Replication of lentiviruses generates different DNA forms, including RNA:DNA hybrids, ssDNA, and dsDNA. Nucleic acids stimulate innate immune responses, and pattern recognition receptors detecting dsDNA have been identified. However, sensors for ssDNA have not been reported, and the ability of RNA:DNA hybrids to stimulate innate immune responses is controversial. Using ssDNAs derived from HIV-1 proviral DNA, we report that this DNA form potently induces the expression of IFNs in primary human macrophages. This response was stimulated by stem regions in the DNA structure and was dependent on IFN-inducible protein 16 (IFI16), which bound immunostimulatory DNA directly and activated the stimulator of IFN genes -TANK-binding kinase 1 - IFN regulatory factors 3/7 (STING-TBK1-IRF3/7) pathway. Importantly, IFI16 colocalized and associated with lentiviral DNA in the cytoplasm in macrophages, and IFI16 knockdown in this cell type augmented lentiviral transduction and also HIV-1 replication. Thus, IFI16 is a sensor for DNA forms produced during the lentiviral replication cycle and regulates HIV-1 replication in macrophages.

Type I interferons (IFNs) are critical mediators of antiviral defense, but their elicitation by bacterial pathogens can be detrimental to hosts. Many intracellular bacterial pathogens, including Mycobacterium tuberculosis, induce type I IFNs following phagosomal membrane perturbations. Cytosolic M. tuberculosis DNA has been implicated as a trigger for IFN production, but the mechanisms remain obscure. We report that the cytosolic DNA sensor, cyclic GMP-AMP synthase (cGAS), is required for activating IFN production via the STING/TBK1/IRF3 pathway during M. tuberculosis and L. pneumophila infection of macrophages, whereas L. monocytogenes short-circuits this pathway by producing the STING agonist, c-di-AMP. Upon sensing cytosolic DNA, cGAS also activates cell-intrinsic antibacterial defenses, promoting autophagic targeting of M. tuberculosis. Importantly, we show that cGAS binds M. tuberculosis DNA during infection, providing direct evidence that this unique host-pathogen interaction occurs in vivo. These data uncover a mechanism by which IFN is likely elicited during active human infections.

Upon detection of viral RNA, the helicases RIG-I and/or MDA5 trigger, via their adaptor Cardif (also known as IPS-1, MAVS, or VISA), the activation of the transcription factors NF-kappaB and IRF3, which collaborate to induce an antiviral type I interferon (IFN) response. FADD and RIP1, known as mediators of death-receptor signaling, are implicated in this antiviral pathway; however, the link between death-receptor and antiviral signaling is not known. Here we showed that TRADD, a crucial adaptor of tumor necrosis factor receptor (TNFRI), was important in RIG-like helicase (RLH)-mediated signal transduction. TRADD is recruited to Cardif and orchestrated complex formation with the E3 ubiquitin ligase TRAF3 and TANK and with FADD and RIP1, leading to the activation of IRF3 and NF-kappaB. Loss of TRADD prevented Cardif-dependent activation of IFN-beta, reduced the production of IFN-beta in response to RNA viruses, and enhanced vesicular stomatitis virus replication. Thus, TRADD is not only an essential component of proinflammatory TNFRI signaling, but is also required for RLH-Cardif-dependent antiviral immune responses.

