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Among these elements, four pseudoknots (PKs) at the viral 3\u2032-terminus, designated as SLII, SLIV, DBI, and DBII, are among the most crucial players in the overall flaviviral lifecycle. While many studies have focused on exploring the behavior of individual PKs, we investigated the collective role of all four PKs in viral growth and subgenomic flaviviral RNA (sfRNA) formation. Through mutational analyses and infectious models, we establish that the four PKs are interdependent and work synergistically to aid in the folding and compaction of the WNV 3\u2032-terminal region. A striking hierarchy is observed in PK contributions to global folding and sfRNA formation, whereby SLIV plays the largest role, followed by DBI, DBII, and SLII. We also discover highly conserved RNA tertiary motifs within the PK assembly that are shared across flaviviruses, suggesting a new type of druggable target that may be of value in the search for pan-flaviviral therapeutics.<\/jats:p>","DOI":"10.1093\/nar\/gkag889","type":"journal-article","created":{"date-parts":[[2026,9,10]],"date-time":"2026-09-10T11:51:34Z","timestamp":1789041094000},"source":"Crossref","is-referenced-by-count":0,"title":["Interdependent RNA structural motifs at the 3\u2032-terminus of the West Nile virus genome regulate viral growth"],"prefix":"10.1093","volume":"54","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4228-8332","authenticated-orcid":false,"given":"Lucille H","family":"Tsao","sequence":"first","affiliation":[{"name":"Department of Chemistry, Yale University , New Haven, CT 06511 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Doug E","family":"Brackney","sequence":"additional","affiliation":[{"name":"Department of Entomology, Connecticut Agricultural Experimental Station, State of Connecticut , New Haven, CT 06511 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9045-8872","authenticated-orcid":false,"given":"Anna Marie","family":"Pyle","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Yale University , New Haven, CT 06511 ,","place":["United States"]},{"name":"Department of Molecular, Cellular, and Developmental Biology, Yale University , New Haven, CT 06511 ,","place":["United States"]},{"name":"Howard Hughes Medical Institute , Chevy Chase, MD 20815 ,","place":["United 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