The RNA-binding protein ZFP36 strengthens innate antiviral signaling by targeting RIG-I for K63-linked ubiquitination.

Innate immunity is the first line of defense against infections, which functions as a significant role in resisting pathogen invasion. Rapid immune response is initiated by pattern recognition receptors (PRRs) quickly distinguishing "self" and "non-self." Upon evolutionarily conserved pathogen-associated molecular pattern (PAMP) is recognized by PRRs, innate immune response against infection is triggered via an orchestration of molecular interaction, cytokines cascades, and immune cells. RIG-I plays a critical role in type I interferon (IFN-I) production by direct recognition of cytoplasmic double-stranded viral RNA. However, the activation mechanism of RIG-I is incompletely understood. In this study, we reported RNA-binding protein ZFP36 as a positive regulator of RIG-I-mediated IFN-I production. ZFP36 is a member of Zinc finger proteins (ZFPs) characterized by the zinc finger (ZnF) motif, which broadly involved gene transcription and signal transduction. However, its role in regulating antiviral innate immune signaling is still unclear. We found that ZFP36 associates with RIG-I and potentiates the FN-ß production induced by SeV. Mechanistically, ZFP36 promotes K63-linked polyubiquitination of RIG-I, mostly at K154/K164/K172, thereby facilitating the activation of RIG-I during infection. While the mutant ZFP36 (C118S/C162S) failed to increase polyubiquitination of RIG-I and SeV induced FN-ß. Our findings collectively demonstrated that ZFP36 acts as a positive regulator in antiviral innate immunity by targeting RIG-I for K63-linked ubiquitination, thus improving our understanding of the activation mechanism of RIG-I.

TRAF7 negatively regulates the RLR signaling pathway by facilitating the K48-linked ubiquitination of TBK1.

TANK-binding kinase 1 (TBK1) is a nodal protein involved in multiple signal transduction pathways. In RNA virus-mediated innate immunity, TBK1 is recruited to the prion-like platform formed by MAVS and subsequently activates the transcription factors IRF3/7 and NF-κB to produce type I interferon (IFN) and proinflammatory cytokines for the signaling cascade. In this study, TRAF7 was identified as a negative regulator of innate immune signaling. TRAF7 interacts with TBK1 and promotes K48-linked polyubiquitination and degradation of TBK1 through its RING domain, impairing the activation of IRF3 and the production of IFN-ß. In addition, we found that the conserved cysteine residues at position 131 of TRAF7 are necessary for its function toward TBK1. Knockout of TRAF7 could facilitate the activation of IRF3 and increase the transcript levels of downstream antiviral genes. These data suggest that TRAF7 negatively regulates innate antiviral immunity by promoting the K48-linked ubiquitination of TBK1.

BAG6 negatively regulates the RLR signaling pathway by targeting VISA/MAVS.

The virus-induced signaling adaptor protein VISA (also known as MAVS, ISP-1, Cardif) is a critical adaptor protein in the innate immune response to RNA virus infection. Upon viral infection, VISA self-aggregates to form a sizeable prion-like complex and recruits downstream signal components for signal transduction. Here, we discover that BAG6 (BCL2-associated athanogene 6, formerly BAT3 or Scythe) is an essential negative regulator in the RIG-I-like receptor signaling pathway. BAG6 inhibits the aggregation of VISA by promoting the K48-linked ubiquitination and specifically attenuates the recruitment of TRAF2 by VISA to inhibit RLR signaling. The aggregation of VISA and the interaction of VISA and TRAF2 are enhanced in BAG6-deficient cell lines after viral infection, resulting in the enhanced transcription level of downstream antiviral genes. Our research shows that BAG6 is a critical regulating factor in RIG-I/VISA-mediated innate immune response by targeting VISA.

SOX9 negatively regulates the RLR antiviral signaling by targeting MAVS.