Poxviruses encode proteins that suppress host immune responses, including secreted decoy receptors for pro-inflammatory cytokines such as interleukin-1 (IL-1) and the vaccinia virus proteins A46R and A52R that inhibit intracellular signaling by members of the IL-1 receptor (IL-1R) and Toll-like receptor (TLR) family. In vivo, the TLRs mediate the innate immune response by serving as pathogen recognition receptors, whose oligomerized intracellular Toll/IL-1 receptor (TIR) domains can initiate innate immune signaling. A family of TIR domain-containing adapter molecules transduces signals from engaged receptors that ultimately activate NF-kappaB and/or interferon regulatory factor 3 (IRF3) to induce pro-inflammatory cytokines. Data base searches detected a significant similarity between the N1L protein of vaccinia virus and A52R, a poxvirus inhibitor of TIR signaling. Compared with other poxvirus virulence factors, the poxvirus N1L protein strongly affects virulence in vivo; however, the precise target of N1L was previously unknown. Here we show that N1L suppresses NF-kappaB activation following engagement of Toll/IL-1 receptors, tumor necrosis factor receptors, and lymphotoxin receptors. N1L inhibited receptor-, adapter-, TRAF-, and IKK-alpha and IKK-beta-dependent signaling to NF-kappaB. N1L associated with several components of the multisubunit I-kappaB kinase complex, most strongly associating with the kinase, TANK-binding kinase 1 (TBK1). Together these findings are consistent with the hypothesis that N1L disrupts signaling to NF-kappaB by Toll/IL-1Rs and TNF superfamily receptors by targeting the IKK complex for inhibition. Furthermore, N1L inhibited IRF3 signaling, which is also regulated by TBK1. These studies define a role for N1L as an immunomodulator of innate immunity by targeting components of NF-kappaB and IRF3 signaling pathways.

Stimulator of interferon genes (STING) is a cytosolic receptor that senses both exogenous and endogenous cytosolic cyclic dinucleotides (CDNs), activating TBK1/IRF3 (interferon regulatory factor 3), NF-κB (nuclear factor κB), and STAT6 (signal transducer and activator of transcription 6) signaling pathways to induce robust type I interferon and proinflammatory cytokine responses. CDN ligands were formulated with granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing cellular cancer vaccines--termed STINGVAX--that demonstrated potent in vivo antitumor efficacy in multiple therapeutic models of established cancer. We found that rationally designed synthetic CDN derivative molecules, including one with an Rp,Rp dithio diastereomer and noncanonical c[A(2',5')pA(3',5')p] phosphate bridge structure, enhanced antitumor efficacy of STINGVAX in multiple aggressive therapeutic models of established cancer in mice. Antitumor activity was STING-dependent and correlated with increased activation of dendritic cells and tumor antigen-specific CD8(+) T cells. Tumors from STINGVAX-treated mice demonstrated marked PD-L1 (programmed death ligand 1) up-regulation, which was associated with tumor-infiltrating CD8(+)IFNγ(+) T cells. When combined with PD-1 (programmed death 1) blockade, STINGVAX induced regression of palpable, poorly immunogenic tumors that did not respond to PD-1 blockade alone.

TANK-binding kinase-1 (TBK1) and the inducible IkappaB kinase (IKK-i) have been shown recently to activate interferon (IFN) regulatory factor-3 (IRF3), the primary transcription factor regulating induction of type I IFNs. Here, we have compared the role and specificity of TBK1 in the type I IFN response to lipopolysaccharide (LPS), polyI:C, and viral challenge by examining IRF3 nuclear translocation, signal transducer and activator of transcription 1 phosphorylation, and induction of IFN-regulated genes. The LPS and polyI:C-induced IFN responses were abolished and delayed, respectively, in macrophages from mice with a targeted disruption of the TBK1 gene. When challenged with Sendai virus, the IFN response was normal in TBK1(-/-) macrophages, but defective in TBK1(-/-) embryonic fibroblasts. Although both TBK1 and IKK-i are expressed in macrophages, only TBK1 but not IKK-i was detected in embryonic fibroblasts by Northern blotting analysis. Furthermore, the IFN response in TBK1(-/-) embryonic fibroblasts can be restored by reconstitution with wild-type IKK-i but not a mutant IKK-i lacking kinase activity. Thus, our studies suggest that TBK1 plays an important role in the Toll-like receptor-mediated IFN response and is redundant with IKK-i in the response of certain cell types to viral infection.

