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

Baum, Alina; García-Sastre, Adolfo

events generated by the T7 polymerase. When T7 products were analyzed by gel electrophoresis and sequencing, it was observed that the RNA mixture contained a significant number of RNA molecules of double-stranded nature. After polyacrylamide gel separation, the products corresponding to the true ssRNA size were no longer capable of inducing an IFN response upon transfection into cells. Further characterization of RIG-I activation requirements showed that dsRNA complementarity of at least 10-18 nt was required at the 5 0 ppp containing end in order to induce RIG-I activity. The nature of these types of RNA molecules fits nicely with the structure of RNA virus panhandles and the ends of copy back defective interfering (DIs) genomes from negative strand RNA viruses, which are characterized as very potent IFN inducers (Strahle et al. 2006 ). However, since very short synthetic (19-24 nt) RNA molecules were analyzed in these studies, it remains to be seen whether dsRNA structures complementary to the 5 0 ppp end of an RNA molecule will be required for RIG-I activation with its natural substrates. Since longer products of T7 transcription also induce IFN, it should also be determined whether these RNAs are produced with similar 5 0 dsRNA characteristics. Based on numerous studies, it is very likely that all dsRNA molecules regardless of length, sequence, phosphates, or overhangs are capable of binding RIG-I. The question of why some of those molecules induce RIG-I mediated IFN induction while others do not has been recently addressed. In a study by Takahasi et al. (2008) , RIG-I was found to be able to bind any dsRNA molecule regardless of presence of 3 0 or 5 0 overhangs, contrary to some earlier findings by Marques et al. (2006) , and ssRNA molecules containing a 5 0 -ppp, but not ssRNA containing a 5 0 -OH group or a 5 0 -monophosphate. This group also found that dsRNA molecules which possessed even a single monophosphate on one RNA strand were still able to activate RIG-I and induce IFN. Taking into account reports by Schlee et al. and Schmidt et al. on the nature of T7 transcribed RNA products it becomes more challenging to interpret this data since it is unclear whether true ssRNA species were analyzed. In addition, the induction of IFN by short dsRNA with a monophosphate is in disagreement with reports of Schlee et al. and Schmidt et al. which showed that a 5 0 monophosphate group on a dsRNA molecule was not sufficient for IFN induction. Therefore, it appears that slight differences between the RNA molecules or cells used in the three studies might account for the discrepancy of whether a 5 0 ppp is required in the context of a short dsRNA, or if a single phosphate is sufficient for RIG-I activation. Nevertheless, it is clear that at least one phosphate group is required for induction of RIG-I signaling, when short dsRNA is used as substrate. The study by Takahasi et al. also provides some insightful information on the discrepancy between poly(I:C) binding and signaling. Poly(I:C) has been shown to bind RIG-I with very high affinity but has been characterized by many groups to signal through MDA5. To address this apparent discrepancy, partial protease digestion of RIG-I/poly(I:C) complex was performed and revealed that this interaction is different from that of RIG-I with 5 0 ppp-RNA, as different cleavage products were generated (Takahasi et al. 2008 ). It has not been established whether any viral RNAs can recapitulate the poly(I:C)