{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T13:00:35Z","timestamp":1785416435669,"version":"3.56.0"},"posted":{"date-parts":[[2025,12,30]]},"group-title":"Microbiology","reference-count":57,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,12,30]],"date-time":"2025-12-30T00:00:00Z","timestamp":1767052800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/publicdomain\/zero\/1.0\/"}],"funder":[{"award":["R01DK048370 (Subaward VUMC95054)"],"award-info":[{"award-number":["R01DK048370 (Subaward VUMC95054)"]}],"id":[{"id":"https:\/\/ror.org\/00adh9b73","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000001","name":"U.S. National Science Foundation","doi-asserted-by":"crossref","award":["STC Award 1231306"],"award-info":[{"award-number":["STC Award 1231306"]}],"id":[{"id":"10.13039\/100000001","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000001","name":"U.S. National Science Foundation","doi-asserted-by":"crossref","award":["NSF RAPID: IIBR Award 2032199"],"award-info":[{"award-number":["NSF RAPID: IIBR Award 2032199"]}],"id":[{"id":"10.13039\/100000001","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000001","name":"U.S. National Science Foundation","doi-asserted-by":"crossref","award":["National Science Foundation (NSF) Cooperative Agreement No. HRD-997886"],"award-info":[{"award-number":["National Science Foundation (NSF) Cooperative Agreement No. HRD-997886"]}],"id":[{"id":"10.13039\/100000001","id-type":"DOI","asserted-by":"crossref"}]},{"award":["R01NS117513"],"award-info":[{"award-number":["R01NS117513"]}],"id":[{"id":"https:\/\/ror.org\/01s5ya894","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,12,30]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>Coronavirus particles assemble at endoplasmic reticulum Golgi intermediate compartment (ERGIC) membranes and exit from host cells via secretory organelles that are not well defined. The interplay between viral components and intracellular transport pathways that facilitate assembly and egress are not fully understood and recent studies suggest that multiple pathways maybe involved. Reverse genetics was used to develop a model system to further understand the assembly and egress processes. Mouse hepatitis coronavirus (MHV-A59) was genetically engineered to express the membrane (M) protein fused to green fluorescent protein (M-GFP), with the chimeric gene cloned in place of the open reading frame (ORF) 4 coding region in the RNA genome. The recovered M-GFP virus also expresses wild-type (WT) M protein (M WT) from its native ORF. The M-GFP virus exhibited morphology and growth properties like WT virus. M-GFP and WT M proteins colocalized early in infection, but less M-GFP trafficked toward the cell surface at later times, suggesting that the fusion protein is incorporated less efficiently into virus particles. M-GFP was stably expressed through at least four virus passages. Early passage virus M-GFP virus particles were visualized by confocal and Total Internal Reflection Fluorescence (TIRF) microscopy in live cells. The fluorescently labeled virus particles represent a new tool for coronavirus intracellular trafficking and egress studies. Such studies can also help provide a more detailed understanding of infection and disease processes to provide new insight for development of new therapeutic strategies.<\/jats:p>\n                <jats:sec>\n                  <jats:title>IMPORTANCE<\/jats:title>\n                  <jats:p>Coronavirus assembly, intracellular transport, egress, and the virus-host interactions involved in these processes are not fully understood. The M protein is the most abundant virion structural component, which forms the scaffold for assembly of the viral envelope. It facilitates incorporation of the viral nucleocapsid and the other viral membrane proteins, spike (S) and envelope (E). In this study we engineered M protein tagged with GFP, which is expressed along with wild-type (WT) M from the viral genome. M-GFP interacted with WT M protein and co-assembled into virus particles. The resulting fluorescently labeled coronavirus particles open opportunities to use state of the art microscopy to monitor virus assembly, trafficking, and egress in live cells to facilitate deeper understanding of the molecular and cell biology of these processes. The ability to visualize particles also opens opportunities to help further understanding of steps in viral replication and pathogenesis for potential therapeutic targets.