{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T18:28:04Z","timestamp":1786991284319,"version":"build-2736575974"},"update-to":[{"DOI":"10.1371\/journal.ppat.1014028","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T00:00:00Z","timestamp":1786924800000}}],"reference-count":46,"publisher":"Public Library of Science (PLoS)","issue":"8","license":[{"start":{"date-parts":[[2026,8,10]],"date-time":"2026-08-10T00:00:00Z","timestamp":1786320000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004488","name":"Hrvatska Zaklada za Znanost","doi-asserted-by":"publisher","award":["2019-04-6048"],"award-info":[{"award-number":["2019-04-6048"]}],"id":[{"id":"10.13039\/501100004488","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004488","name":"Hrvatska Zaklada za Znanost","doi-asserted-by":"publisher","award":["IP-2025-02-3470"],"award-info":[{"award-number":["IP-2025-02-3470"]}],"id":[{"id":"10.13039\/501100004488","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100011033","name":"Agencia Estatal de Investigaci\u00f3n","doi-asserted-by":"publisher","award":["PID2022-136456NB-I00\/AEI\/10.13039\/501100011033"],"award-info":[{"award-number":["PID2022-136456NB-I00\/AEI\/10.13039\/501100011033"]}],"id":[{"id":"10.13039\/501100011033","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100010665","name":"H2020 Marie Sk\u0142odowska-Curie Actions","doi-asserted-by":"publisher","award":["101129778"],"award-info":[{"award-number":["101129778"]}],"id":[{"id":"10.13039\/100010665","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plospathogens.org"],"crossmark-restriction":false},"short-container-title":["PLoS Pathog"],"abstract":"<jats:p>Understanding intracellular trafficking is central to decoding viral pathogenesis and engineering optimized viral vectors. How a virus or vector is routed through the endocytic pathway directly dictates its genome release, immune sensing, and overall transduction efficiency. Human adenovirus type 26 (HAdV-D26) presents a promising platform for vector design due to its low preexisting immunity, potent immune stimulation, scalable production, and versatile genetic engineering capacity. Although increasingly significant, the fundamental mechanisms governing HAdV-D26 intracellular trafficking are still not fully understood. Our study demonstrates that compared to well-described human adenovirus type 5 (HAdV-C5), HAdV-D26 undergoes prolonged intracellular trafficking, transiently localizing to early endosomes before residing in late endosomes\/lysosomes for up to four hours post-infection. Inhibition of lysosomal acidification modestly enhances HAdV-D26 transduction efficiency, whereas blocking transport from early to late endosomes\/lysosomes does not. Strikingly, Rab9 knockdown reduces HAdV-D26 late endosomal\/lysosomal localization while increasing both virus internalization and genome delivery to the host cell nucleus. These findings indicate that late endosomal sorting pathways actively influence HAdV-D26 infection outcomes. By identifying a previously unappreciated role for Rab9 in adenovirus transduction, our results provide new mechanistic insight into HAdV-D26 intracellular trafficking, highlight serotype-specific differences in adenovirus entry pathways, and identify endosomal trafficking steps that may be targeted to improve adenoviral vector performance.<\/jats:p>","DOI":"10.1371\/journal.ppat.1014028","type":"journal-article","created":{"date-parts":[[2026,8,10]],"date-time":"2026-08-10T17:39:09Z","timestamp":1786383549000},"page":"e1014028","update-policy":"https:\/\/doi.org\/10.1371\/journal.ppat.corrections_policy","source":"Crossref","is-referenced-by-count":0,"title":["Rab9 depletion enhances human adenovirus type 26 transduction efficiency through increased internalization and reduced late endosomal\/lysosomal retention"],"prefix":"10.1371","volume":"22","author":[{"given":"Isabela","family":"Dra\u0161kovi\u0107","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Davor","family":"Nesti\u0107","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lucija","family":"Luli\u0107 