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 B (NF-B) 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-B as well as IRF3 to promote proinflammatory gene transcription. The triggering of NF-B function was noted to require TRAF6 activation. Our findings detail a novel dsDNA-mediated NF-B 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. IMPORTANCE The IKK complex, which is composed of two catalytic subunits, IKK␣ and IKK␤, has been suggested to be essential for the activation of canonical NF-B signaling in response to various stimuli, including cytokines (e.g., interleukin-1␣ [IL-1␣] and tumor necrosis factor alpha [TNF-␣]), 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-B signaling. In the present study, we demonstrate that TBK1, identified as an IKK-related kinase, may predominantly control the activation of NF-B in response to dsDNA signaling via STING through the IKK␣␤ activation loop. Thus, our results establish TBK1 as a downstream kinase controlling dsDNA-mediated IRF3 and NF-B signaling dependent on STING.

The innate immune system detects pathogen- and host-derived double-stranded DNA exposed to the cytosol and induces type I interferon (IFN) and other cytokines. Here, we identified interferon-inducible tripartite-motif (TRIM) 56 as a regulator of double-stranded DNA-mediated type I interferon induction. TRIM56 overexpression enhanced IFN-β promoter activation after double-stranded DNA stimulation whereas TRIM56 knockdown abrogated it. TRIM56 interacted with STING and targeted it for lysine 63-linked ubiquitination. This modification induced STING dimerization, which was a prerequisite for recruitment of the antiviral kinase TBK1 and subsequent induction of IFN-β. Taken together, these results indicate that TRIM56 is an interferon-inducible E3 ubiquitin ligase that modulates STING to confer double-stranded DNA-mediated innate immune responses.

Hepatitis C virus (HCV) is a major cause of chronic liver disease that frequently leads to steatosis, cirrhosis, and eventually hepatocellular carcinoma (HCC). HCV core protein is not only a component of viral particles but also a multifunctional protein because liver steatosis and HCC are developed in HCV core gene-transgenic (CoreTg) mice. Proteasome activator PA28␥/REG␥ regulates host and viral proteins such as nuclear hormone receptors and HCV core protein. Here we show that a knockout of the PA28␥ gene induces the accumulation of HCV core protein in the nucleus of hepatocytes of CoreTg mice and disrupts development of both hepatic steatosis and HCC. Furthermore, the genes related to fatty acid biosynthesis and srebp-1c promoter activity were up-regulated by HCV core protein in the cell line and the mouse liver in a PA28␥-dependent manner. Heterodimer composed of liver X receptor ␣ (LXR␣) and retinoid X receptor ␣ (RXR␣) is known to up-regulate srebp-1c promoter activity. Our data also show that HCV core protein enhances the binding of LXR␣/RXR␣ to LXR-response element in the presence but not the absence of PA28␥. These findings suggest that PA28␥ plays a crucial role in the development of liver pathology induced by HCV infection.fatty acid ͉ proteasome ͉ sterol regulatory element-binding protein (SREBP) ͉ RXR␣ ͉ LXR␣ H epatitis C virus (HCV) belongs to the Flaviviridae family, and it possesses a positive, single-stranded RNA genome that encodes a single polyprotein composed of Ϸ3,000 aa. The HCV polyprotein is processed by host and viral proteases, resulting in 10 viral proteins. Viral structural proteins, including the capsid (core) protein and two envelope proteins, are located in the N-terminal one-third of the polyprotein, followed by nonstructural proteins.HCV infects Ͼ170 million individuals worldwide, and then it causes liver disease, including hepatic steatosis, cirrhosis, and eventually hepatocellular carcinoma (HCC) (1). The prevalence of fatty infiltration in the livers of chronic hepatitis C patients has been reported to average Ϸ50% (2, 3), which is higher than the percentage in patients infected with hepatitis B virus and other liver diseases. However, the precise functions of HCV proteins in the development of fatty liver remain unknown because of the lack of a system sufficient to investigate the pathogenesis of HCV. HCV core protein expression has been shown to induce lipid droplets in cell lines and hepatic steatosis and HCC in transgenic mice (4-6). These reports suggest that HCV core protein plays an important role in the development of various types of liver failure, including steatosis and HCC.Recent reports suggest that lipid biosynthesis affects HCV replication (7-9). Involvement of a geranylgeranylated host protein, FBL2, in HCV replication through the interaction with NS5A suggests that the cholesterol biosynthesis pathway is also important for HCV replication (9). Increases in saturated and monounsaturated fatty acids enhance HCV RNA replication, whereas increases in polyunsaturat...

We have previously shown that mice inoculated intranasally with a wild-type baculovirus (Autographa californica nuclear polyhedrosis virus [AcNPV]) are protected from a lethal challenge by influenza virus. However, the precise mechanism of induction of this protective immune response by the AcNPV treatment remained unclear. Here we show that AcNPV activates immune cells via the Toll-like receptor 9 (TLR9)/ MyD88-dependent signaling pathway. The production of inflammatory cytokines was severely reduced in peritoneal macrophages (PECs) and splenic CD11c؉ dendritic cells (DCs) derived from mice deficient in MyD88 or TLR9 after cultivation with AcNPV. In contrast, a significant amount of alpha interferon (IFN-␣) was still detectable in the PECs and DCs of these mice after stimulation with AcNPV, suggesting that a TLR9/MyD88-independent signaling pathway might also participate in the production of IFN-␣ by AcNPV. Since previous work showed that TLR9 ligands include bacterial DNA and certain oligonucleotides containing unmethylated CpG dinucleotides, we also examined the effect of baculoviral DNA on the induction of innate immunity. Transfection of the murine macrophage cell line RAW264.7 with baculoviral DNA resulted in the production of the inflammatory cytokine, while the removal of envelope glycoproteins from viral particles, UV irradiation of the virus, and pretreatment with purified baculovirus envelope proteins or endosomal maturation inhibitors diminished the induction of the immune response by AcNPV. Together, these results indicate that the internalization of viral DNA via membrane fusion mediated by the viral envelope glycoprotein, as well as endosomal maturation, which releases the viral genome into TLR9-expressing cellular compartments, is necessary for the induction of the innate immune response by AcNPV.

Summary How the cell recognizes cytosolic DNA including DNA based microbes to trigger host defense related gene activation remains to be fully resolved. Here, we demonstrate that STING (for Stimulator of Interferon Genes), an endoplasmic reticulum (ER) translocon associated transmembrane protein, acts to detect cytoplasmic DNA species. STING homodimers were able to complex with self (apoptotic, necrotic) or pathogen related ssDNA and dsDNA and were indispensible for HSV-1-mediated transcriptional activation of a wide array of innate immune and pro-inflammatory genes in addition to type I IFN. Our data indicates that STING instigates cytoplasmic DNA-mediated cellular defense gene transcription and facilitates adoptive responses that are required for protection of the host. In contrast, chronic STING activation may manifest inflammatory responses and possibly autoimmune disease triggered by self-DNA.