{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,18]],"date-time":"2026-07-18T09:40:30Z","timestamp":1784367630730,"version":"3.55.0"},"update-to":[{"DOI":"10.1371\/journal.ppat.1014224","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2026,5,27]],"date-time":"2026-05-27T00:00:00Z","timestamp":1779840000000}}],"reference-count":47,"publisher":"Public Library of Science (PLoS)","issue":"5","license":[{"start":{"date-parts":[[2026,5,18]],"date-time":"2026-05-18T00:00:00Z","timestamp":1779062400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Touro University System"}],"content-domain":{"domain":["www.plospathogens.org"],"crossmark-restriction":false},"short-container-title":["PLoS Pathog"],"abstract":"<jats:p>Human cytomegalovirus (HCMV) is a widespread beta-herpesvirus that establishes lifelong infection and can cause severe disease in immunocompromised individuals as well as congenital abnormalities. While HCMV entry into fibroblasts is classically described as plasma membrane fusion, accumulating evidence indicates that a fraction of virions undergo endocytic uptake, notably via macropinocytosis, and traffic through the endosomal system. However, the mechanisms by which internalized viral components reach the nucleus are still being elucidated. Here, we investigated whether HCMV exploits type II nuclear envelope invaginations (NEIs), rare and discrete folds of the nuclear membrane that extend into the nucleoplasm, and the associated VAP-A\u2013ORP3\u2013Rab7 (VOR) complex to mediate nuclear delivery of viral components, a mechanism previously described for HIV-1. Using primary human foreskin fibroblasts (HFFs), we tracked the tegument protein pp65 and immediate-early proteins IE1\/2 during early infection. We show that HCMV infection induces a rapid increase in NEI formation within the first hour of infection, accompanied by the accumulation of pp65 within Rab7\u2009\u207a\u2009endosomal structures that localize to NEIs. Pharmacological inhibition of the VOR complex with an ORP3-targeting drug significantly reduced NEI formation, decreased the association of pp65 with NEIs, and impaired its nuclear accumulation by approximately 2.5-fold. In contrast, inhibition of this pathway did not affect immediate-early gene expression at 24 hours post-infection. Functionally, disruption of the VOR complex resulted in a 3-fold reduction in viral replication, highlighting the contribution of this pathway to efficient infection. Together, these findings support a model in which HCMV tegument proteins, but not the viral genome, access the nucleus via a NEI\/VOR-dependent trafficking route. This work identifies a previously unrecognized nuclear delivery pathway exploited by HCMV and suggests that targeting nuclear\u2013endosomal communication may represent a novel antiviral strategy.<\/jats:p>","DOI":"10.1371\/journal.ppat.1014224","type":"journal-article","created":{"date-parts":[[2026,5,18]],"date-time":"2026-05-18T17:39:21Z","timestamp":1779125961000},"page":"e1014224","update-policy":"https:\/\/doi.org\/10.1371\/journal.ppat.corrections_policy","source":"Crossref","is-referenced-by-count":1,"title":["Nuclear transport of human cytomegalovirus tegument protein pp65 through nucleoplasmic reticulum"],"prefix":"10.1371","volume":"22","author":[{"given":"Maysa","family":"Azzeh","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mark F.","family":"Santos","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexandra M. K.","family":"Yokomizo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Victoria","family":"Cha","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Denis","family":"Corbeil","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0644-7375","authenticated-orcid":true,"given":"Aurelio","family":"Lorico","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2026,5,18]]},"reference":[{"issue":"3","key":"ppat.1014224.ref001","doi-asserted-by":"crossref","DOI":"10.1002\/rmv.2034","article-title":"Estimation of the worldwide seroprevalence of cytomegalovirus: a systematic review and meta-analysis","volume":"29","author":"M Zuhair","year":"2019","journal-title":"Rev Med 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Kotton","year":"2018","journal-title":"Transplantation"},{"issue":"1","key":"ppat.1014224.ref005","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1002\/bdr2.2098","article-title":"Congenital cytomegalovirus surveillance in the United States","volume":"115","author":"K Raines","year":"2023","journal-title":"Birth Defects Res"},{"issue":"2","key":"ppat.1014224.ref006","doi-asserted-by":"crossref","first-page":"249","DOI":"10.1128\/MMBR.00040-07","article-title":"Tegument proteins of human cytomegalovirus","volume":"72","author":"RF Kalejta","year":"2008","journal-title":"Microbiol Mol Biol Rev"},{"issue":"1","key":"ppat.1014224.ref007","doi-asserted-by":"crossref","first-page":"37","DOI":"10.1016\/j.coviro.2012.01.001","article-title":"Human cytomegalovirus entry into cells","volume":"2","author":"AL Vanarsdall","year":"2012","journal-title":"Curr Opin 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mechanism","volume":"89","author":"M Zhou","year":"2015","journal-title":"J Virol"},{"issue":"50","key":"ppat.1014224.ref011","doi-asserted-by":"crossref","first-page":"20037","DOI":"10.1073\/pnas.0709704104","article-title":"Human cytomegalovirus uses two