How the innate immune system tailors specific responses to diverse microbial infections is not well understood. Cells use a limited number of host receptors and signaling pathways to both discriminate among extracellular and intracellular microbes, and also to generate responses commensurate to each threat. Here, we have addressed these questions by using DNA microarrays to monitor the macrophage transcriptional response to the intracellular bacterial pathogen Listeria monocytogenes. By utilizing combinations of host and bacterial mutants, we have defined the host transcriptional responses to vacuolar and cytosolic bacteria. These compartment-specific host responses induced significantly different sets of target genes, despite activating similar transcription factors. Vacuolar signaling was entirely MyD88-dependent, and induced the transcription of pro-inflammatory cytokines. The IRF3-dependent cytosolic response induced a distinct set of target genes, including IFNbeta. Many of these cytosolic response genes were induced by secreted cytokines, so we further identified those host genes induced independent of secondary signaling. The host response to cytosolic bacteria was reconstituted by the cytosolic delivery of L. monocytogenes genomic DNA, but we observed an amplification of this response by NOD2 signaling in response to MDP. Correspondingly, the induction of IFNbeta was reduced in nod2-/- macrophages during infection with either L. monocytogenes or Mycobacterium tuberculosis. Combinatorial control of IFNbeta induction by recognition of both DNA and MDP may highlight a mechanism by which the innate immune system integrates the responses to multiple ligands presented in the cytosol by intracellular pathogens.

In mammals, one of the most pronounced consequences of viral infection is the induction of type I interferons, cytokines with potent antiviral activity. Schlafen (Slfn) genes are a subset of interferon-stimulated early response genes (ISGs) that are also induced directly by pathogens via the interferon regulatory factor 3 (IRF3) pathway. However, many ISGs are of unknown or incompletely understood function. Here we show that human SLFN11 potently and specifically abrogates the production of retroviruses such as human immunodeficiency virus 1 (HIV-1). Our study revealed that SLFN11 has no effect on the early steps of the retroviral infection cycle, including reverse transcription, integration and transcription. Rather, SLFN11 acts at the late stage of virus production by selectively inhibiting the expression of viral proteins in a codon-usage-dependent manner. We further find that SLFN11 binds transfer RNA, and counteracts changes in the tRNA pool elicited by the presence of HIV. Our studies identified a novel antiviral mechanism within the innate immune response, in which SLFN11 selectively inhibits viral protein synthesis in HIV-infected cells by means of codon-bias discrimination.

We have previously shown that double-stranded RNA-triggered, Toll-like receptor 3 (TLR3)-mediated signaling is independent of MyD88, IRAK4, and IRAK. Instead, TRAF6, TAK1, and TAB2 are recruited to TLR3 on poly(I.C) stimulation. TRAF6-TAK1-TAB2 are then translocated to the cytosol where TAK1 is phosphorylated and activated, leading to the activation of IkappaB kinase and NFkappaB. The present study addressed two important questions: (i) How are TRAF6, TAK1, and TAB2 recruited to TLR3? (ii) Are TRAF6, TAK1, and TAB2 also required for TLR3-mediated IRF3 activation? Recently, a novel Toll-IL-1 receptor (TIR)-containing adapter, TIR domain-containing adapter inducing IFN-beta (TRIF), was shown to play a critical role in TLR3-mediated activation of NF-kappaB and IRF3. We found that TLR3 recruits TRAF6 via adapter TRIF through a TRAF6-binding sequence in TRIF (PEEMSW, amino acids 250-255). Mutation of this TRAF6-binding sequence abolished the interaction of TRIF with TRAF6, but not with TLR3. Interestingly, mutation of the TRAF6-binding site of TRIF only abolished its ability to activate NF-kappaB but not IRF3, suggesting that TLR3-mediated activation of NF-kappaB and IRF3 might bifurcate at TRIF. In support of this finding, we showed that DN-TRAF6 and DN-TAK1 blocked poly(I.C)-induced NF-kappaB but not IRF3 activation. Furthermore, whereas poly(I.C)-induced NF-kappaB activation is completely abolished inTRAF6-/- MEFs, the signal-induced activation of IRF3 is TRAF6 independent. In conclusion, TRIF recruits TRAF6-TAK1-TAB2 to TLR3 through its TRAF6-binding site, which is required for NF-kappaB but not IRF3 activation. Therefore, double-stranded RNA-induced TLR3/TRIF-mediated NF-kappaB and IRF3 activation diverge at TRIF.

