{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T15:07:28Z","timestamp":1785424048150,"version":"3.56.0"},"posted":{"date-parts":[[2026,6,30]]},"group-title":"Microbiology","reference-count":31,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,6,30]],"date-time":"2026-06-30T00:00:00Z","timestamp":1782777600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"DOI":"10.13039\/100000060","name":"National Institute of Allergy and Infectious Diseases","doi-asserted-by":"crossref","award":["R01AI133348"],"award-info":[{"award-number":["R01AI133348"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000060","name":"National Institute of Allergy and Infectious Diseases","doi-asserted-by":"crossref","award":["F31AI189061"],"award-info":[{"award-number":["F31AI189061"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"crossref"}]},{"id":[{"id":"https:\/\/ror.org\/04yhme281","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,6,30]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  RNA molecules form specific 3D structures that facilitate a variety of functions through interactions with other macromolecules. Many RNA viral genomes maintain these structures to interact with and evade host immunity machinery. One such element, the competitive inhibitor RNA (ciRNA), discovered in the protein coding region of the poliovirus serotype 1 (PV1) genome, inhibits a host antiviral protein, ribonuclease L (RNase L). Although some functionally essential structural motifs of the PV1 ciRNA have been studied, the extent of its evolutionary conservation and other structural requirements remained unexplored. Here we combined bioinformatic and biochemical techniques to further define the requirements of a functional ciRNA and assess its phylogenetic distribution. We systematically mutated ciRNA structural features, verifying that ciRNA inhibitory activity requires a conserved loop E motif and a long-range base-pairing interaction, but its peripheral stems are dispensable and in fact a circularly permuted version maintains function. A structure-based homology search identified potential ciRNAs across the\n                  <jats:italic>Picornaviridae<\/jats:italic>\n                  family, but only a subset of those tested were functional\u2014all are in\n                  <jats:italic>Enterovirus coxsackiepol<\/jats:italic>\n                  . When structural features needed for function were transposed from PV1 ciRNA to an RNA unable to inhibit RNase L, the chimeric RNAs did not gain wild-type function, and chemical probing data revealed that these nonfunctional RNAs are unable to form the correct secondary structure. Overall, the dual constraints of encoding a protein and forming a specific functional structure appear to not only limit the sequence diversity, but also the phylogenetic distribution, of ciRNAs.\n                <\/jats:p>","DOI":"10.64898\/2026.06.29.735259","type":"posted-content","created":{"date-parts":[[2026,6,30]],"date-time":"2026-06-30T18:38:00Z","timestamp":1782844680000},"source":"Crossref","is-referenced-by-count":0,"title":["Enterovirus RNase L inhibiting RNAs are highly conserved with limited phylogenetic distribution"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3264-9678","authenticated-orcid":false,"given":"Samantha I.","family":"Zangari","sequence":"first","affiliation":[{"name":"Department of Biochemistry & Molecular Biophysics, Columbia University, New York, NY, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6471-4243","authenticated-orcid":false,"given":"Madeline E.","family":"Sherlock","sequence":"additional","affiliation":[{"name":"New York Structural Biology Center, New York, NY, USA"},{"name":"Department of Molecular, Cell & Developmental Biology, University of California Santa Cruz, Santa Cruz, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3718-1891","authenticated-orcid":false,"given":"Jeffrey S.","family":"Kieft","sequence":"additional","affiliation":[{"name":"Department of Biochemistry & Molecular Biophysics, Columbia University, New York, NY, USA"},{"name":"New York Structural Biology Center, New York, NY, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026070313301150000_2026.06.29.735259v1.1","doi-asserted-by":"publisher","DOI":"10.1038\/273684a0"},{"key":"2026070313301150000_2026.06.29.735259v1.2","doi-asserted-by":"publisher","DOI":"10.3390\/v16030474"},{"key":"2026070313301150000_2026.06.29.735259v1.3","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.272.31.19193"},{"key":"2026070313301150000_2026.06.29.735259v1.4","doi-asserted-by":"publisher","DOI":"10.1089\/jir.2010.0120"},{"key":"2026070313301150000_2026.06.29.735259v1.5","doi-asserted-by":"crossref","first-page":"131","DOI":"10.1016\/S0079-6603(08)60008-2","article-title":"Structural elements in RNA","volume":"41","year":"1991","journal-title":"Prog Nucleic Acid Res Mol 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2\u2032-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) for direct, versatile and accurate RNA structure analysis","volume":"10","year":"2015","journal-title":"Nature Protocols 2015 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