NF-κB functions together with another transcription factor, IRF3, to induce type-I interferon production upon viral infection. After viral infection, viral RNA is detected by the RIG-I family of RNA helicases. In 2005, we identified the protein MAVS (also known as IPS-1, VISA or CARDIF) as a key adaptor for RIG-I signaling. MAVS is localized on the mitochondrial outer membrane and this localization is indispensable for its function, as underscored by the finding that viral protease NS3/4A of hepatitis C virus efficiently cleaves MAVS off the mitochondrial membrane to suppress interferon induction. The essential role of MAVS in defense against RNA virus is further confirmed in MAVS knock out mice. Recently, we found that the RIG-I pathway is also important for detecting commensal bacterial RNA and maintaining intestinal homeostasis.
Mechanism of signal transduction in the RIG-I–MAVS pathway
Our recent work has focused on the mechanism of signal transduction in the RIG-I pathway. Through the development of a cell-free system that recapitulates the RIG-I pathway from detection of viral RNA to activation of IRF3, we found that ubiquitination also plays a key role in RIG-I activation. Specifically, after binding to RNA, RIG-I undergoes a conformational change that exposes its N-terminal CARD domains, which then binds to unanchored K63 polyubiquitin chains. This binding promotes the formation of RIG-I tetramer, which interacts with MAVS and promotes MAVS aggregation. Strikingly, MAVS aggregates catalyze the polymerization of other MAVS on the mitochondria through a prion-like mechanism. The structure of MAVS aggregates is solved by electron microscopy and the prion-like mechanism turns out to be a universal mechanism in cell signaling. These prion-like fibers of MAVS recruits multiple ubiquitin E3 ligases, including TRAF2 TRAF5 and TRAF6, to potently activate the cytosolic kinases IKK and TBK1, leading to the activation of NF-κB and IRF3, respectively. Interestingly, MAVS is phosphorylated by IKK and TBK1, which provides a binding site for IRF3. This signal induced phosphorylation adds another layer of precise regulation for IRF3 activation.
Interferon induction by cytosolic DNA
We are also interested in how cytosolic DNA induces type-I interferons, which are important for immune defense against DNA viruses and intracellular bacteria. Furthermore, inappropriate presence of cytosolic self-DNA could trigger autoimmune diseases. We have found that AT-rich DNAs are transcribed by RNA polymerase III into RNAs bearing 5’-triphosphates, which then induce interferons through the RIG-I pathway. However, most DNA induces interferons in a sequence-independent manner that depends on the endoplasmic reticulum protein STING (also known as MITA). We have recently shown that after stimulation, STING recruits both TBK1 and IRF3, thereby specifying IRF3 phosphorylation by TBK1.
Discovery of cGAS-cGAMP pathway and its role in immune defense
We identified cGAS, which directly binds to cytosolic DNA, forms a dimer and synthesizes cGAMP from GTP and ATP. cGAMP with mixed phosphodiester linkages binds to STING with high-affinity and induces STING conformational change and interferon expression. We generated cGAS knockout mice and cell lines. Using these reagents we found that cGAS-cGAMP pathway is important for immune response against DNA viruses, retroviruses and bacteria. In addition, we found that cGAS is responsible for the severe inflammation found in DNAse deficient mice, suggesting cGAS can also be activated by self DNA and may cause inflammation in some lupus patients.
Novel functions and regulations of TLRs
Our recent work on commensal bacteria led us to discover the role of TLR13 in detecting the bacterial 23S ribosomal RNA (rRNA). Remarkably, we found that TLR13 recognizes a specific sequence of about 13 nucleotides near the active site of the 23S rRNA, which catalyzes peptide bond synthesis. Thus, unlike other innate immune sensors that detect a ‘pattern’ of microbial components, TLR13 detects bacterial RNA with exquisite sequence specificity. IRF5 is a transcription factor that are essential for immune signaling of multiple TLRs, we recently discovered that IKKb phosphorylated IRF5 and induces IRF5 dimerization and nuclear translocation.
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- Collins, A.C., Cai, H., Li, T., Franco, L.H., Li, X-D., Nair, V.R., Scharn, C.R., Stamm, C.E., Levine, B., Chen, Z.J*., Shiloh, M.U*. (2015) Cyclic GMP-AMP synthase is an innate immune sensor of Mycobacterium tuberculosis DNA. Cell Host Microbe 17, 820-828. *co-corresponding author
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