While the recognition of microbial infection often occurs at the cell surface via Toll-like receptors, the cytosol of the cell is also under surveillance for microbial products that breach the cell membrane. An important outcome of cytosolic recognition is the induction of IFNalpha and IFNbeta, which are critical mediators of immunity against both bacteria and viruses. Like many intracellular pathogens, a significant fraction of the transcriptional response to Mycobacterium tuberculosis infection depends on these type I interferons, but the recognition pathways responsible remain elusive. In this work, we demonstrate that intraphagosomal M. tuberculosis stimulates the cytosolic Nod2 pathway that responds to bacterial peptidoglycan, and this event requires membrane damage that is actively inflicted by the bacterium. Unexpectedly, this recognition triggers the expression of type I interferons in a Tbk1- and Irf5-dependent manner. This response is only partially impaired by the loss of Irf3 and therefore, differs fundamentally from those stimulated by bacterial DNA, which depend entirely on this transcription factor. This difference appears to result from the unusual peptidoglycan produced by mycobacteria, which we show is a uniquely potent agonist of the Nod2/Rip2/Irf5 pathway. Thus, the Nod2 system is specialized to recognize bacteria that actively perturb host membranes and is remarkably sensitive to mycobacteria, perhaps reflecting the strong evolutionary pressure exerted by these pathogens on the mammalian immune system.

Memory CD8(+) T cells induced upon immunization exhibit improved functional features that contribute to protection of immunized hosts. Although both cognate antigen recognition and inflammation are important for memory CD8(+) T cell reactivation, the relative contribution of these factors and the cell types providing these signals in vivo are poorly defined. Here, we show that Ly6C(+)CCR2(+) inflammatory monocytes, a subset of monocytes, largely orchestrate memory CD8(+) T and NK lymphocytes activation by differentiating into interleukin-18 (IL-18)- and IL-15-producing cells in an inflammasome and type I interferon-IRF3-dependent manner. Memory CD8(+) T cells became potent effector cells by sensing inflammation from monocytes independently of their cognate antigen. Like NK cells, they underwent rapid mobilization, upregulated intense and sustained effector functions during bacterial, viral, and parasitic infections, and contributed to innate responses and protection in vivo. Thus, inflammatory monocyte-derived IL-18 and IL-15 are critical to initiate memory CD8(+) T and NK lymphocytes differentiation into antimicrobial effector cells.

Although Toll-like receptor 9 (TLR9) has been implicated in cytokine and type I interferon (IFN) production during malaria in humans and mice, the high AT content of the Plasmodium falciparum genome prompted us to examine the possibility that malarial DNA triggered TLR9-independent pathways. Over 6000 ATTTTTAC ("AT-rich") motifs are present in the genome of P. falciparum, which we show here potently induce type I IFNs. Parasite DNA, parasitized erythrocytes and oligonucleotides containing the AT-rich motif induce type I IFNs via a pathway that did not involve the previously described sensors TLR9, DAI, RNA polymerase-III or IFI16/p204. Rather, AT-rich DNA sensing involved an unknown receptor that coupled to the STING, TBK1 and IRF3-IRF7 signaling pathway. Mice lacking IRF3, IRF7, the kinase TBK1 or the type I IFN receptor were resistant to otherwise lethal cerebral malaria. Collectively, these observations implicate AT-rich DNA sensing via STING, TBK1 and IRF3-IRF7 in P. falciparum malaria.