Horvat","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jelena","family":"Martin\u010di\u0107","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mario","family":"Stojanovi\u0107","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gabriela N.","family":"Condezo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Klara","family":"Ka\u0161nar","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2071-8365","authenticated-orcid":true,"given":"Esther","family":"Gonz\u00e1lez-Almela","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3420-758X","authenticated-orcid":true,"given":"Dar\u00edo","family":"Lago","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carmen","family":"San Mart\u00edn","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jerome","family":"Custers","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0385-0900","authenticated-orcid":true,"given":"Dragomira","family":"Majhen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2026,8,10]]},"reference":[{"issue":"5","key":"ppat.1014028.ref001","doi-asserted-by":"crossref","first-page":"2192","DOI":"10.1016\/j.ymthe.2025.03.062","article-title":"Four decades of adenovirus gene transfer vectors: history and current use","volume":"33","author":"NR Hackett","year":"2025","journal-title":"Mol Ther"},{"issue":"1","key":"ppat.1014028.ref002","doi-asserted-by":"crossref","first-page":"2475831","DOI":"10.1080\/22221751.2025.2475831","article-title":"The seroprevalence of adenoviruses since 2000","volume":"14","author":"L Hong","year":"2025","journal-title":"Emerg Microbes Infect"},{"issue":"6","key":"ppat.1014028.ref003","doi-asserted-by":"crossref","DOI":"10.1002\/rmv.2338","article-title":"Human adenovirus type 26 basic biology and its usage as vaccine vector","volume":"32","author":"D Majhen","year":"2022","journal-title":"Rev Med Virol"},{"issue":"1","key":"ppat.1014028.ref004","doi-asserted-by":"crossref","DOI":"10.1128\/JVI.01474-18","article-title":"\u03b1v\u03b23 integrin is required for efficient infection of epithelial cells with human adenovirus type 26","volume":"93","author":"D Nesti\u0107","year":"2018","journal-title":"J Virol"},{"issue":"4","key":"ppat.1014028.ref005","doi-asserted-by":"crossref","DOI":"10.1128\/spectrum.01097-22","article-title":"Human adenovirus type 26 infection mediated by \u03b1v\u03b23 integrin is caveolin-1-dependent","volume":"10","author":"D Nesti\u0107","year":"2022","journal-title":"Microbiol Spectr"},{"issue":"18","key":"ppat.1014028.ref006","doi-asserted-by":"crossref","first-page":"10354","DOI":"10.1128\/JVI.00936-14","article-title":"Late endosomal trafficking of alternative serotype adenovirus vaccine vectors augments antiviral innate immunity","volume":"88","author":"JE Teigler","year":"2014","journal-title":"J Virol"},{"issue":"4","key":"ppat.1014028.ref007","doi-asserted-by":"crossref","first-page":"1992","DOI":"10.1128\/JVI.79.4.1992-2000.2005","article-title":"Adenovirus protein VI mediates membrane disruption following capsid disassembly","volume":"79","author":"CM Wiethoff","year":"2005","journal-title":"J Virol"},{"key":"ppat.1014028.ref008","doi-asserted-by":"crossref","first-page":"591","DOI":"10.1016\/j.virol.2015.03.006","article-title":"Adenovirus membrane penetration: tickling the tail of a sleeping dragon","author":"CM Wiethoff","year":"2015","journal-title":"Virology"},{"issue":"8","key":"ppat.1014028.ref009","doi-asserted-by":"crossref","first-page":"3661","DOI":"10.1128\/jvi.64.8.3661-3673.1990","article-title":"Human adenovirus-host cell interactions: comparative study with members of subgroups B and C","volume":"64","author":"C Defer","year":"1990","journal-title":"J Virol"},{"key":"ppat.1014028.ref010","doi-asserted-by":"crossref","first-page":"4464","DOI":"10.1016\/j.csbj.2022.08.016","article-title":"Rab GTPases: the principal players in crafting the regulatory landscape of endosomal trafficking","volume":"20","author":"J Zhang","year":"2022","journal-title":"Comput Struct Biotechnol J"},{"issue":"12","key":"ppat.1014028.ref011","doi-asserted-by":"crossref","first-page":"2265","DOI":"10.1111\/j.1600-0854.2008.00835.x","article-title":"Infectious