Mitochondrial virus-induced signal adaptor (MAVS), also known as VISA, IPS-1, and Cardif, is a crucial adaptor protein in the RIG-I-like receptor (RLR) signaling pathway. Upon viral infection, RIG-I recognizes viral dsRNA and further transfers it to mitochondria, where it binds to MAVS through its CARD domain, generating a series of signal cascades. Transduction through this signaling cascade leads to phosphorylation and nuclear translocation of interferon regulatory factor 3/7 (IRF3/IRF7) and activation of NF-κB, which ultimately produces type I interferon (IFN) and proinflammatory cytokines. Here, our experiments demonstrated that overexpression of SRY-related high-mobility group protein 9 (SOX9) significantly inhibited Sendai virus (SeV)-induced and MAVS-mediated activation of the IFN-ß promoter and ISRE. However, knocking out the expression of SOX9 in cells promoted SeV-induced IFN-ß promoter and ISRE activation. Further studies have shown that SOX9 interacts with MAVS and targets MAVS to inhibit the association of MAVS-TRAF2, thereby inhibiting MAVS-mediated TRAF2 ubiquitination. Taken together, these results indicate that SOX9 downregulates IFN-ß expression and antiviral signal transduction by targeting MAVS.

The Kinase MAP4K1 Inhibits Cytosolic RNA-Induced Antiviral Signaling by Promoting Proteasomal Degradation of TBK1/IKKε.

TANK-binding kinase 1 (TBK1)/IκB kinase-ε (IKKε) mediates robust production of type I interferons (IFN-I) and proinflammatory cytokines in response to acute viral infection. However, excessive or prolonged production of IFN-I is harmful and even fatal to the host by causing autoimmune disorders. In this study, we identified mitogen-activated protein kinase kinase kinase kinase 1 (MAP4K1) as a negative regulator in the RIG-I-like receptor (RLR) signaling pathway. MAP4K1, a member of Ste20-like serine/threonine kinases, was previously known as a prominent regulator in adaptive immunity by downregulating T-cell receptor (TCR) signaling and B-cell receptor (BCR) signaling. However, its role in regulating antiviral innate immune signaling is still unclear. This study reports an undiscovered role of MAP4K1, which inhibits RLR signaling by targeting TBK1/IKKε for proteasomal degradation via the ubiquitin ligase DTX4. We initially identify MAP4K1 as an interacting partner of TBK1 by yeast two-hybrid screens and subsequently investigate its function in RLR-mediated antiviral signaling pathways. Overexpression of MAP4K1 significantly inhibits RNA virus-triggered activation of IFN-ß and the production of proinflammatory cytokines. Consistently, knockdown or knockout experiments show opposite effects. Furthermore, MAP4K1 promotes the degradation of TBK1/IKKε by K48-linked ubiquitination via DTX4. Knockdown of DTX4 abrogated the ubiquitination and degradation of TBK1/IKKε. Collectively, our results identify that MAP4K1 acts as a negative regulator in antiviral innate immunity by targeting TBK1/IKKε, discover a novel TBK1 inhibitor, and extend a novel functional role of MAP4K1 in immunity. IMPORTANCE TANK-binding kinase 1 (TBK1)/IκB kinase-ε (IKKε) mediates robust production of type I interferons (IFN-I) and proinflammatory cytokines to restrict the spread of invading viruses. However, excessive or prolonged production of IFN-I is harmful to the host by causing autoimmune disorders. In this study, we identified that mitogen-activated protein kinase kinase kinase kinase 1 (MAP4K1) is a negative regulator in the RLR signaling pathway. Notably, MAP4K1 promotes the degradation of TBK1/IKKε by K48-linked ubiquitination via the ubiquitin ligase DTX4, leading to the negative regulation of the IFN signaling pathway. Previous studies showed that MAP4K1 has a pivotal function in adaptive immune responses. This study identifies that MAP4K1 also plays a vital role in innate immunity and outlines a novel mechanism by which the IFN signaling pathway is tightly controlled to avoid excessive inflammation. Our study documents a novel TBK1 inhibitor, which serves as a potential therapeutic target for autoimmune diseases, and elucidated a significant function for MAP4K1 linked to innate immunity in addition to subsequent adaptive immunity.

