Mechanism of Ubiquitin Induced Activation of RIG-I Like Receptors in Antiviral Innate Immunity

The innate immune response is the first line of defense against viral infections. Innate immune recognition is mediated by a set of host pattern recognition receptors that can recognize pathogen associated molecular patterns, and trigger the expression of immune response genes. Two proteins of the RIG-I like receptor family, RIG-I and MDA5, are essential for the detection of viral RNA in the cytoplasm. Upon viral recognition, RIG-I and MDA5 activate the mitochondrial signaling protein AVS, and initiate downstream signaling pathways that activate the transcription factors NF-kappaB and IRF3. These transcription factors regulate the production of type I interferons and proinflammatory cytokines, which serve to keep virus infections under control. RIG-I and MDA5 contain N-terminal tandem CARD domains. CARD domains in other proteins are known to mediate protein-protein interactions. MAVS also contain one CARD domain. It has been proposed that the activation of MAVS by RIG-I is mediated by homotypic interaction between their CARD domains. However, it is hard to detect stable interaction between the CARD domains of RIG-I and that of MAVS. Therefore, the underlying biochemical mechanisms of how activation signals are transduced from a cytosolic receptor, RIG-I, to a mitochondria signaling protein, MAVS, is not clear. Protein ubiquitination have pivotal roles in diverse cell signaling pathways, including those in the immune system. A ubiquitin E3 ligase, TRIM25, promotes Lysine 63-linked polyubiquitination of RIG-I, and is essential for the RIG-I antiviral pathway. However, the biochemical mechanisms by which RIG-I is activated by ubiquitin remain poorly understood. In addition, whether ubiquitination is involved in MDA5 activation is unknown. Here, I describe the development of an in vitro biochemical assay to detect MAVS activation by a cytosolic activator. Purification of this activator revealed an important role of ubiquitin in RIG-I activation. Biochemical characterization of the ubiquitin induced RIG-I activation process revealed a new mechanism by which ubiquitin regulates cell signaling that involves the formation of a polyubiquitin-induced tetrameric complex. In addition, polyubiquitin-induced oligomerization appears to be a conserved mechanism for both MDA5 and RIG-I activation.