{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T05:25:31Z","timestamp":1787030731877,"version":"build-2736575974"},"reference-count":48,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2026,8,15]],"date-time":"2026-08-15T00:00:00Z","timestamp":1786752000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000057","name":"US National Institute of General Medical Sciences","doi-asserted-by":"crossref","award":["R01GM130950"],"award-info":[{"award-number":["R01GM130950"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"crossref"}]},{"award":["R01GM130950"],"award-info":[{"award-number":["R01GM130950"]}],"id":[{"id":"https:\/\/ror.org\/04q48ey07","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/100002590","name":"American Lung Association","doi-asserted-by":"publisher","award":["IA-831472"],"award-info":[{"award-number":["IA-831472"]}],"id":[{"id":"10.13039\/100002590","id-type":"DOI","asserted-by":"publisher"}]},{"award":["IA-831472"],"award-info":[{"award-number":["IA-831472"]}],"id":[{"id":"https:\/\/ror.org\/01p3rh655","id-type":"ROR","asserted-by":"publisher"}]},{"name":"SECM4 Consortium at Florida State University","award":["NIH U24 GM116788"],"award-info":[{"award-number":["NIH U24 GM116788"]}]},{"award":["NIH U24 GM116788"],"award-info":[{"award-number":["NIH U24 GM116788"]}],"id":[{"id":"https:\/\/ror.org\/05g3dte14","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Midwest Consortium at Purdue University","award":["NIH U24 GM116789"],"award-info":[{"award-number":["NIH U24 GM116789"]}]},{"name":"NIH Common Fund Transformative High-Resolution Cryo-Electron Microscopy program","award":["U24 GM129539"],"award-info":[{"award-number":["U24 GM129539"]}]},{"award":["U24 GM129539"],"award-info":[{"award-number":["U24 GM129539"]}],"id":[{"id":"https:\/\/ror.org\/001d55x84","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000893","name":"Simons Foundation","doi-asserted-by":"publisher","award":["SF349247"],"award-info":[{"award-number":["SF349247"]}],"id":[{"id":"10.13039\/100000893","id-type":"DOI","asserted-by":"publisher"}]},{"award":["SF349247"],"award-info":[{"award-number":["SF349247"]}],"id":[{"id":"https:\/\/ror.org\/01cmst727","id-type":"ROR","asserted-by":"publisher"}]},{"name":"New York State Assembly"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Microorganisms"],"abstract":"<jats:p>Respiratory syncytial virus (RSV) RNA synthesis relies on RNA-dependent RNA polymerase, a complex composed of the L and P proteins. We recently resolved four major structural states of the RSV nucleotide addition cycle (NAC), establishing a framework for viral RNA elongation. However, reaction-derived complexes are influenced by catalytic efficiency and intermediate occupancy and may incompletely sample the NAC conformational landscape. Here, we report two cryo-electron microscopy (cryo-EM) structures of unreacted RSV polymerase bound to a post-translocation RNA product mimic. One structure closely recapitulates the previously determined reaction-derived post-translocation state, demonstrating that the canonical architecture can be recovered independently of nucleotide incorporation. The second structure reveals an additional post-translocation intermediate that retains the same RNA register and vacant +1 product-binding site while adopting a two-domain L organization. Structural comparisons further identify localized rearrangements near the catalytic center, including changes in the motif A loop and supporting helix. Together, these findings expand the structural landscape of the RSV post-translocation stage and refine the current NAC model. More broadly, our results highlight unreacted state-matched complexes as a complementary approach to reaction-derived structures for capturing more complete structural snapshots of dynamic reaction cycles.<\/jats:p>","DOI":"10.3390\/microorganisms14081801","type":"journal-article","created":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T08:05:41Z","timestamp":1786953941000},"page":"1801","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Unreacted RSV Polymerase Structures Expand the Post-Translocation Landscape of the Nucleotide Addition Cycle"],"prefix":"10.3390","volume":"14","author":[{"given":"Dongdong","family":"Cao","sequence":"first","affiliation":[{"name":"Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eleanore","family":"Zhou","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jun","family":"Sim","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5617-9228","authenticated-orcid":false,"given":"Bo","family":"Liang","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,15]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"ofab159","DOI":"10.1093\/ofid\/ofab159","article-title":"The Global Epidemiology of RSV in Community and Hospitalized Care: Findings from 15 Countries","volume":"8","author":"Staadegaard","year":"2021","journal-title":"Open Forum Infect. Dis."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"77","DOI":"10.15585\/mmwr.mm7506a2","article-title":"Respiratory Virus Activity\u2014United States, July 1, 2024-June 30, 2025","volume":"75","author":"Silk","year":"2026","journal-title":"MMWR Morb. Mortal. Wkly. Rep."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"946","DOI":"10.1016\/S0140-6736(17)30938-8","article-title":"Global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in young children in 2015: A systematic review and modelling study","volume":"390","author":"Shi","year":"2017","journal-title":"Lancet"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"986","DOI":"10.1136\/thoraxjnl-2018-212212","article-title":"Respiratory syncytial virus (RSV): A scourge from infancy to old age","volume":"74","author":"Coultas","year":"2019","journal-title":"Thorax"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"29","DOI":"10.1016\/j.ccm.2016.11.010","article-title":"Respiratory Syncytial Virus Infection: An Illness for All Ages","volume":"38","author":"Walsh","year":"2017","journal-title":"Clin. Chest Med."