Although the transcription factors IRF-3 and IRF-7 are considered master regulators of type I interferon (IFN) induction and IFN stimulated gene (ISG) expression, Irf3(-/-)×Irf7(-/-) double knockout (DKO) myeloid dendritic cells (mDC) produce relatively normal levels of IFN-β after viral infection. We generated Irf3(-/-)×Irf5(-/-)×Irf7(-/-) triple knockout (TKO) mice to test whether IRF-5 was the source of the residual induction of IFN-β and ISGs in mDCs. In pathogenesis studies with two unrelated positive-sense RNA viruses (West Nile virus (WNV) and murine norovirus), TKO mice succumbed at rates greater than DKO mice and equal to or approaching those of mice lacking the type I IFN receptor (Ifnar(-/-)). In ex vivo studies, after WNV infection or exposure to Toll-like receptor agonists, TKO mDCs failed to produce IFN-β or express ISGs. In contrast, this response was sustained in TKO macrophages following WNV infection. To define IRF-regulated gene signatures, we performed microarray analysis on WNV-infected mDC from wild type (WT), DKO, TKO, or Ifnar(-/-) mice, as well as from mice lacking the RIG-I like receptor adaptor protein MAVS. Whereas the gene induction pattern in DKO mDC was similar to WT cells, remarkably, almost no ISG induction was detected in TKO or Mavs(-/-) mDC. The relative equivalence of TKO and Mavs(-/-) responses suggested that MAVS dominantly regulates ISG induction in mDC. Moreover, we showed that MAVS-dependent induction of ISGs can occur through an IRF-5-dependent yet IRF-3 and IRF-7-independent pathway. Our results establish IRF-3, -5, and -7 as the key transcription factors responsible for mediating the type I IFN and ISG response in mDC during WNV infection and suggest a novel signaling link between MAVS and IRF-5.

Toll-like receptors (TLRs) form a major group of transmembrane receptors that are involved in the detection of invading pathogens. Double-stranded RNA is a marker for viral infection that is recognized by TLR3. TLR3 triggering activates specific signaling pathways that culminate in the activation of NF-kappaB and IRF3 transcription factors, as well as apoptosis, enabling the host to mount an effective innate immune response through the induction of cytokines, chemokines, and other proinflammatory mediators. In this review, we describe the paradoxical role of TLR3 in innate immunity against different viruses and in viral pathogenesis but also the evidence for TLR3 as a "danger" receptor in nonviral diseases. We also discuss the structure and cellular localization of TLR3, as well as the complex signaling and regulatory events that contribute to TLR3-mediated immune responses.

The outcome of a viral infection is regulated in part by the complex coordination of viral and host interactions that compete for the control and optimization of virus replication. Severe acute respiratory syndrome coronavirus (SARS-CoV) intimately engages and regulates the host innate immune responses during infection. Using a novel interferon (IFN) antagonism screen, we show that the SARS-CoV proteome contains several replicase, structural, and accessory proteins that antagonize the IFN pathway. In this study, we focus on the SARS-CoV papain-like protease (PLP), which engages and antagonizes the IFN induction and NF-kappaB signaling pathways. PLP blocks these pathways by affecting activation of the important signaling proteins in each pathway, IRF3 and NF-kappaB. We also show that the ubiquitin-like domain of PLP is necessary for pathway antagonism but not sufficient by itself to block these pathways regardless of the enzymatic activity of the protease. The potential mechanism of PLP antagonism and its role in pathogenesis are discussed.

IFN regulatory factor 3 (IRF3), a constitutively expressed protein localizing largely to the cytoplasm, is a primary effector of the innate immune response. Infection can trigger the phosphorylation, dimerization, and nuclear translocation of IRF3, where the factor stimulates the expression and release of IFN. In this study, we determined that the rotavirus gene 5 product, nonstructural protein 1 (NSP1), interacts with IRF3 in the infected cell. To understand the importance of the interaction, we compared IRF3 activation by rotaviruses expressing wild-type and C-truncated forms of NSP1. The analysis showed that IRF3 underwent dimerization and nuclear translocation and stimulated IFN promoter activity in infected cells expressing truncated NSP1. In contrast, infected cells expressing wild-type NSP1 were characterized by the rapid degradation of IRF3 during the replication cycle, severe decreases in IRF3 dimerization and nuclear translocation, and lack of IFN promoter activity. The implication of these results, that wild-type NSP1 is an antagonist of the IFN-signaling pathway, was confirmed in transient expression assays, which showed that wild-type NSP1, but not the C-truncated protein, induced the degradation of IRF3 fusion proteins. Related experiments indicated that NSP1 mediates IRF3 degradation through a proteasome-dependent pathway. The critical role of NSP1 in promoting cell-to-cell spread of rotavirus was demonstrated by using gene 5-specific short interfering RNAs in plaque assays. Although several viruses have been described that subvert the innate immune response by preventing IRF3 activation, rotavirus is identified as one that accomplishes this task by inducing the degradation of IRF3.

