{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,6,23]],"date-time":"2026-06-23T11:49:18Z","timestamp":1782215358129,"version":"3.54.5"},"posted":{"date-parts":[[2026,6,19]]},"group-title":"Microbiology","reference-count":29,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,6,19]],"date-time":"2026-06-19T00:00:00Z","timestamp":1781827200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,6,19]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Packaging of viral genomes into progeny virions is a critical step in the viral life cycle. Coronaviruses such as SARS-CoV-2 possess unusually large RNA genomes (\u223c30 kb), yet the mechanism by which these genomes are selectively condensed and incorporated into virions remains poorly understood. Here, we demonstrate that the SARS-CoV-2 genome contains multiple dispersed RNA structures, termed packaging signals (PSs), which cooperate with a dominant central PS to direct genomic RNA incorporation into infectious particles. We identify the dominant PS within the coding region of the\n                  <jats:italic>nsp15<\/jats:italic>\n                  gene, downstream of a packaging signal previously described in Embecoviruses. Using virus-like particles (VLPs), we investigate its role in virion assembly and selective genomic RNA packaging, and show that it promotes the formation of ribonucleoprotein (RNP) complexes with the viral nucleocapsid protein (N), as revealed by mass photometry. Notably, we uncover a unique N\u2013induced conformational rearrangement of the PS RNA, from an extended structure to a double stem-loop architecture. This dominant PS acts together with a nearby stem-loop element to assemble a higher-order RNP complex containing 12 N-protein dimers. Using a SARS-CoV-2 replicon system, we further demonstrate functional cooperativity between the dominant PS, its proximal partner stem-loop, and additional packaging elements located approximately 10 kb upstream. Collectively, our findings support a highly dynamic and cooperative mechanism of SARS-CoV-2 genome packaging that relies on multiple dispersed packaging signals organized around a dominant central PS. These insights provide a mechanistic framework for understanding coronavirus genome packaging and reveal new opportunities for antiviral intervention through disruption of this process. They also have important implications for vaccine development and may enable the design of membrane-based vector systems capable of efficiently delivering large nucleic acid cargoes, expanding the potential of bionanotechnology and genetic medicine.\n                <\/jats:p>","DOI":"10.64898\/2026.06.18.733228","type":"posted-content","created":{"date-parts":[[2026,6,20]],"date-time":"2026-06-20T04:10:14Z","timestamp":1781928614000},"source":"Crossref","is-referenced-by-count":0,"title":["Pandemic Coronavirus Genome Packaging Relies on Multiple Dispersed Packaging Signals"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7344-7490","authenticated-orcid":false,"given":"Taha Y.","family":"Taha","sequence":"first","affiliation":[{"name":"Gladstone Institutes, San Francisco, CA, USA"},{"name":"Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6098-3633","authenticated-orcid":false,"given":"Nikesh","family":"Patel","sequence":"additional","affiliation":[{"name":"Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6865-4452","authenticated-orcid":false,"given":"Sam","family":"Clark","sequence":"additional","affiliation":[{"name":"Departments of Mathematics and Biology, University of York, YO10 5GE, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Julia","family":"Rosecrans","sequence":"additional","affiliation":[{"name":"Gladstone Institutes, San Francisco, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9156-9662","authenticated-orcid":false,"given":"Abdullah","family":"Syed","sequence":"additional","affiliation":[{"name":"Gladstone Institutes, San Francisco, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1225-4601","authenticated-orcid":false,"given":"Rebecca","family":"Chandler-Bostock","sequence":"additional","affiliation":[{"name":"Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6419-038X","authenticated-orcid":false,"given":"Erik R.","family":"Farquhar","sequence":"additional","affiliation":[{"name":"CWRU Center for Synchrotron Biosciences, NSLS-II, Brookhaven National Laboratory, Upton, NY 11973, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lekshmi B.G.","family":"Nair","sequence":"additional","affiliation":[{"name":"Departments of Mathematics and Biology, University of York, YO10 5GE, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Abid","family":"Javed","sequence":"additional","affiliation":[{"name":"Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK"},{"name":"Francis Crick Institute, 1 Midland Road, London. UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9161-999X","authenticated-orcid":false,"given":"Jennifer","family":"Doudna","sequence":"additional","affiliation":[{"name":"Gladstone Institutes, San Francisco, CA, USA"},{"name":"Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA"},{"name":"Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA"},{"name":"Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA USA"},{"name":"Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1360-2751","authenticated-orcid":false,"given":"Peter G.","family":"Stockley","sequence":"additional","affiliation":[{"name":"Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, 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