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

Baum, Alina; García-Sastre, Adolfo

In line with RIG-I's prominent role in innate antiviral immunity, the quest for its physiological substrates has been a major focus of research in recent years. Based on the fact that RIG-I is an RNA helicase and was shown early onto directly interact with poly(I:C), the substrate responsible for its induction was initially assumed to be viral dsRNA. However, following up on earlier observations that siRNAs produced by in vitro transcription were capable of inducing an antiviral response, two independent groups showed that in vitro transcribed (IVT) RNA molecules, of at least 19nt, bearing a 5 0 ppp end can efficiently induce RIG-I (Hornung et al. 2006; Pichlmair et al. 2006) . The importance of the 5 0 ppp was demonstrated by the loss of IFN induction following Calf Intestinal Alkaline Phosphotase (CIP) treatment of the RNA and by the fact that a synthetic ssRNA (with a 5 0 OH group) of same sequence was not capable of inducing IFN. Furthermore, RNA isolated from influenza A or rabies viruses also lost IFN stimulation activity following CIP treatment and was unable to induce an IFN response in RIG-I -/-MEFs, thereby confirming that the presence of phosphates on an RNA molecule is a physiologically relevant, RIG-I specific PAMP . Small RNA generated by measles virus polymerase in vitro (likely containing short leader RNA) also induced IFN-b when transfected into cells (Plumet et al. 2007 ). Thus, it appeared that the presence of a 5 0 ppp on an RNA molecule, either single-or double-stranded was sufficient for activation of RIG-I. The concept of a 5 0 ppp PAMP is appealing in that most RNA viruses contain 5 0 ppp in their genomes and antigenomes, making this motif an inherent part of a viral lifecycle and unlikely to be mutated under immune system pressure. On the other hand, cellular RNAs lack exposed 5 0 ppp as a result of mRNA capping or processing of 5 0 ppp into monophosphates (Nallagatla et al. 2008 ). In addition to removal of phosphate groups, cellular RNAs are also extensively modified as a result of incorporation of modified nucleotides or methylation. These modifications likely play a role in prevention of an antiviral response to cellular RNA. In fact, incorporation of pseudourudine, 2-thiouridine, or 2 0 O-methylated uridine into T7 transcripts strongly inhibited IFN production by those RNA molecules (Hornung et al. 2006) . As a way to prevent exposure of the 5 0 ppp to the cell, viruses are thought to hide these molecules in tightly packed nucleocapsids or replicate in cellular compartments physically removed from the sensors (Nallagatla et al. 2008) . Members of the bunyaviridae family were shown to remove the triphosphate group from their genomes, thereby creating genomes that no longer interact with RIG-I in vitro (Habjan et al. 2008) . Since the discovery of the 5 0 ppp as a PAMP in 2006, it has become clearly established as a RIG-I specific recognition motif. However, challenging the earlier notion that the 5 0 ppp moiety was sufficient for RIG-I induction are two recent reports that illustrate a requirement of a double-stranded component in addition to the triphosphate. Both studies made use of previously unavailable, synthetic 5 0 ppp ssRNA and observed that this molecule was not capable of inducing IFN when introduced into cells. However, the same RNA molecule when generated by T7 in vitro transcription served as a competent activator of the IFN response. The discrepancy appears to result from aberrant transcription