Yoneyama and colleagues identified the interferon-inducing cytoplasmic DexD/ H box RNA helicase RIG-I, containing a caspase recruitment domain (CARD) (Yoneyama et al. 2004). Expression of the CARD domain sensitized cells to activate the transcription factor IRF3, leading to the induction of the IFN-ß promoter. Later on it was shown that this pathway involves the IRF3 kinase TBK1, which is activated by the newly characterized adaptor protein IPS-1, also known as Cardif, MAVS, or VISA (Kawai et al. 2005; Meylan et al. 2005; Seth et al. 2005; Xu et al. 2005; reviewed in Sen and Sarkar 2005). In overexpression experiments, RIG-I was shown to bind poly I:C. However, overexpression of a dominant negative mutant of RIG-I impaired IRF3 activation by Newcastle disease virus (NDV), a negative-strand RNA virus, while IRF3 activation by poly I:C was not inhibited. Subsequent studies with RIG-I-/- mice and MEFs (Kato et al. 2006) showed no defect in the response to poly I:C.
Hornung and colleagues (2006) demonstrated that RIG-I detects in vitro transcribed RNA. RNA with a triphosphate at the 5' end (now termed 3pRNA), which is generated during in vitro transcription, was identified to be the ligand for RIG-I. The minimal length of 3pRNA was 19 nucleotides. The activity of 3pRNA was independent of double-strand formation. Both exogenous 3pRNA trans-fected into the cell and endogenously formed 3pRNA (expression of T7 RNA polymerase) activated RIG-I. Genomic RNA prepared from a negative-strand RNA virus and RNA prepared from virus-infected cells, but not RNA from non-infected cells, triggered a potent IFN-a response in a 5'-triphosphate-dependent manner. Binding studies of RIG-I and 3pRNA revealed a direct molecular interaction. The 5' capping or incorporation of modified nucleotides such as pseu-douridine, 2-thiouridine, and 2'-O-methylated uridine in place of uridine in short 3pRNA strongly diminished IFN-a induction. In a parallel study, Pichlmair et al. (2006) attributed the inhibitory effect of the influenza virus protein NS1 to its binding and inhibition of the RIG-I triphosphate RNA complex.
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