N4BP3 Regulates RIG-I-Like Receptor Antiviral Signaling Positively by Targeting Mitochondrial Antiviral Signaling Protein.

Mitochondrial antiviral signaling protein (MAVS), an adaptor protein, is activated by RIG-I, which is critical for an effective innate immune response to infection by various RNA viruses. Viral infection causes the RIG-I-like receptor (RLR) to recognize pathogen-derived dsRNA and then becomes activated to promote prion-like aggregation and activation of MAVS. Subsequently, through the recruitment of TRAF proteins, MAVS activates two signaling pathways mediated by TBK1-IRF3 and IKK- NF-κb, respectively, and turns on type I interferon and proinflammatory cytokines. This study discovered that NEDD4 binding protein 3 (N4BP3) is a positive regulator of the RLR signaling pathway by targeting MAVS. Overexpression of N4BP3 promoted virus-induced activation of the interferon-ß (IFN-ß) promoter and interferon-stimulated response element (ISRE). Further experiments showed that knockdown or knockout N4BP3 impaired RIG-I-like receptor (RLR)-mediated innate immune response, induction of downstream antiviral genes, and cellular antiviral responses. We also detected that N4BP3 could accelerate the interaction between MAVS and TRAF2. Related experiments revealed that N4BP3 could facilitate the ubiquitination modification of MAVS. These findings suggest that N4BP3 is a critical component of the RIG-I-like receptor (RLR)-mediated innate immune response by targeting MAVS, which also provided insight into the mechanisms of innate antiviral responses.

HSPBP1 facilitates cellular RLR-mediated antiviral response by inhibiting the K48-linked ubiquitination of RIG-I.

Retinoic acid-inducible gene I (RIG-I) plays a critical role in the recognition of intracytoplasmic viral RNA. Upon binding to the RNA of invading viruses, the activated RIG-I translocates to mitochondria, where it recruits adapter protein MAVS, causing a series of signaling cascades. In this study, we demonstrated that Hsp70 binding protein 1 (HSPBP1) promotes RIG-I-mediated signal transduction. The overexpression of HSPBP1 can increase the stability of RIG-I protein by inhibiting its K48-linked ubiquitination, and promote the activation of IRF3 and the production of IFN-ß induced by Sendai virus. Knockdown and knockout of HSPBP1 leads to down-regulation of virus-induced RIG-I expression, inhibits IRF3 activation, and reduces the production of IFNB1. These results indicate that HSPBP1 positively regulates the antiviral signal pathway induced by inhibiting the K48-linked ubiquitination of RIG-I.

Mitochondrial DUT-M potentiates RLR-mediated antiviral signaling by enhancing VISA and TRAF2 association.

Upon recognition of intracytoplasmic viral RNA, activated RIG-I is recruited to the mitochondrion-located adaptor protein VISA (also known as MAVS, CARDIF, and IPS-1). VISA then acts as a central signaling platform for linking RIG-I and downstream signaling components, such as TRAF2, 5, and 6, TBK1, and IKK, leading to activation of the kinases TBK1 and IKK. These activated kinases further phosphorylate the transcription factors IRF3/7 and NF-κB, leading to the induction of downstream antiviral genes. Here, we report a mitochondrial isoform, deoxyuridine triphosphate nucleotidohydrolase (dUTPase), DUT-M, as a positive regulator in RLR-VISA-mediated antiviral signaling. DUT-M interacts with VISA and RIG-I to facilitate the assembly of the VISA-TRAF2 complex and to augment the polyubiquitination of TRAF2, leading to potentiated activation of IRF3 dimerization and phosphorylation of P65, and enhanced VISA-mediated innate immune response. RLR-VISA-mediated IRF3 dimerization and P65 phosphorylation, were inhibited in DUT-knockdown and DUT-deficient 293 cells. Thus, DUT-M is a positive regulator of the RIG-I-VISA-mediated innate immune response to RNA viruses.

SNX5 inhibits RLR-mediated antiviral signaling by targeting RIG-I-VISA signalosome.

