{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T01:07:49Z","timestamp":1789088869979,"version":"build-2803163510"},"posted":{"date-parts":[[2026,7,3]]},"group-title":"Microbiology","reference-count":38,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,7,3]],"date-time":"2026-07-03T00:00:00Z","timestamp":1783036800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"name":"Singapore MOE Academic Research Fund (AcRF) Tier 2 Award"},{"name":"Singapore Nucleic Acid Therapeutics Initiative","award":["H25J5a0061"],"award-info":[{"award-number":["H25J5a0061"]}]},{"name":"National Institute of Allergy and Infectious Diseases of the NIH","award":["R01AI187483"],"award-info":[{"award-number":["R01AI187483"]}]},{"award":["PRG1154"],"award-info":[{"award-number":["PRG1154"]}],"id":[{"id":"https:\/\/ror.org\/00jjeja18","id-type":"ROR","asserted-by":"publisher"}]},{"award":["PRG3145"],"award-info":[{"award-number":["PRG3145"]}],"id":[{"id":"https:\/\/ror.org\/00jjeja18","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,7,3]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>Membrane-associated replication complexes (RCs) are a hallmark of alphaviruses, yet the mechanisms by which viral RNA is delivered into these compartments and coordinated with RNA synthesis remain incompletely understood. Here, we present a cryo\u2013electron microscopy (cryo-EM) structure of the Chikungunya virus (CHIKV) RC core, consisting of nsP1, nsP2, and nsP4, in complex with the replicative RNA substrates, revealing two spatially distinct RNA-binding sites. A single-stranded RNA (ssRNA) engages the nsP2 helicase and extends toward a pore formed at the nsP1\u2013nsP4 interface, whereas a double-stranded RNA (dsRNA) is accommodated within the central catalytic pocket of the nsP4 polymerase in an elongation configuration. Structural analysis suggests a helicase-assisted RNA threading model in which the 5\u2032 end of the viral genome is guided toward the spherule lumen through the RC pore. Concurrently, the nsP4 polymerase engages dsRNA in a manner consistent with RNA synthesis. This work provides a structural framework for understanding RNA trafficking and enzymatic coordination in viral replication organelles.<\/jats:p>\n                <jats:sec>\n                  <jats:title>Significance Statement<\/jats:title>\n                  <jats:p>Like many positive-sense RNA viruses, alphavirus replication occurs within membrane-bound spherules, but how viral RNA is delivered into these compartments and coupled to RNA synthesis has remained unclear. Here, we show that the CHIKV RC core contains two functionally distinct RNA-engagement modules: an nsP2-associated ssRNA path directed toward the nsP1\u2013nsP4 pore, and an nsP4-bound duplex RNA positioned in an elongation-like polymerase channel. These observations support a model in which alphavirus RCs integrate genome import and RNA synthesis within a single membrane-associated molecular machine. This work provides a structural framework for understanding RNA trafficking, replication-organelle function, and potential antiviral targeting of alphavirus replication.<\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.07.03.736289","type":"posted-content","created":{"date-parts":[[2026,7,4]],"date-time":"2026-07-04T01:50:11Z","timestamp":1783129811000},"source":"Crossref","is-referenced-by-count":1,"title":["Distinct RNA engagements define genome import and replication elongation in alphaviruses"],"prefix":"10.64898","author":[{"given":"Yaw Bia","family":"Tan","sequence":"first","affiliation":[{"name":"Lee Kong Chian School of Medicine, Nanyang Technological University, 59 Nanyang Drive, Singapore 636921"},{"name":"Institute of Structural Biology, Nanyang Technological University, 59 Nanyang Drive, Singapore 636921"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yongbo","family":"Luo","sequence":"additional","affiliation":[{"name":"Lee Kong Chian School of Medicine, Nanyang Technological University, 59 Nanyang Drive, Singapore 636921"},{"name":"Institute of Structural Biology, Nanyang Technological University, 59 Nanyang Drive, Singapore 636921"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ping","family":"Liu","sequence":"additional","affiliation":[{"name":"Lee Kong Chian School of Medicine, Nanyang Technological University, 59 Nanyang Drive, Singapore 636921"},{"name":"Institute of Structural Biology, Nanyang Technological University, 59 Nanyang Drive, Singapore 636921"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andres","family":"Mertis","sequence":"additional","affiliation":[{"name":"University of Tartu, Institute of Bioengineering, Tartu, Estonia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7637-7275","authenticated-orcid":false,"given":"Dahai","family":"Luo","sequence":"additional","affiliation":[{"name":"Lee Kong Chian School of Medicine, Nanyang Technological University, 59 Nanyang Drive, Singapore 636921"},{"name":"Institute of Structural Biology, Nanyang Technological University, 59 Nanyang Drive, Singapore 636921"},{"name":"National Centre for Infectious Diseases, 16 Jln Tan Tock Seng, Singapore 308442"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026070702000476000_2026.07.03.736289v1.1","doi-asserted-by":"publisher","DOI":"10.1017\/S0950268808001167"},{"key":"2026070702000476000_2026.07.03.736289v1.2","doi-asserted-by":"publisher","DOI":"10.1016\/j.medmal.2015.05.007"},{"key":"2026070702000476000_2026.07.03.736289v1.3","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pntd.0000389"},{"key":"2026070702000476000_2026.07.03.736289v1.4","doi-asserted-by":"crossref","unstructured":"Freedman, D.O. , A new non-live chikungunya vaccine for travellers. 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