{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,2]],"date-time":"2026-10-02T09:11:21Z","timestamp":1790932281120,"version":"4.1.0"},"reference-count":44,"publisher":"American Society for Microbiology","issue":"11","license":[{"start":{"date-parts":[[2025,11,25]],"date-time":"2025-11-25T00:00:00Z","timestamp":1764028800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2025,11,25]],"date-time":"2025-11-25T00:00:00Z","timestamp":1764028800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["32270155"],"award-info":[{"award-number":["32270155"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Shanghai Municipal Science and Technology Major Project","award":["ZD2021CY001"],"award-info":[{"award-number":["ZD2021CY001"]}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["J Virol"],"published-print":{"date-parts":[[2025,11,25]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>Coronaviruses induce the formation of double-membrane vesicles (DMVs) to facilitate viral RNA replication and transcription by the replication-transcription complexes (RTCs), comprising non-structural proteins (nsps) 2-16 and nucleocapsid protein. Nsp3 and nsp4 are the minimal components necessary for DMV formation and assemble a molecular pore that spans the DMV double membrane, connecting the DMV interior to the cytosol. However, the recruitment mechanisms of additional RTC components and the roles of other viral proteins in DMV assembly remain poorly understood. To dissect these processes independently of viral replication, we sought to establish a surrogate expression system using severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) polypeptides to recapitulate DMV formation and RTC recruitment. We characterized DMVs formed in cells expressing various nsp combinations and assessed the localization of RTC components using proteinase K (PK) protection assays following cell permeabilization. Compared to nsp3-4, expression of nsp3-8 and nsp3-10 resulted in larger and more morphologically heterogeneous DMVs. Portions of nsp5, nsp7, and nsp8 are associated with DMV-enriched membrane fractions, with nsp5 and nsp8 showing partial resistance to proteinase K digestion, suggesting that these proteins are at least partially localized within the DMV interior or protected by the DMV membrane architecture. Notably, mutations in the membrane-associated element (MAE) of nsp6 impaired nsp5-mediated proteolytic processing, abrogated DMV formation, and induced a cross-linked endoplasmic reticulum (ER) phenotype. These results highlight the essential role of nsp6 in the DMV biogenesis and demonstrate the utility of this surrogate system for mechanistic studies of coronavirus-induced membrane remodeling.<\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>Coronaviruses remodel host membranes through the action of non-structural proteins to generate double-membrane vesicles (DMVs), which serve as platforms for viral replication-transcription complexes (RTCs). Deciphering the molecular mechanisms governing DMV assembly and RTC recruitment is critical for understanding coronavirus replication and identifying novel antiviral targets. Here, we developed a surrogate system that recapitulates DMV formation in the absence of viral replication, enabling genetic manipulation and functional dissection of individual proteins. Using this system, we demonstrate that expression of the SARS-CoV-2 nsp3-10 polyprotein is sufficient to drive DMV formation and reveal a pivotal role for the membrane-associated element (MAE) of nsp6 in this process. These findings establish a tractable model for investigating coronavirus-induced membrane remodeling and underscore the essential contributions of nsp6 to DMV biogenesis.<\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/jvi.01385-25","type":"journal-article","created":{"date-parts":[[2025,11,6]],"date-time":"2025-11-06T14:01:21Z","timestamp":1762437681000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":6,"title":["SARS-CoV-2 polyprotein expression and the induction of double-membrane vesicles"],"prefix":"10.1128","volume":"99","author":[{"given":"Meng","family":"Zhao","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/013q1eq08","id-type":"ROR","asserted-by":"publisher"}],"name":"Key Laboratory of Medical Molecular Virology (MOE\/NHC\/CAMS), Shanghai Institute of Infectious Disease and Biosecurity, Shanghai Frontiers Science Center of Pathogenic Microorganisms and Infection, School of Basic Medical Sciences, Fudan University","place":["Shanghai, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yajie","family":"Zhang","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/013q1eq08","id-type":"ROR","asserted-by":"publisher"}],"name":"Key Laboratory of Medical Molecular Virology (MOE\/NHC\/CAMS), Shanghai Institute of Infectious Disease and Biosecurity, Shanghai Frontiers Science Center of Pathogenic Microorganisms and Infection, School of Basic Medical Sciences, Fudan University","place":["Shanghai, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yakun","family":"Liang","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/0220qvk04","id-type":"ROR","asserted-by":"publisher"}],"name":"Shanghai Institute of Precision Medicine, Shanghai Ninth People\u2019s Hospital, Shanghai Jiaotong University School of Medicine","place":["Shanghai, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fuzhi","family":"Lei","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/013q1eq08","id-type":"ROR","asserted-by":"publisher"}],"name":"Key Laboratory of Medical Molecular Virology (MOE\/NHC\/CAMS), Shanghai Institute of Infectious Disease and Biosecurity, Shanghai Frontiers Science Center of Pathogenic Microorganisms and Infection, School of Basic Medical Sciences, Fudan University","place":["Shanghai, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuying","family":"Han","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/013q1eq08","id-type":"ROR","asserted-by":"publisher"}],"name":"Key Laboratory of Medical Molecular Virology (MOE\/NHC\/CAMS), Shanghai Institute of Infectious Disease and Biosecurity, Shanghai Frontiers Science Center of Pathogenic Microorganisms and Infection, School of Basic Medical Sciences, Fudan University","place":["Shanghai, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0268-4891","authenticated-orcid":false,"given":"Zhenghong","family":"Yuan","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/013q1eq08","id-type":"ROR","asserted-by":"publisher"}],"name":"Key Laboratory of Medical Molecular Virology (MOE\/NHC\/CAMS), Shanghai Institute of Infectious Disease and Biosecurity, Shanghai Frontiers Science Center of Pathogenic Microorganisms and Infection, School of Basic Medical Sciences, Fudan University","place":["Shanghai, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4560-4970","authenticated-orcid":true,"given":"Zhigang","family":"Yi","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/013q1eq08","id-type":"ROR","asserted-by":"publisher"}],"name":"Key Laboratory of Medical Molecular Virology (MOE\/NHC\/CAMS), Shanghai Institute of Infectious Disease and Biosecurity, Shanghai Frontiers Science Center of Pathogenic Microorganisms and Infection, School of Basic Medical Sciences, Fudan University","place":["Shanghai, China"]},{"name":"Shanghai public health clinical center, Fudan University","place":["Shanghai, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_4_2_2","doi-asserted-by":"publisher","DOI":"10.1186\/s12889-023-17491-w"},{"key":"e_1_3_4_3_2","unstructured":"COVID-19 cases | WHO COVID-19 dashboard. 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