Upon invading the cell, the viral RNA is recognized by the RIG-I receptor located in the cytoplasm, causing the RIG-I receptor to be activated. The activated RIG-I receptor transmits downstream antiviral signals by interacting with the adaptor protein VISA located on the mitochondria, leading to the production of type Ⅰ interferons and crude inflammatory cytokine genes. Although there have been many studies on antiviral signal transduction of RIG-I receptors in recent years, the mechanism of RIG-I-VISA-mediated antiviral regulation is still not fully understood. In this study, we identified SNX5 as a negative regulator of RLR-mediated antiviral signaling. Our results show that overexpression of SNX5 inhibits viral-induced activation of the IFN-ß promoter, ISRE, NF-κB, and IRF3, whereas RNAi knockdown of SNX5 expression shows opposite results. We also found that overexpression of SNX5 enhanced RIG-I's K48 ubiquitination and attenuated its K63 ubiquitination, resulting in inhibition of virus-induced RIG-I expression. Besides, further studies show that SNX5 overexpression weakens the interaction between VISA and TRAF2/5. Our findings suggest that SNX5 negatively regulates RLR-mediated antiviral signaling by targeting the RIG-I-VISA signalosome and provide new evidence for the negative regulation of RIG-I-mediated innate immune response mechanisms.

CHID1 positively regulates RLR antiviral signaling by targeting the RIG-I/VISA signalosome.

Retinoic acid-inducible gene-I (RIG-I) belongs to the RIGI-like receptors (RLRs), a class of primary pattern recognition receptors. It senses viral double-strand RNA in the cytoplasm and delivers the activated signal to its adaptor virus-induced signaling adapter (VISA), which then recruits the downstream TNF receptor-associated factors and kinases, triggering a downstream signal cascade that leads to the production of proinflammatory cytokines and antiviral interferons (IFNs). However, the mechanism of RIG-I-mediated antiviral signaling is not fully understood. Here, we demonstrate that chitinase domain-containing 1 (CHID1), a member of the chitinase family, positively regulates the RLR antiviral signaling pathway by targeting the RIG-I/VISA signalosome. CHID1 overexpression enhances the activation of nuclear factor κB (NF-кB) and interferon regulatory factor 3 (IRF3) triggered by Sendai virus (SeV) by promoting the polyubiquitination of RIG-I and VISA, thereby potentiating IFN-ß production. CHID1 knockdown in human 239T cells inhibits SeV-induced activation of IRF3 and NF-κB and the induction of IFN-ß. These results indicate that CHID1 positively regulates RLR antiviral signal, revealing the novel mechanism of the RIG-I antiviral signaling pathway.

TARBP2 inhibits IRF7 activation by suppressing TRAF6-mediated K63-linked ubiquitination of IRF7.

Interferon regulatory factor 7 (IRF7), a crucial regulator of type I interferons (IFNs), plays a crucial role in resistance to viral infection. The abnormal production of type I IFNs is associated with many types of disease, such as cancer and inflammatory disorders. Thus, understanding the post-translational modifications of IRF7 is essential to promoting an appropriate immune response. We have recently showed that the TAR RNA binding protein 2 (TARBP2) suppresses IFN-ß production and the innate antiviral response by targeting MAVS. Here, we further identified TARBP2 as a novel inhibitor of IRF7, which inhibits IRF7-mediated IFN-ß production triggered by the Sendai virus in 293 T cells. Overexpression of TARBP2 inhibits the phosphorylation as well as the K63-linked ubiquitination of IRF7, whilst TARBP2 also impairs the stability of endogenous TRAF6. Furthermore, TARBP2 participates in the interaction between IRF7 and TRAF6, thereby suppressing TRAF6-mediated K63-linked ubiquitination of IRF7, which is a prerequisite of IRF7 phosphorylation. Our findings further reveal the mechanism by which TARBP2 regulates the antiviral signaling pathways of the innate immune system.

THO Complex Subunit 7 Homolog Negatively Regulates Cellular Antiviral Response against RNA Viruses by Targeting TBK1.