Two different CpG oligonucleotides (ODN) were used to study the regulation of type I IFN in human plasmacytoid dendritic cells (PDC): ODN 2216, a CpG-A ODN, known to induce high amounts of IFN-alpha in PDC, and ODN 2006, a CpG-B ODN, which is potent at stimulating B cells. CpG-A ODN showed higher and prolonged kinetics of type I IFN production compared with that of CpG-B ODN. In contrast, CpG-B ODN was more active than CpG-A ODN in stimulating IL-8 production and increasing costimulatory and Ag-presenting molecules, suggesting that CpG-A and CpG-B trigger distinct regulatory pathways in PDC. Indeed, CpG-A ODN, but not CpG-B ODN, activated the type I IFNR-mediated autocrine feedback loop. PDC were found to express high constitutive levels of IFN regulatory factor (IRF)7. IRF7 and STAT1, but not IRF3, were equally up-regulated by both CpG-A and CpG-B. CD40 ligand synergistically increased CpG-B-induced IFN-alpha independent of the IFNR but did not affect CpG-B-induced IFN-beta. In conclusion, our studies provide evidence for the existence of two distinct regulatory pathways of type I IFN synthesis in human PDC, one dependent on and one independent of the IFNR-mediated feedback loop. The alternate use of these pathways is based on the type of stimulus rather than the quantity of IFN-alphabeta available to trigger the IFNR. Constitutive expression of IRF7 and the ability to produce considerable amounts of IFN-alpha independent of the IFNR seem to represent characteristic features of PDC.

Zika virus (ZIKV) can be transmitted sexually between humans. However, it is unknown whether ZIKV replicates in the vagina and impacts the unborn fetus. Here, we establish a mouse model of vaginal ZIKV infection and demonstrate that, unlike other routes, ZIKV replicates within the genital mucosa even in wild-type (WT) mice. Mice lacking RNA sensors or transcription factors IRF3 and IRF7 resulted in higher levels of local viral replication. Furthermore, mice lacking the type I interferon (IFN) receptor (IFNAR) became viremic and died of infection after a high-dose vaginal ZIKV challenge. Notably, vaginal infection of pregnant dams during early pregnancy led to fetal growth restriction and infection of the fetal brain in WT mice. This was exacerbated in mice deficient in IFN pathways, leading to abortion. Our study highlights the vaginal tract as a highly susceptible site of ZIKV replication and illustrates the dire disease consequences during pregnancy.

Research into the five Toll/IL1 receptor (TIR) adaptor proteins involved in innate immunity continues to advance. Here we outline some of the more recent findings. MyD88 has a key role in signalling by the IL1 receptor complex and TLRs. However, a MyD88-independent pathway of IL1beta signalling in neurons has been described which involves the protein kinase Akt, and which has an anti-apoptotic effect. This pathway may also be important for the mechanism whereby Alum exerts its adjuvant effect since this depends on IL1beta but is MyD88-independent. MyD88 is also involved in tumourigenesis in models of hepatocarcinoma and familial associated polyposis (FAP); negative regulation of TLR3 signalling and in PKCepsilon activation. The adaptor Mal is regulated by phosphorylation and caspase-1 cleavage. A variant form of Mal in humans termed S180L confers protection in multiple infectious diseases. TRAM is controlled by myristoylation and phosphorylation and the localisation of TRAM with TLR4 to endosomes is required for activation of IRF3 and induction of IFNbeta. Finally SARM has been shown to regulate TRIF and also appears to be involved in neuronal injury mediated by oxidative stress in mouse neurons. These advances confirm the importance for the TIR domain-containing adapters in host defence and inflammation.

