dsRNA regulation of the cytosolic innate immune system

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Precise control of protein synthesis is essential for maintenance of normal cellular function and is central to innate antiviral responses within the cell. For example, the protein 2’-5’-oligoadenylate synthetase (OAS) family of proteins detects cytosolic double-stranded (ds)RNA and initiates a translational control response via activation of the latent ribonuclease L (RNase L), to limit viral protein synthesis and thus replication. Structures of OAS1 and OAS1-dsRNA complexes have revealed important insights into OAS1 activation: dsRNA binding drives a functionally essential reorganization of the OAS1 active site. For OAS3, individual domain structures suggest that key elements of the activation process are similar but functionally divided among its three OAS domains, with one (catalytically inactive) domain primarily dictating dsRNA binding and a another distant (active) OAS domain responsible for 2’-5’-oligoadenylate synthesis. However, our published studies on OAS1-activating RNA sequence and structural motifs, and extensive new preliminary data presented here, strongly argue that we still have an incomplete understanding of how specific RNA features and their contexts combine to drive potent activation of OAS1 and OAS3. In particular, our unpublished studies indicate that the extent of OAS3 activation is dependent on the action of some of the same RNA molecular signatures (sequence and structural motifs) that we have defined for OAS1. This proposal continues our innovative, multidisciplinary study with a specific focus on defining the RNA features and contexts responsible for driving OAS1 and OAS3 activation and their resultant impacts on the cellular antiviral response. In Aim 1, we will continue deciphering the “rules” that govern OAS1 activation using in vitro biochemical and human cell-based assays, coupled with biophysical and structural approaches. Specifically, we will define: 1) how placement of common RNA structural motifs affects the function of OAS1-activating RNA signatures; 2) the molecular mechanism of action of dsRNA terminal motifs capable of strongly enhancing OAS1 activation; 3) the proposed role for OAS1 oligomerization on dsRNA for full enzyme activation; and 4) the basis of OAS1 activation by the SARS-CoV-2 5’-UTR and the impact of disrupting the OAS/RNase L pathway on viral propagation. In Aim 2, we will use high- resolution single-particle cryo-EM to determine the structures of full-length OAS3 bound to two cellular non- coding RNAs. These studies will reveal the molecular basis for OAS3 activation by 1) a defined dsRNA region larger than the accepted 50 base pair (bp) minimum length, and 2) an RNA with less than 50bp but containing a unique RNA tertiary motif that we hypothesize drives a distinct mode of OAS3 activation. These studies will also provide a launch point to begin defining OAS3’s dependence on specific RNA sequence and structural motifs for its potent activation as we have done for OAS1. Collectively, these studies will substantially deepen our fundamental understanding of OAS/RNase L pathway regulation by RNA and may offer an essential foundation for development of novel, generally applicable anti-viral therapeutic approaches.