RNA virus invasion induces a cytosolic RIG-I-like receptor (RLR) signaling pathway by promoting assembly of the Mitochondrial antiviral-signaling protein (MAVS) signalosome and triggers the rapid production of type I interferons (IFNs) and proinflammatory cytokines. During this process, the pivotal kinase TANK binding kinase 1 (TBK1) is recruited to the MAVS signalosome to transduce a robust innate antiviral immune response by phosphorylating transcription factors interferon regulatory factor 3 (IRF3) and nuclear factor (NF)-κB and promoting their nuclear translocation. However, the molecular mechanisms underlying the negative regulation of TBK1 are largely unknown. In the present study, we found that THO complex subunit 7 homolog (THOC7) negatively regulated the cellular antiviral response by promoting the proteasomal degradation of TBK1. THOC7 overexpression potently inhibited Sendai virus- or polyI:C-induced IRF3 dimerization and phosphorylation and IFN-ß production. In contrast, THOC7 knockdown had the opposite effects. Moreover, we simulated a node-activated pathway to show that THOC7 regulated the RIG-I-like receptors (RLR)-/MAVS-dependent signaling cascade at the TBK1 level. Furthermore, THOC7 was involved in the MAVS signalosome and promoted TBK1 degradation by increasing its K48 ubiquitin-associated polyubiquitination. Together, these findings suggest that THOC7 negatively regulates type I IFN production by promoting TBK1 proteasomal degradation, thus improving our understanding of innate antiviral immune responses.

RACK1 attenuates RLR antiviral signaling by targeting VISA-TRAF complexes.

Virus-induced signaling adaptor (VISA), which mediates the production of type I interferon, is crucial for the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) signaling pathway. Upon viral infection, RIG-I recognizes double-stranded viral RNA and interacts with VISA to mediate antiviral innate immunity. However, the mechanisms underlying RIG/VISA-mediated antiviral regulation remain unclear. In this study, we confirmed that receptor for activated C kinase 1 (RACK1) interacts with VISA and attenuates the RIG/VISA-mediated antiviral innate immune signaling pathway. Overexpression of RACK1 inhibited the interferon-ß (IFN-ß) promoter; interferon-stimulated response element (ISRE); nuclear factor kappa B (NF-κB) activation; and dimerization of interferon regulatory factor 3 (IRF3) mediated by RIG-I, VISA, and TANK-binding kinase 1 (TBK1). A reduction in RACK1 expression level upon small interfering RNA knockdown increased RIG/VISA-mediated antiviral transduction. Additionally, RACK1 disrupted formation of the VISA-tumor necrosis factor receptor-associated factor 2 (TRAF2), VISA-TRAF3, and VISA-TRAF6 complexes during RIG-I/VISA-mediated signal transduction. Additionally, RACK1 enhanced K48-linked ubiquitination of VISA, attenuated its K63-linked ubiquitination, and decreased VISA-mediated antiviral signal transduction. Together, these results indicate that RACK1 interacts with VISA to repress downstream signaling and downregulates virus-induced IFN-ß production in the RIG-I/VISA signaling pathway.

FKBP8 inhibits virus-induced RLR-VISA signaling.

The mitochondrial antiviral signal protein mitochondrial antiviral signaling protein, also known as virus-induced signaling adaptor (VISA), plays a key role in regulating host innate immune signaling pathways. This study identifies FK506 binding protein 8 (FKBP8) as a candidate interacting protein of VISA through the yeast two-hybrid technique. The interaction of FKBP8 with VISA, retinoic acid inducible protein 1 (RIG-I), and IFN regulatory factor 3 (IRF3) was confirmed during viral infection in mammalian cells by coimmunoprecipitation. Overexpression of FKBP8 using a eukaryotic expression plasmid significantly attenuated Sendai virus-induced activation of the promoter interferons ß (IFN-ß), and transcription factors nuclear factor κ-light chain enhancer of activated B cells (NF-κB) and IFN-stimulated response element (ISRE). Overexpression of FKBP8 also decreased dimer-IRF3 activity, but enhanced virus replication. Conversely, knockdown of FKBP8 expression by RNA interference showed opposite effects. Further studies indicated that FKBP8 acts as a negative interacting partner to regulate RLR-VISA signaling by acting on VISA and TANK binding kinase 1 (TBK1). Additionally, FKBP8 played a negative role on virus-induced signaling by inhibiting the formation of TBK1-IRF3 and VISA-TRAF3 complexes. Notably, FKBP8 also promoted the degradation of TBK1, RIG-I, and TRAF3 resulting from FKBP8 reinforced Sendai virus-induced endogenous polyubiquitination of RIG-I, TBK1, and TNF receptor-associated factor 3 (TRAF3). Therefore, a novel function of FKBP8 in innate immunity antiviral signaling regulation was revealed in this study.