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1967","DOI":"10.1007\/s00705-019-04247-4","article-title":"Taxonomy of the order Mononegavirales: Update 2019","volume":"164","author":"Amarasinghe","year":"2019","journal-title":"Arch. Virol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"002134","DOI":"10.1099\/jmm.0.002134","article-title":"Respiratory syncytial virus\u2014From discovery to vaccines","volume":"75","author":"Lopez","year":"2026","journal-title":"J. Med. Microbiol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"25151355241310601","DOI":"10.1177\/25151355241310601","article-title":"The recent landscape of RSV vaccine research","volume":"13","author":"Kelleher","year":"2025","journal-title":"Ther. Adv. Vaccines Immunother."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Liu, H., Zhong, H., and Liu, H. (2026). Respiratory syncytial virus (RSV) antibody and small-molecule drugs: Current status of clinical translation and challenges. Front. Microbiol., 17.","DOI":"10.3389\/fmicb.2026.1745808"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"130365","DOI":"10.1016\/j.bmcl.2025.130365","article-title":"Recent advances of the respiratory syncytial virus inhibitors","volume":"129","author":"Shi","year":"2025","journal-title":"Bioorg. Med. Chem. Lett."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"303","DOI":"10.1038\/s41579-020-00501-8","article-title":"Structural insights into RNA polymerases of negative-sense RNA viruses","volume":"19","author":"Grimes","year":"2021","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Cao, D., Gao, Y., and Liang, B. (2021). Structural Insights into the Respiratory Syncytial Virus RNA Synthesis Complexes. Viruses, 13.","DOI":"10.3390\/v13050834"},{"key":"ref_13","first-page":"61","article-title":"Transcription and replication of nonsegmented negative-strand RNA viruses","volume":"283","author":"Whelan","year":"2004","journal-title":"Curr. Top. Microbiol. Immunol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"368","DOI":"10.1038\/s41467-019-14246-3","article-title":"Cryo-EM structure of the respiratory syncytial virus RNA polymerase","volume":"11","author":"Cao","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"193","DOI":"10.1016\/j.cell.2019.08.014","article-title":"Structure of the Respiratory Syncytial Virus Polymerase Complex","volume":"179","author":"Gilman","year":"2019","journal-title":"Cell"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"611","DOI":"10.1038\/s41586-023-06867-y","article-title":"Structures of the promoter-bound respiratory syncytial virus polymerase","volume":"625","author":"Cao","year":"2024","journal-title":"Nature"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"5854","DOI":"10.1038\/s41467-026-72519-0","article-title":"Orchestrated and dynamic nucleotide addition cycle during respiratory syncytial virus early-stage elongation","volume":"17","author":"Cao","year":"2026","journal-title":"Nat. Commun."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"290","DOI":"10.1038\/nmeth.4169","article-title":"cryoSPARC: Algorithms for rapid unsupervised cryo-EM structure determination","volume":"14","author":"Punjani","year":"2017","journal-title":"Nat. Methods"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"188","DOI":"10.1016\/j.jsb.2015.08.007","article-title":"Alignment of cryo-EM movies of individual particles by optimization of image translations","volume":"192","author":"Rubinstein","year":"2015","journal-title":"J. Struct. Biol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1605","DOI":"10.1002\/jcc.20084","article-title":"UCSF Chimera--a visualization system for exploratory research and analysis","volume":"25","author":"Pettersen","year":"2004","journal-title":"J. Comput. Chem."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"486","DOI":"10.1107\/S0907444910007493","article-title":"Features and development of Coot","volume":"66","author":"Emsley","year":"2010","journal-title":"Acta Crystallogr. D Biol. Crystallogr."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1107\/S0907444909052925","article-title":"PHENIX: A comprehensive Python-based system for macromolecular structure solution","volume":"66","author":"Adams","year":"2010","journal-title":"Acta Crystallogr. D Biol. Crystallogr."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"12","DOI":"10.1107\/S0907444909042073","article-title":"MolProbity: All-atom structure validation for macromolecular crystallography","volume":"66","author":"Chen","year":"2010","journal-title":"Acta Crystallogr. D Biol. Crystallogr."},{"key":"ref_24","unstructured":"Schrodinger, LLC (2022). The PyMOL Molecular Graphics System, Version 2.5.4, Schrodinger, LLC."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"439","DOI":"10.1107\/S2059798324004881","article-title":"Introduction of the Capsules environment to support further growth of the SBGrid structural biology software collection","volume":"80","author":"Herre","year":"2024","journal-title":"Acta Crystallogr. D Struct. Biol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"275","DOI":"10.1038\/s41586-019-1759-1","article-title":"Structure of the human metapneumovirus polymerase phosphoprotein complex","volume":"577","author":"Pan","year":"2020","journal-title":"Nature"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1016\/j.celrep.2019.12.024","article-title":"Structure of the Vesicular Stomatitis Virus L Protein in Complex with Its Phosphoprotein Cofactor","volume":"30","author":"Jenni","year":"2020","journal-title":"Cell Rep."