Selective Budding of SARS-CoV-Like Particles from Glycolipid-Enriched Membrane Lipid Rafts and Host Gene Modulation

Manoj K. Pastey; Yue Huang; Barney Graham

Manoj K Pastey, Yue Huang, Barney Graham

Microorganisms, doi:10.3390/microorganisms14010159

Severe acute respiratory syndrome coronavirus (SARS-CoV) assembles and buds from the Golgi apparatus or the ER membrane, but the specific membrane microdomains utilized during this process remain underexplored. Here, we show that co-expression of the SARS-CoV structural proteins S, M, and N in HEK-293T cells is sufficient to generate genomefree SARS-CoV-like virus-like particles (VLPs), which preferentially bud from glycolipidenriched membrane lipid raft microdomains. Immunofluorescence microscopy using raft-selective dyes (DiIC16) and spike-specific antibodies revealed strong co-localization of VLPs with lipid rafts. Detergent-resistant membrane analysis and sucrose gradient centrifugation further confirmed the presence of S protein in buoyant, raft-associated fractions alongside the raft marker CD44. Importantly, pharmacological disruption of rafts with methyl-β-cyclodextrin reduced VLP budding and S protein partitioning into raft domains, underscoring the requirement for intact lipid rafts in assembly. Additionally, our data support lipid raft-associated proteins' (e.g., FNRA, VIM, CD59, RHOA) roles in modulating cellular responses conducive to viral replication and assembly. These findings highlight lipid rafts as crucial platforms for SARS-CoV morphogenesis and suggest new avenues for vaccine and antiviral development using VLPs and raft-targeting therapeutics.

Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/microorganisms14010159/s1 , Figure S1 . Formation of coronaviruslike particles by inclusion of S glycoprotein expression vector. Electron micrograph of virus particles in HEK-293T cells transfected by the calcium phosphate method (total of 8 µg of DNA per transfection), with plasmids encoding SARS-CoV S, M, and N proteins. Panel (A) shows a high-magnification view of VLPs forming in the cytoplasm adjacent to the nuclear membrane (×30,000). Panel (B) shows a VLP with a corona-like structure emerging from an intracellular membrane (×200,000). (Adapted with permission from co-author [12] ; doi: 10.1128/JVI.78.22.12557-12565); Figure S2 . Comparison of microarray and quantitative real-time PCR (qPCR) fold-change values for host genes significantly upregulated in HEK-293T cells co-transfected with SARS-CoV S, M, and N structural protein expression vectors. Bars represent mean fold-change ± standard deviation (SD) from three independent experiments (p < 0.01). Microarray data (yellow) and qPCR data (orange) show strong concordance, with key raft-associated genes-VIM, RHOA, FNRA, and CD59-exhibiting marked induction. These genes encode proteins involved in membrane dynamics, cytoskeletal anchoring, and immune modulation, consistent with lipid raft-mediated viral assembly; Table S1 . Microarray and quantitative real-time PCR (qPCR) fold-change values for..

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DOI record: { "DOI": "10.3390/microorganisms14010159", "ISSN": [ "2076-2607" ], "URL": "http://dx.doi.org/10.3390/microorganisms14010159", "abstract": "Severe acute respiratory syndrome coronavirus (SARS-CoV) assembles and buds from the Golgi apparatus or the ER membrane, but the specific membrane microdomains utilized during this process remain underexplored. Here, we show that co-expression of the SARS-CoV structural proteins S, M, and N in HEK-293T cells is sufficient to generate genome-free SARS-CoV-like virus-like particles (VLPs), which preferentially bud from glycolipid-enriched membrane lipid raft microdomains. Immunofluorescence microscopy using raft-selective dyes (DiIC16) and spike-specific antibodies revealed strong co-localization of VLPs with lipid rafts. Detergent-resistant membrane analysis and sucrose gradient centrifugation further confirmed the presence of S protein in buoyant, raft-associated fractions alongside the raft marker CD44. Importantly, pharmacological disruption of rafts with methyl-β-cyclodextrin reduced VLP budding and S protein partitioning into raft domains, underscoring the requirement for intact lipid rafts in assembly. Additionally, our data support lipid raft-associated proteins’ (e.g., FNRA, VIM, CD59, RHOA) roles in modulating cellular responses conducive to viral replication and assembly. These findings highlight lipid rafts as crucial platforms for SARS-CoV morphogenesis and suggest new avenues for vaccine and antiviral development using VLPs and raft-targeting therapeutics.", "alternative-id": [ "microorganisms14010159" ], "author": [ { "affiliation": [ { "name": "Department of Veterinary Biomedical Sciences, Oregon State University, Corvallis, OR 97330, USA" } ], "family": "Pastey", "given": "Manoj K.", "sequence": "first" }, { "affiliation": [ { "name": "Vaccine Research Center, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA" } ], "family": "Huang", "given": "Yue", "sequence": "additional" }, { "affiliation": [ { "name": "Vaccine Research Center, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA" } ], "family": "Graham", "given": "Barney", "sequence": "additional" } ], "container-title": "Microorganisms", "container-title-short": "Microorganisms", "content-domain": { "crossmark-restriction": false, "domain": [] }, "created": { "date-parts": [ [ 2026, 1, 12 ] ], "date-time": "2026-01-12T07:30:34Z", "timestamp": 1768203034000 }, "deposited": { "date-parts": [ [ 2026, 1, 14 ] ], "date-time": "2026-01-14T05:16:02Z", "timestamp": 1768367762000 }, "funder": [ { "DOI": "10.13039/100018634", "award": [ "2501" ], "award-info": [ { "award-number": [ "2501" ] } ], "doi-asserted-by": "publisher", "id": [ { "asserted-by": "publisher", "id": "10.13039/100018634", "id-type": "DOI" } ], "name": "Carlson College of Veterinary Medicine" } ], "indexed": { "date-parts": [ [ 2026, 1, 15 ] ], "date-time": "2026-01-15T02:26:08Z", "timestamp": 1768443968373, "version": "3.49.0" }, "is-referenced-by-count": 0, "issue": "1", "issued": { "date-parts": [ [ 2026, 1, 10 ] ] }, "journal-issue": { "issue": "1", "published-online": { "date-parts": [ [ 2026, 1 ] ] } }, "language": "en", "license": [ { "URL": "https://creativecommons.org/licenses/by/4.0/", "content-version": "vor", "delay-in-days": 0, "start": { "date-parts": [ [ 2026, 1, 10 ] ], "date-time": "2026-01-10T00:00:00Z", "timestamp": 1768003200000 } } ], "link": [ { "URL": "https://www.mdpi.com/2076-2607/14/1/159/pdf", "content-type": "unspecified", "content-version": "vor", "intended-application": "similarity-checking" } ], "member": "1968", "original-title": [], "page": "159", "prefix": "10.3390", "published": { "date-parts": [ [ 2026, 1, 10 ] ] }, "published-online": { "date-parts": [ [ 2026, 1, 10 ] ] }, "publisher": "MDPI AG", "reference": [ { "DOI": "10.1038/42408", "article-title": "Functional rafts in cell membranes", "author": "Simons", "doi-asserted-by": "crossref", "first-page": "569", "journal-title": "Nature", "key": "ref_1", "volume": "387", "year": "1997" }, { "DOI": "10.1074/jbc.274.4.2038", "article-title": "Influenza viruses select ordered lipid domains during budding from the plasma membrane", "author": "Scheiffele", "doi-asserted-by": "crossref", "first-page": "2038", "journal-title": "J. 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