distinct pathways to enter retinal pigmented epithelial cells","volume":"104","author":"D Wang","year":"2007","journal-title":"Proc Natl Acad Sci U S A"},{"issue":"2","key":"ppat.1014224.ref012","doi-asserted-by":"crossref","first-page":"710","DOI":"10.1128\/JVI.80.2.710-722.2006","article-title":"Human cytomegalovirus entry into epithelial and endothelial cells depends on genes UL128 to UL150 and occurs by endocytosis and low-pH fusion","volume":"80","author":"BJ Ryckman","year":"2006","journal-title":"J Virol"},{"issue":"4","key":"ppat.1014224.ref013","doi-asserted-by":"crossref","first-page":"351","DOI":"10.1111\/tra.12355","article-title":"HCMV Induces Macropinocytosis for Host Cell Entry in Fibroblasts","volume":"17","author":"S 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Zhuang","year":"2024","journal-title":"bioRxiv"},{"issue":"1","key":"ppat.1014224.ref026","doi-asserted-by":"crossref","first-page":"4588","DOI":"10.1038\/s41467-023-40227-8","article-title":"HIV-1-induced nuclear invaginations mediated by VAP-A, ORP3, and Rab7 complex explain infection of activated T cells","volume":"14","author":"MF Santos","year":"2023","journal-title":"Nat Commun"},{"issue":"3","key":"ppat.1014224.ref027","doi-asserted-by":"crossref","first-page":"529","DOI":"10.1042\/BST20253005","article-title":"Extracellular membrane particles en route to the nucleus - exploring the VOR complex","volume":"53","author":"A Lorico","year":"2025","journal-title":"Biochem Soc Trans"},{"key":"ppat.1014224.ref028","doi-asserted-by":"crossref","first-page":"107589","DOI":"10.1016\/j.bioorg.2024.107589","article-title":"Triazole derivatives inhibit the VOR complex-mediated nuclear transport of extracellular particles: potential application in cancer and HIV-1 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An update of its use in the prevention of cytomegalovirus infection and disease in transplant recipients","volume":"56","author":"S Noble","year":"1998","journal-title":"Drugs"},{"key":"ppat.1014224.ref034","first-page":"101","article-title":"Functions of human cytomegalovirus tegument proteins prior to immediate early gene expression","volume":"325","author":"RF Kalejta","year":"2008","journal-title":"Curr Top Microbiol Immunol"},{"issue":"2","key":"ppat.1014224.ref035","doi-asserted-by":"crossref","first-page":"1228","DOI":"10.1128\/JVI.02606-13","article-title":"Human cytomegalovirus major immediate early 1 protein targets host chromosomes by docking to the acidic pocket on the nucleosome surface","volume":"88","author":"K M\u00fccke","year":"2014","journal-title":"J Virol"},{"issue":"2","key":"ppat.1014224.ref036","doi-asserted-by":"crossref","first-page":"1357","DOI":"10.1128\/JVI.77.2.1357-1367.2003","article-title":"Mouse cytomegalovirus immediate-early protein 1 binds with host 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noninfectious virion-like particle released from cells infected with human strains of cytomegalovirus","volume":"130","author":"A Irmiere","year":"1983","journal-title":"Virology"},{"issue":"5","key":"ppat.1014224.ref040","doi-asserted-by":"crossref","first-page":"1110","DOI":"10.1093\/infdis\/168.5.1110","article-title":"Ultrastructural analysis of circulating cytomegalic cells in patients with active cytomegalovirus infection: evidence for virus production and endothelial origin","volume":"168","author":"A Grefte","year":"1993","journal-title":"J Infect Dis"},{"issue":"13","key":"ppat.1014224.ref041","doi-asserted-by":"crossref","first-page":"6132","DOI":"10.1128\/JVI.74.13.6132-6146.2000","article-title":"Dense bodies of human cytomegalovirus induce both humoral and cellular immune responses in the absence of viral gene expression","volume":"74","author":"S Pepperl","year":"2000","journal-title":"J Virol"},{"issue":"1","key":"ppat.1014224.ref042","doi-asserted-by":"crossref","first-page":"517","DOI":"10.1038\/s41597-023-02418-2","article-title":"Proteome changes of fibroblasts and endothelial cells upon incubation with human cytomegalovirus subviral Dense Bodies","volume":"10","author":"I Penner","year":"2023","journal-title":"Sci Data"},{"issue":"6","key":"ppat.1014224.ref043","doi-asserted-by":"crossref","first-page":"1333","DOI":"10.3390\/v15061333","article-title":"Subviral dense bodies of human cytomegalovirus enhance interferon-beta responses in infected cells and impair progeny production","volume":"15","author":"I Penner","year":"2023","journal-title":"Viruses"},{"issue":"11","key":"ppat.1014224.ref044","doi-asserted-by":"crossref","first-page":"5686","DOI":"10.3390\/v7112900","article-title":"3D analysis of HCMV induced-nuclear membrane structures by FIB\/SEM tomography: insight into an unprecedented membrane morphology","volume":"7","author":"C 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Methods"}],"updated-by":[{"DOI":"10.1371\/journal.ppat.1014224","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2026,5,27]],"date-time":"2026-05-27T00:00:00Z","timestamp":1779840000000}}],"container-title":["PLOS Pathogens"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.ppat.1014224","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,27]],"date-time":"2026-05-27T17:52:37Z","timestamp":1779904357000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.ppat.1014224"}},"subtitle":[],"editor":[{"given":"Eain 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