TARBP2 negatively regulates IFN-ß production and innate antiviral response by targeting MAVS.

MAVS as an essential receptor protein for anti-virus innate immunity plays an important role in the production of virus-induced typeⅠ interferon and regulation of interferon regulatory factor 3/7. Understanding the MAVS-mediated antiviral signaling pathway can provide detailed insights. In this study, we identify transactivation response element RNA-binding protein (TARBP2), as an inhibitor of the cellular protein kinase PKR, negatively regulates virus -induced IFN-ß production by targets MAVS. Overexpression of TARBP2 inhibits virus-induced IFN-ß production as well as cellular antiviral response. Then knockdown of TARBP2 inhibited virus-induced IFN-ß signaling. Further studies demonstrated that TARBP2 interacted with MAVS and targeted MAVS to abrogate MAVS-RIG-I and MAVS-TRAF3 association. Our findings suggest that TARBP2 is an important non-redundant virus-mediated negative regulator of typeⅠ interferon.

HAUS8 regulates RLR­VISA antiviral signaling positively by targeting VISA.

Mitochondrial anti­viral signaling protein (VISA), additionally termed MAVS, IPS­1 and Cardif, is located at the outer membrane of mitochondria and is an essential adaptor in the Rig­like receptor (RLRs) signaling pathway. Upon viral infection, activated RLRs interact with VISA on mitochondria, forming a RLR­VISA platform, leading to the recruitment of different TRAF family members, including TRAF3, TRAF2 and TRAF6. This results in the phosphorylation and nuclear translocation of interferon regulatory factors 3 and 7 (IRF3/IRF7) by TANK binding kinase 1 (TBK1) and/or IKKε, as well as activation of NF­κB, to induce type I interferons (IFNs) and pro­inflammatory cytokines. It remains to be elucidated how VISA functions as a scaffold for protein complex assembly in mitochondria to regulate RLR­VISA antiviral signaling. In the present study, it was demonstrated that HAUS augmin like complex subunit 8 (HAUS8) augments the RLR­VISA­dependent antiviral signaling pathway by targeting the VISA complex. Co­immunoprecipitation verified that HAUS8 was associated with VISA and the VISA signaling complex components retinoic acid­inducible gene I (RIG­I) and TBK1 when the RLR­VISA signaling pathway was activated. The data demonstrated that overexpression of HAUS8 significantly promoted the activity of the transcription factors NF­κB, IRF3 and the IFN­ß promoter induced by Sendai virus­mediated RLR­VISA signaling. HAUS8 increased the polyubiquitination of VISA, RIG­I and TBK1. Knockdown of HAUS8 inhibited the activation of the transcription factors IRF­3, NF­κB and the IFN­ß promoter triggered by Sendai virus. Collectively, these results demonstrated that HAUS8 may function as a positive regulator of RLR­VISA dependent antiviral signaling by targeting the VISA complex, providing a novel regulatory mechanism of antiviral responses.

Sec13 is a positive regulator of VISA-mediated antiviral signaling.