Induction of interferon-alpha (IFNalpha) gene expression in virus-infected cells requires phosphorylation-induced activation of the transcription factors IRF3 and IRF7. However, the kinase(s) that targets these proteins has not been identified. Using a combined pharmacological and genetic approach, we found that none of the kinases tested was responsible for IRF phosphorylation in cells infected with Newcastle disease virus (NDV). Although the broad-spectrum kinase inhibitor staurosporine potently blocked IRF3 and -7 phosphorylation, inhibitors for protein kinase C, protein kinase A, MEK, SAPK, IKK, and protein kinase R (PKR) were without effect. Both IkappaB kinase and PKR have been implicated in IFN induction, but cells genetically deficient in IkappaB kinase, PKR, or the PKR-related genes PERK, IRE1, or GCN2 retained the ability to phosphorylate IRF7 and induce IFNalpha. Interestingly, PKR mutant cells were defective for response to double-stranded (ds) RNA but not to virus infection, suggesting that dsRNA is not the only activating viral component. Consistent with this notion, protein synthesis was required for IRF7 phosphorylation in virus-infected cells, and the kinetics of phosphorylation and viral protein production were similar. Despite evidence for a lack of involvement of dsRNA and PKR, vaccinia virus E3L protein, a dsRNA-binding protein capable of inhibiting PKR, was an effective IRF3 and -7 phosphorylation inhibitor. These results suggest that a novel cellular protein that is activated by viral products in addition to dsRNA and is sensitive to E3L inhibition is responsible for IRF activation and reveal a novel mechanism for the anti-IFN effect of E3L distinct from its inhibition of PKR.

Induction of type I IFNs is a fundamental cellular response to both viral and bacterial infection. The role of the transcription factor IRF3 is well established in driving this process. However, equally as important are cellular mechanisms for turning off type I IFN production to limit this response. In this respect, IRF3 has previously been shown to be targeted for ubiquitin-mediated degradation postviral detection to turn off the IFN-beta response. In this study, we provide evidence that the E3 ligase Ro52 (TRIM21) targets IRF3 for degradation post-pathogen recognition receptor activation. We demonstrate that Ro52 interacts with IRF3 via its C-terminal SPRY domain, resulting in the polyubiquitination and proteasomal degradation of the transcription factor. Ro52-mediated IRF3 degradation significantly inhibits IFN-beta promoter activity, an effect that is reversed in the presence of the proteasomal inhibitor MG132. Specific targeting of Ro52 using short hairpin RNA rescues IRF3 degradation following polyI:C-stimulation of HEK293T cells, with a subsequent increase in IFN-beta production. Additionally, shRNA targeting of murine Ro52 enhances the production of the IRF3-dependent chemokine RANTES following Sendai virus infection of murine fibroblasts. Collectively, this demonstrates a novel role for Ro52 in turning off and thus limiting IRF3-dependent type I IFN production by targeting the transcription factor for polyubiquitination and subsequent proteasomal degradation.

STING (stimulator of interferon genes) is known to control the induction of innate immune genes in response to the recognition of cytosolic DNA species, including the genomes of viruses such as herpes simplex virus 1 (HSV-1). However, while STING is essential for protection of the host against numerous DNA pathogens, sustained STING activity can lead to lethal inflammatory disease. It is known that STING utilizes interferon regulatory factor 3 (IRF3) and nuclear factor κB (NF-κB) pathways to exert its effects, although the signal transduction mechanisms remain to be clarified fully. Here we demonstrate that in addition to the activation of these pathways, potent induction of the Jun N-terminal protein kinase/stress-activated protein kinase (JNK/SAPK) pathway was similarly observed in response to STING activation by double-stranded DNA (dsDNA). Furthermore, TANK-binding kinase 1 (TBK1) associated with STING was found to facilitate dsDNA-mediated canonical activation of NF-κB as well as IRF3 to promote proinflammatory gene transcription. The triggering of NF-κB function was noted to require TRAF6 activation. Our findings detail a novel dsDNA-mediated NF-κB activation pathway facilitated through a STING-TRAF6-TBK1 axis and suggest a target for therapeutic intervention to plausibly stimulate antiviral activity or, alternatively, avert dsDNA-mediated inflammatory disease.

