Induction of type I interferon by RNA viruses: cellular receptors and their substrates

Baum, Alina; García-Sastre, Adolfo

The carboxy-terminal regulatory domain (RD), also referred to as the repressor domain or carboxy-terminal domain (CTD) of RIG-I has proven to contain multiple diverse functions critical to RIG-I activity. Through mutational analysis, this domain was identified to posses the repressor activity responsible for self-inhibition, and constructs lacking the RD are constitutively active. The repression of signaling likely occurs through intramolecular association between the RD and both the CARD and helicase domains (Saito et al. 2007b; Takahasi et al. 2008 LGP2) . RD is also required for homo-(RIG-I, MDA5) and hetero-(LGP2) dimer formation, necessary for signaling by these receptors. The RD of RIG-I additionally provides a unique function of autorepression, and RIG-I constructs lacking the RD domain constitutively induce IFN in the absence of RNA stimuli. *Activity has not been shown directly and is assumed based on sequence similarity to the helicase domain of RIG-I the unfolding of the molecule and exposure of the CARD allowing for downstream signaling. Surprisingly, the RD domain and not the helicase domain was also identified as the primary RNA recognition domain of RIG-I Takahasi et al. 2008) . Structural studies of the RD have revealed a basic groove located on one side of this domain and an acidic surface on the opposite side. The basic groove is believed to serve as a site of 5 0 ppp-RNA recognition since mutation of key residues (K858, K888, and H830) within this region led to a loss of RNA binding in vitro and inability of the mutant RIG-I to rescue the phenotype of RIG-I -/-MEFs (Takahasi et al. 2008 ). The acidic surface on the opposite side of the RD presents a suitable area for interaction with the CARD domain of RIG-I. In addition to signaling repression and RNA recognition, the RD domain has also been characterized as being required for RIG-I dimerization. Like full length RIG-I, the RD alone forms dimers in vitro in the presence of 5 0 ppp-RNA; unlike RIG-I-DRD which is unable to dimerize in the presence of 5 0 ppp-RNA or synthetic dsRNA. Complex formation is also observed between wild-type RIG-I and the RD alone or in conjunction with the helicase domain, providing a mechanism for the dominant negative phenotype of those mutants Saito et al. 2007b; Yoneyama et al. 2004 ). The exact role of the helicase domain in RIG-I activity has been the most challenging to elucidate. The biochemical roles of this domain can be separated into two related but separate enzymatic functions, ATPase activity and helicase/ translocase activity. As with other helicases of the DExD/H family, ATP hydrolysis is required for the helicase function of RIG-I. In support of the ATPase requirement are mutational studies illustrating that walker-type ATP binding site mutants possess a dominant-negative phenotype Yoneyama et al. 2004 ). Additionally, a direct relationship between ATPase activity and immunostimulatory potential of RNA molecules is illustrated by in vitro biochemical analysis with purified RIG-I protein.