Viral infection triggers the innate antiviral immune response that rapidly produces type I interferons in most cell types to combat viruses invading. Upon viral infection, the cytoplasmic RNA sensors RIG-I/MDA5 recognize viral RNA, and then RIG-I/MDA5 is transported to mitochondria interacting with VISA through the CARD domain. From there, VISA recruits downstream antiviral signaling pathways molecules, such as TRAFs and TBK1. Eventually, IRF3 is phosphorylated and type I IFNs are induced to fight as the first line of defense against viruses. However, it remains unclear how VISA acts as a scaffold to assemble the signalosome in RIG-I-mediated antiviral signaling. Here, we demonstrated Sec13 as a novel component that was involved in VISA-mediated antiviral signaling pathway. The co-immunoprecipitation assays showed that Sec13 specifically interacts with VISA. Overexpression of Sec13 increases VISA's aggregation and ubiquitination and significantly enhances the phosphorylation and dimerization of IRF3, facilitating the IFN-ß production. Conversely, the knockdown of Sec13 attenuates Sendai virus-induced and VISA-mediated IRF3 activation and the production of IFNß, thus weakens antiviral immune activity.

RELT family members activate p38 and induce apoptosis by a mechanism distinct from TNFR1.

Receptor Expressed in Lymphoid Tissues (RELT) is a human Tumor Necrosis Factor Receptor (TNFR) family member that has two identified homologous binding partners, RELL1 and RELL2. This study sought to further understand the pattern of RELT expression, the functional role of RELT family members, and the mechanism of RELT-induced apoptosis. RELT protein expression was detected in the spleen, lymph node, brain, breast and peripheral blood leukocytes (PBLs). A smaller than expected size of RELT was observed in PBLs, suggesting a proteolytically cleaved form of RELT. RELL1 and RELL2 overexpression activated the p38 MAPK pathway more substantially than RELT in HEK-293 cells, and this activation of p38 by RELT family members was blocked by dominant-negative mutant forms of OSR1 or TRAF2, implicating these molecules in RELT family member signaling. RELT was previously shown to induce apoptosis in human epithelial cells despite lacking the characteristic death domain (DD) found in other TNFRs. Seven deletion mutants of RELT that lacked differing portions of the intracellular domain were created to assess whether RELT possesses a novel DD. None of the deletion mutants induced apoptosis as efficiently as full-length RELT, a result that is consistent with a novel DD being located at the carboxyl-terminus. Interestingly, induction of apoptotic morphology by RELT overexpression was not prevented when signaling by FADD or Caspase-8 was blocked, indicating RELT induces apoptosis by a pathway distinct from other death-inducing TNFRs such as TNFR1. Collectively, this study provides more insights into RELT expression, RELT family member function, and the mechanism of RELT-induced death.

VISA is required for B cell expression of TLR7.

B cells play a critical role in the initialization and development of the systemic lupus erythematosus that is dependent on the expression of the endosomal ssRNA receptor TLR7. Previous studies have established that B cell expression of TLR7 is controlled by the type I IFN secreted by plasmacytoid dendritic cells. In this article, we report that VISA, also known as MAVS, IPS-1, and CardIf, essential for RIG-I/MDA5-mediated signaling following sensing of cytosolic RNA, regulate B cell expression of TLR7 and CD23. We found that B cells from a VISA(-/-) mouse express reduced TLR7 but normal basal levels of type I IFN. We also show that although IFN-ß and TLR7 agonists synergize to promote TLR7 expression in VISA(-/-) B cells, they do not fully complement the defect seen in VISA(-/-) cells. Cell transfer experiments revealed that the observed effects of VISA(-/-) are B cell intrinsic. The reduced TLR7 expression in B cells is correlated with impaired TLR7 agonist-induced upregulation of activation markers CD69 and CD86, cell proliferation, production of IFN-α, TNF, and IL-12, and NF-κB activation. Finally, studies indicate that genetic background may influence the observed phenotype of our VISA(-/-) mice, because VISA(-/-) B cells differ in CD23 and TLR7 expression when on C57BL/6 versus 129Sv-C57BL/6 background. Thus, our findings suggest an unexpected link between VISA-mediated cytosolic RLR signaling and autoimmunity.