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The control GST protein failed to protect polyUb from digestion by IsoT or vOTU (Figures S6A and S6B). Thus, RIG-I(N) binds to K63-polyUb chains and protects these chains from digestion by DUBs. In this report, we demonstrate that RNA with characteristics of?viral RNA (5��-pppRNA and dsRNA) can activate the RIG-I pathway in?vitro in a reconstitution system with exquisite specificity and sensitivity. To our knowledge, this is the first in?vitro reconstitution of an immune signaling cascade from a microbial ligand (viral RNA) to activation of a transcription selleck chemical factor (IRF3). Our success in reconstituting the RIG-I pathway in?vitro is beneficiary of at least three previous discoveries that (1) RNAs containing 5��-triphosphate are direct functional ligands of RIG-I (Hornung et?al., 2006?and?Pichlmair et?al., 2006); (2) mitochondria containing MAVS are essential for RIG-I signaling (Seth et?al., 2005); and (3) ubiquitination Biperiden HCl is important for RIG-I activation (Gack et?al., 2007). The in?vitro reconstitution of the RIG-I pathway sets the stage for delineating the biochemical mechanism of RIG-I activation and subsequent steps of downstream signaling. Testimonial to the power of this in?vitro system are the discoveries revealed in this study that the tandem CARDs of RIG-I represent a new class of ubiquitin-binding domain with specificity for K63-polyUb chains and that unanchored K63-polyUb chains are potent intracellular signaling molecules www.selleckchem.com/products/Rapamycin.html that activate the RIG-I pathway. A major determinant that distinguishes viral RNA from the host RNA in mammalian cells is the presence of 5��-triphosphate in viral RNA but not in host RNA (Fujita, 2009). 5��-pppRNA is generated in the course of RNA replication by viral RNA polymerases. However, RIG-I may have very limited access to viral 5��-pppRNA for the following reasons: (1) viral RNA replication normally occurs within a membrane compartment (e.g., vesicles) that is sequestered from the cytosolic immune sensors such as RIG-I; (2) some viruses such as influenza replicate in the nucleus rather than in the cytosol; (3) the majority of viral RNA is in complex with viral capsid proteins to form ribonucleoprotein particles (RNPs), and the 5�� end of viral RNA can be blocked by viral proteins; (4) like their mammalian counterparts, most viral mRNAs also contain 5��-cap or other 5��-modifications. Thus, RIG-I must be capable of detecting very tiny amounts of 5��-pppRNA that may be inadvertently or transiently exposed during the course of viral?replication and intracellular trafficking. By reconstituting RNA-mediated activation of the RIG-I cascade in?vitro, we demonstrate that RIG-I can detect a few 5��-pppRNA molecules (