of siRNA and DNA, respectively. Co-immunoprecipitation analysis further revealed that RIG-I and IFI16 formed a complex via siRNA, and the dissociation of IFI16 from this complex in the presence of DNA activated the downstream STING-TBK1-IRF3 (stimulator of interferon genes – tank-binding kinase 1 – interferon regulatory factor 3) pathway, shedding light on a new physiological signalling pathway to activate innate immunity. Collectively, these findings may provide rational information for siRNA-induced innate immunity, with important implications for developing siRNA-based reagents to control human diseases.

TANK-binding kinase 1 (TBK1) plays an essential role in Toll-like receptor (TLR)- and retinoic acid-inducible gene I (RIG-I)-mediated induction of type I interferon (IFN; IFN-α/β) and host antiviral responses. How TBK1 activity is negatively regulated remains largely unknown. We report that TNF receptor-associated factor (TRAF)-interacting protein (TRIP) promotes proteasomal degradation of TBK1 and inhibits TLR3/4- and RIG-I-induced IFN-β signaling. TRIP knockdown resulted in augmented activation of IFN regulatory factor 3 (IRF3) and enhanced expression of IFN-β in TLR3/4- and RIG-I-activated primary peritoneal macrophages, whereas overexpression of TRIP had opposite effects. Consistently, TRIP impaired Sendai virus (SeV) infection-induced IRF3 activation and IFN-β production and promoted vesicular stomatitis virus (VSV) replication. As an E3 ubiquitin ligase, TRIP negatively regulated the cellular levels of TBK1 by directly binding to and promoting K48-linked polyubiquitination of TBK1. Therefore, we identified TRIP as a negative regulator in TLR3/4- and RIG-I-triggered antiviral responses and suggested TRIP as a potential target for the intervention of diseases with uncontrolled IFN-β production.

ABSTRACTIntroduction: TANK-binding kinase 1 (TBK1) is vital for the induction of antiviral innate immune responses. Both RNA and DNA viral infection induces TBK1 activation, triggers phosphorylation of interferon regulatory factor (IRF) 3 and subsequent expression of type I interferons (IFNs; IFN-α/β). Type I IFNs can induce the expression of numerous antiviral genes called interferon-stimulated genes (ISGs) to build a remarkable antiviral state and limit viral replication. Thus, optimal TBK1 activity is crucial for IRF3-induced type I IFNs expression and ISGs-mediated viral elimination.Areas covered: This review provides an overview of the diverse roles of TBK1 in antiviral innate immune responses, the regulatory mechanisms of TBK1 activity and the implication in antiviral development.Expert opinion: TBK1 is a key kinase against antiviral infection via inducing type I IFNs expression. Multiple types of post-translational modifications of TBK1 tightly regulate TBK1 activity and subsequent TBK1-dependent antiviral responses. The identified regulators of TBK1 unveil regulatory mechanisms of host antiviral innate immunity and immuno-escape mechanism of virus provide strategies to control viral diseases by modulating TBK1 activity.