SARS-CoV-2 5'-UTR stem-loops activate the antiviral protein oligoadenylate synthetase 1 (OAS1).

The innate immune system relies on pathogen recognition receptors, such as the 2',5'-oligoadenylate synthetase (OAS) proteins, to detect pathogen-associated molecular patterns like viral double-stranded (ds)RNA. A specific splicing variant of OAS1 (OAS1-p46) has been implicated in initiating an immune response that leads to decreased disease severity during SARS-CoV-2 infection. OAS1-p46 has a C-terminal lipid modification motif that allows for anchoring of the protein to intracellular membranes and thus potential colocalization with immunogenic viral RNA regions such as the SARS-CoV-2 5'-untranslated region (5'-UTR). Here, we show that OAS proteins can detect the 5'-structured elements (5'-SE)-comprising the 5'-UTR and three additional stem-loop structures-and activate the cellular RNase L pathway. Through systematic 3'-end truncations of the 5'-SE, we show that the smallest 5'-SE fragment capable of potently activating OAS1 is the conserved and highly structured SL1-4b region, containing the first four stem-loop structures and their intervening linking sequences (SL1-4), as well as an unstructured region ("SL4b") to the 3'-side of SL4. Analyses of OAS1 activation and RNA secondary structure probing using selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) of additional SL1-4b RNA variants suggests a model in which SL4 acts as the primary OAS1 interaction site, while the unstructured SL4b region and two other stem-loops (SL1 and SL3) are necessary for optimal presentation of this region for OAS1 activation. Our findings reveal a structurally complex viral RNA region that potently activates OAS1, underscoring the potential complexity of RNAs that can strongly activate this innate immune sensor.