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"314","DOI":"10.1016\/j.cell.2015.06.018","article-title":"Structure of the L Protein of Vesicular Stomatitis Virus from Electron Cryomicroscopy","volume":"162","author":"Liang","year":"2015","journal-title":"Cell"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"2099","DOI":"10.1073\/pnas.1918809117","article-title":"Structure of a rabies virus polymerase complex from electron cryo-microscopy","volume":"117","author":"Horwitz","year":"2020","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"3080","DOI":"10.1038\/s41467-025-58308-1","article-title":"Structural insights into the RNA-dependent RNA polymerase complexes from highly pathogenic Marburg and Ebola viruses","volume":"16","author":"Li","year":"2025","journal-title":"Nat. Commun."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"603","DOI":"10.1038\/s41586-023-06608-1","article-title":"Molecular mechanism of de novo replication by the Ebola virus polymerase","volume":"622","author":"Peng","year":"2023","journal-title":"Nature"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"394","DOI":"10.1038\/s41586-022-05271-2","article-title":"Structure of the Ebola virus polymerase complex","volume":"610","author":"Yuan","year":"2022","journal-title":"Nature"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"7508","DOI":"10.1038\/s41467-025-62906-4","article-title":"The structure of the mammalian bornavirus polymerase complex","volume":"16","author":"Carrique","year":"2025","journal-title":"Nat. Commun."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"gkaf1413","DOI":"10.1093\/nar\/gkaf1413","article-title":"Structure and function of the RNA polymerase complex of Borna disease virus, a nuclear-replicating non-segmented negative-strand RNA virus","volume":"54","author":"Gibbs","year":"2026","journal-title":"Nucleic Acids Res."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"e2504779122","DOI":"10.1073\/pnas.2504779122","article-title":"Structural insights into the dynamic mechanism of bornavirus polymerase","volume":"122","author":"Yang","year":"2025","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"563","DOI":"10.1038\/s44318-024-00321-z","article-title":"Structure of the Nipah virus polymerase complex","volume":"44","author":"Balikci","year":"2025","journal-title":"EMBO J."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"688","DOI":"10.1016\/j.cell.2024.12.021","article-title":"Structural and functional analysis of the Nipah virus polymerase complex","volume":"188","author":"Hu","year":"2025","journal-title":"Cell"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"10524","DOI":"10.1038\/s41467-024-54994-5","article-title":"Cryo-EM structure of Nipah virus L-P polymerase complex","volume":"15","author":"Peng","year":"2024","journal-title":"Nat. Commun."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"2261","DOI":"10.1038\/s41467-025-57219-5","article-title":"Structural basis of Nipah virus RNA synthesis","volume":"16","author":"Sala","year":"2025","journal-title":"Nat. Commun."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"eadr7116","DOI":"10.1126\/sciadv.adr7116","article-title":"Cryo-EM structure of Nipah virus RNA polymerase complex","volume":"10","author":"Wang","year":"2024","journal-title":"Sci. Adv."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"705","DOI":"10.1093\/procel\/pwaf014","article-title":"Cryo-EM structures of Nipah virus polymerase complex reveal highly varied interactions between L and P proteins among paramyxoviruses","volume":"16","author":"Xue","year":"2025","journal-title":"Protein Cell"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"8673","DOI":"10.1038\/s41467-024-52701-y","article-title":"Structure of the Nipah virus polymerase phosphoprotein complex","volume":"15","author":"Yang","year":"2024","journal-title":"Nat. Commun."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"6655","DOI":"10.1038\/s41467-025-61764-4","article-title":"Cryo-EM structures of Nipah virus polymerases and high-throughput RdRp assay development enable anti-NiV drug discovery","volume":"16","author":"Chen","year":"2025","journal-title":"Nat. Commun."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"4931","DOI":"10.1073\/pnas.1919837117","article-title":"Structure of a paramyxovirus polymerase complex reveals a unique methyltransferase-CTD conformation","volume":"117","author":"Abdella","year":"2020","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"3163","DOI":"10.1038\/s41467-024-47470-7","article-title":"Structural basis for dimerization of a paramyxovirus polymerase complex","volume":"15","author":"Xie","year":"2024","journal-title":"Nat. Commun."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"4189","DOI":"10.1038\/s41467-024-48389-9","article-title":"Structures of the mumps virus polymerase complex via cryo-electron microscopy","volume":"15","author":"Li","year":"2024","journal-title":"Nat. Commun."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"3819","DOI":"10.1038\/s41467-025-58985-y","article-title":"Structure of the measles virus ternary polymerase complex","volume":"16","author":"Wang","year":"2025","journal-title":"Nat. Commun."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"1324","DOI":"10.1038\/s41467-023-37012-y","article-title":"Structure of the Newcastle Disease Virus L protein in complex with tetrameric phosphoprotein","volume":"14","author":"Cong","year":"2023","journal-title":"Nat. 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