<\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2025.12.29.696688","type":"posted-content","created":{"date-parts":[[2025,12,31]],"date-time":"2025-12-31T02:35:19Z","timestamp":1767148519000},"source":"Crossref","is-referenced-by-count":1,"title":["Coronavirus membrane protein with a fluorescent protein tag enables tracking of virus particles in live cells"],"prefix":"10.64898","author":[{"given":"Bereket","family":"Stefanos","sequence":"first","affiliation":[{"name":"School of Life Sciences, Arizona State University, Tempe, Arizona, USA"},{"name":"Biodesign Institute, Arizona State University, Tempe, Arizona, USA"},{"name":"Center for Applied Structural Discovery, Arizona State University, Tempe, Arizona USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Noe R. B.","family":"Green","sequence":"additional","affiliation":[{"name":"School of Life Sciences, Arizona State University, Tempe, Arizona, USA"},{"name":"Biodesign Institute, Arizona State University, Tempe, Arizona, USA"},{"name":"Center for Applied Structural Discovery, Arizona State University, Tempe, Arizona USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Isha","family":"Nadig","sequence":"additional","affiliation":[{"name":"School of Life Sciences, Arizona State University, Tempe, Arizona, USA"},{"name":"Biodesign Institute, Arizona State University, Tempe, Arizona, USA"},{"name":"Center for Applied Structural Discovery, Arizona State University, Tempe, Arizona USA"},{"name":"School of Life Sciences Undergraduate Research Program"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lynn A.","family":"Lapierre","sequence":"additional","affiliation":[{"name":"Department of Surgery, Vanderbilt University School of Medicine, Nashville, TN, USA"},{"name":"Epithelial Biology Center, Vanderbilt University School of Medicine, Nashville, TN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7833-2940","authenticated-orcid":false,"given":"James R.","family":"Goldenring","sequence":"additional","affiliation":[{"name":"Department of Surgery, Vanderbilt University School of Medicine, Nashville, TN, USA"},{"name":"Epithelial Biology Center, Vanderbilt University School of Medicine, Nashville, TN, USA"},{"name":"Nashville VA Medical Center, Nashville, TN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1977-5573","authenticated-orcid":false,"given":"Ian B.","family":"Hogue","sequence":"additional","affiliation":[{"name":"School of Life Sciences, Arizona State University, Tempe, Arizona, USA"},{"name":"Biodesign Institute, Arizona State University, Tempe, Arizona, USA"},{"name":"ASU-Banner Neurodegenerative Disease Research Center, Arizona State University, Tempe, Arizona, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8148-4224","authenticated-orcid":false,"given":"Honor L.","family":"Glenn","sequence":"additional","affiliation":[{"name":"School of Life Sciences, Arizona State University, Tempe, Arizona, USA"},{"name":"Biodesign Institute, Arizona State University, Tempe, Arizona, USA"},{"name":"Center for Applied Structural Discovery, Arizona State University, Tempe, Arizona USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4545-9208","authenticated-orcid":false,"given":"Brenda G.","family":"Hogue","sequence":"additional","affiliation":[{"name":"School of Life Sciences, Arizona State University, Tempe, Arizona, USA"},{"name":"Biodesign Institute, Arizona State University, Tempe, Arizona, USA"},{"name":"Center for Applied Structural Discovery, Arizona State University, Tempe, Arizona USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026010211351142000_2025.12.29.696688v1.1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-micro-110520-023212"},{"key":"2026010211351142000_2025.12.29.696688v1.2","doi-asserted-by":"publisher","DOI":"10.1038\/S41579-020-00468-6"},{"key":"2026010211351142000_2025.12.29.696688v1.3","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.124.1.55"},{"key":"2026010211351142000_2025.12.29.696688v1.4","first-page":"108","article-title":"Laminated cisternae of the rough endoplasmic reticulum induced by coronavirus MHV-A59 infection","volume":"36","year":"1985","journal-title":"Eur J Cell Biol"},{"key":"2026010211351142000_2025.12.29.696688v1.5","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-020-19619-7"},{"key":"2026010211351142000_2025.12.29.696688v1.6","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2020.09.018"},{"key":"2026010211351142000_2025.12.29.696688v1.7","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.74.17.8127-8134.2000"},{"key":"2026010211351142000_2025.12.29.696688v1.8","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.75.19.9059-9067.2001"},{"key":"2026010211351142000_2025.12.29.696688v1.9","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.77.5.2922-2927.2003"},{"key":"2026010211351142000_2025.12.29.696688v1.10","doi-asserted-by":"crossref","first-page":"e2513552122","DOI":"10.1073\/pnas.2513552122","article-title":"Definition of the components required for selective packaging of coronavirus genomic RNA","volume":"122","year":"2025","journal-title":"Proc Natl Acad Sci U S A"},{"key":"2026010211351142000_2025.12.29.696688v1.11","doi-asserted-by":"crossref","unstructured":"Hogue BG , Machamer CM . 2008. 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