OBJECTIVE

The IKK complex, which is composed of two catalytic subunits, IKKα and IKKβ, has been suggested to be essential for the activation of canonical NF-κB signaling in response to various stimuli, including cytokines (e.g., interleukin-1α [IL-1α] and tumor necrosis factor alpha [TNF-α]), Toll-like receptor (TLR) ligands (e.g., lipopolysaccharide [LPS]), and dsRNAs derived from viruses, or a synthetic analog. STING has been identified as a critical signaling molecule required for the detection of cytosolic dsDNAs derived from pathogens and viruses. However, little is known about how cytosolic dsDNA triggers NF-κB signaling. In the present study, we demonstrate that TBK1, identified as an IKK-related kinase, may predominantly control the activation of NF-κB in response to dsDNA signaling via STING through the IKKαβ activation loop. Thus, our results establish TBK1 as a downstream kinase controlling dsDNA-mediated IRF3 and NF-κB signaling dependent on STING.

The innate immune system responds to unique molecular signatures that are widely conserved among microbes but that are not normally present in host cells. Compounds that stimulate innate immune pathways may be valuable in the design of novel adjuvants, vaccines, and other immunotherapeutics. The cyclic dinucleotide cyclic-di-guanosine monophosphate (c-di-GMP) is a recently appreciated second messenger that plays critical regulatory roles in many species of bacteria but is not produced by eukaryotic cells. In vivo and in vitro studies have previously suggested that c-di-GMP is a potent immunostimulatory compound recognized by mouse and human cells. We provide evidence that c-di-GMP is sensed in the cytosol of mammalian cells via a novel immunosurveillance pathway. The potency of cytosolic signaling induced by c-di-GMP is comparable to that induced by cytosolic delivery of DNA, and both nucleic acids induce a similar transcriptional profile, including triggering of type I interferons and coregulated genes via induction of TBK1, IRF3, nuclear factor kappaB, and MAP kinases. However, the cytosolic pathway that senses c-di-GMP appears to be distinct from all known nucleic acid-sensing pathways. Our results suggest a novel mechanism by which host cells can induce an inflammatory response to a widely produced bacterial ligand.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication and host immune response determine coronavirus disease 2019 (COVID-19), but studies evaluating viral evasion of immune response are lacking. Here, we use unbiased screening to identify SARS-CoV-2 proteins that antagonize type I interferon (IFN-I) response. We found three proteins that antagonize IFN-I production via distinct mechanisms: nonstructural protein 6 (nsp6) binds TANK binding kinase 1 (TBK1) to suppress interferon regulatory factor 3 (IRF3) phosphorylation, nsp13 binds and blocks TBK1 phosphorylation, and open reading frame 6 (ORF6) binds importin Karyopherin α 2 (KPNA2) to inhibit IRF3 nuclear translocation. We identify two sets of viral proteins that antagonize IFN-I signaling through blocking signal transducer and activator of transcription 1 (STAT1)/STAT2 phosphorylation or nuclear translocation. Remarkably, SARS-CoV-2 nsp1 and nsp6 suppress IFN-I signaling more efficiently than SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). Thus, when treated with IFN-I, a SARS-CoV-2 replicon replicates to a higher level than chimeric replicons containing nsp1 or nsp6 from SARS-CoV or MERS-CoV. Altogether, the study provides insights on SARS-CoV-2 evasion of IFN-I response and its potential impact on viral transmission and pathogenesis.

Keywords: COVID-19; SARS-CoV-2; coronavirus disease 2019; immune evasion; interferon; replicon; severe acute respiratory syndrome coronavirus 2.