Abstract
Arenavirus RNA genomes are initiated by a "prime and realign" mechanism, such that the initiating GTP is found as a single unpaired (overhanging) nucleotide when the complementary genome ends anneal to form double-stranded (ds) RNA panhandle structures. dsRNAs modeled on these structures do not induce interferon (IFN), as opposed to blunt-ended (5' ppp)dsRNA. This study examines whether these viral structures can also act as decoys, by trapping RIG-I in inactive dsRNA complexes. We examined the ability of various dsRNAs to activate the RIG-I ATPase (presumably a measure of helicase translocation on dsRNA) relative to their ability to induce IFN. We found that there is no simple relationship between these two properties, as if RIG-I can translocate on short dsRNAs without inducing IFN. Moreover, we found that (5' ppp)dsRNAs with a single unpaired 5' ppp-nucleotide can in fact competitively inhibit the ability of blunt-ended (5' ppp)dsRNAs to induce IFN when co-transfected into cells and that this inhibition is strongly dependent on the presence of the 5' ppp. In contrast, (5' ppp)dsRNAs with a single unpaired 5' ppp-nucleotide does not inhibit poly(I-C)-induced IFN activation, which is independent of the presence of a 5' ppp group.
Highlights
Interaction with the synonymous domain of IPS-1 via conformational changes in RIG-I induced upon binding of PAMP RNAs to the CTD [8]
This study examines the binding of various RNAs to RIG-I and their ability to stimulate its ATPase, and it examines whether 5Ј pppdsRNAs whose 5Ј ppp-nucleotide is unpaired can competitively inhibit the ability of blunt-ended 5Ј pppdsRNA to induce IFN
Because modified nucleosides were shown to interfere with the ability of 5Ј pppRNAs to induce IFN via RIG-I [4], a 54-nt-long RNA1 was designed to contain 46 nt of spacer between the patch of biotinylated uridines at the 3Ј-end and the 5Ј ppp “business end” of the RNA, to minimize the effect of these modified nucleosides on RIG-I interaction
Summary
Interaction with the synonymous domain of IPS-1 (the central adaptor of the pathway to IFN activation) via conformational changes in RIG-I induced upon binding of PAMP RNAs to the CTD [8]. Given that this 3-nt bulge reduces the ability of blunt-ended 5Ј pppdsRNA to activate the IFN promoter ϳ2-fold (Fig. 5A) and that 5Ј OHdsRNA is inactive in this respect, there does not appear to be a simple relationship between the ability of dsRNAs to stimulate RIG-I ATPase activity and to induce IFN.
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