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Previous studies presented controversial results regarding the composition of the viral envelope, suggesting trans-Golgi and endosomal origins, as well as intersections with the exosomal and endocytic pathways. Here, we investigated the role of endocytic membranes for the secondary envelopment of HCMV by using wheat germ agglutinin (WGA) pulse labeling to label glycoproteins at the plasma membrane and to follow their trafficking during HCMV infection by light microscopy and transmission electron microscopy (TEM). WGA labeled different membrane compartments within the cVAC, including early endosomes, multivesicular bodies, trans-Golgi, and recycling endosomes. Furthermore, TEM analysis showed that almost 90% of capsids undergoing secondary envelopment and 50% of enveloped capsids were WGA-positive within 90 min. Our data reveal extensive remodeling of the endocytic compartment in the late stage of HCMV infection, where the endocytic compartment provides an optimized environment for virion morphogenesis and serves as the primary membrane source for secondary envelopment. Furthermore, we show that secondary envelopment is a rapid process in which endocytosed membranes are transported from the plasma membrane to the cVAC within minutes to be utilized by capsids for envelopment.<\/jats:p>","DOI":"10.3390\/biom14091149","type":"journal-article","created":{"date-parts":[[2024,9,12]],"date-time":"2024-09-12T02:30:23Z","timestamp":1726108223000},"page":"1149","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Secondary Envelopment of Human Cytomegalovirus Is a Fast Process Utilizing the Endocytic Compartment as a Major Membrane Source"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6411-1071","authenticated-orcid":false,"given":"Tim","family":"Bergner","sequence":"first","affiliation":[{"name":"Central Facility for Electron Microscopy, Ulm University, 89081 Ulm, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Laura","family":"Cortez Rayas","sequence":"additional","affiliation":[{"name":"Institute of Virology, Ulm University Medical Center, 89081 Ulm, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-4448-9831","authenticated-orcid":false,"given":"Gesa","family":"Freimann","sequence":"additional","affiliation":[{"name":"Central Facility for Electron Microscopy, Ulm University, 89081 Ulm, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4632-2684","authenticated-orcid":false,"given":"Clarissa","family":"Read","sequence":"additional","affiliation":[{"name":"Central Facility for Electron Microscopy, Ulm University, 89081 Ulm, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7508-4702","authenticated-orcid":false,"given":"Jens","family":"von Einem","sequence":"additional","affiliation":[{"name":"Institute of Virology, Ulm University Medical Center, 89081 Ulm, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,9,12]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"11861","DOI":"10.1128\/JVI.01077-07","article-title":"Three-Dimensional Structure of the Human Cytomegalovirus Cytoplasmic Virion Assembly Complex Includes a Reoriented Secretory Apparatus","volume":"81","author":"Das","year":"2007","journal-title":"J. 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