adenovirus type 2 transport through early but not late endosomes","volume":"9","author":"M Gastaldelli","year":"2008","journal-title":"Traffic"},{"issue":"8","key":"ppat.1014028.ref012","doi-asserted-by":"crossref","first-page":"1766","DOI":"10.1002\/j.1460-2075.1996.tb00525.x","article-title":"The role of the adenovirus protease on virus entry into cells","volume":"15","author":"UF Greber","year":"1996","journal-title":"EMBO J"},{"issue":"3","key":"ppat.1014028.ref013","doi-asserted-by":"crossref","first-page":"1387","DOI":"10.1128\/JVI.75.3.1387-1400.2001","article-title":"Adenovirus serotype 7 retention in a late endosomal compartment prior to cytosol escape is modulated by fiber protein","volume":"75","author":"N Miyazawa","year":"2001","journal-title":"J Virol"},{"issue":"1","key":"ppat.1014028.ref014","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1080\/15548627.2020.1797280","article-title":"Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1","volume":"17","author":"DJ Klionsky","year":"2021","journal-title":"Autophagy"},{"issue":"4","key":"ppat.1014028.ref015","doi-asserted-by":"crossref","first-page":"309","DOI":"10.1111\/mmi.14909","article-title":"Adenovirus entry: stability, uncoating, and nuclear import","volume":"118","author":"UF Greber","year":"2022","journal-title":"Mol Microbiol"},{"issue":"22","key":"ppat.1014028.ref016","doi-asserted-by":"crossref","first-page":"12367","DOI":"10.1128\/JVI.01833-13","article-title":"A direct and versatile assay measuring membrane penetration of adenovirus in single cells","volume":"87","author":"M Suomalainen","year":"2013","journal-title":"J Virol"},{"issue":"12","key":"ppat.1014028.ref017","doi-asserted-by":"crossref","first-page":"923","DOI":"10.1038\/s41573-019-0036-1","article-title":"Lysosomes as a therapeutic target","volume":"18","author":"SR Bonam","year":"2019","journal-title":"Nat Rev Drug Discov"},{"key":"ppat.1014028.ref018","doi-asserted-by":"crossref","first-page":"1144127","DOI":"10.3389\/fimmu.2023.1144127","article-title":"Disruption of endosomal trafficking with EGA alters TLR9 cytokine response in human plasmacytoid dendritic cells","volume":"14","author":"MJ Wiest","year":"2023","journal-title":"Front Immunol"},{"issue":"2","key":"ppat.1014028.ref019","doi-asserted-by":"crossref","first-page":"462","DOI":"10.1128\/jvi.17.2.462-471.1976","article-title":"Genetic analysis of adenovirus type 2 III. Temperature sensitivity of processing viral proteins","volume":"17","author":"J Weber","year":"1976","journal-title":"J Virol"},{"issue":"2","key":"ppat.1014028.ref020","article-title":"Tools of the trade: use of dominant-inhibitory mutants of Ras-family GTPases","volume":"1","author":"LA Feig","year":"1999","journal-title":"Nat Cell Biol"},{"issue":"12","key":"ppat.1014028.ref021","doi-asserted-by":"crossref","first-page":"6247","DOI":"10.1128\/JVI.02460-08","article-title":"A dominant-negative mutant of rab5 inhibits infection of cells by foot-and-mouth disease virus: implications for virus entry","volume":"83","author":"HL Johns","year":"2009","journal-title":"J Virol"},{"issue":"10","key":"ppat.1014028.ref022","doi-asserted-by":"crossref","first-page":"1585","DOI":"10.3390\/pharmaceutics13101585","article-title":"The revolving door of adenovirus cell entry: not all pathways are equal","volume":"13","author":"D Nesti\u0107","year":"2021","journal-title":"Pharmaceutics"},{"key":"ppat.1014028.ref023","doi-asserted-by":"crossref","first-page":"434","DOI":"10.3181\/00379727-107-26648","article-title":"Four newly recognized adenoviruses","volume":"107","author":"L Rosen","year":"1961","journal-title":"Proc Soc Exp Biol Med"},{"issue":"3","key":"ppat.1014028.ref024","doi-asserted-by":"crossref","DOI":"10.1128\/JVI.00826-21","article-title":"Ex vivo and In vivo CD46 receptor utilization by species D human adenovirus serotype 26 (HAdV26)","volume":"96","author":"JR Hemsath","year":"2022","journal-title":"J Virol"},{"issue":"9","key":"ppat.1014028.ref025","doi-asserted-by":"crossref","DOI":"10.1126\/sciadv.aax3567","article-title":"Human adenovirus type 26 uses sialic acid-bearing glycans as a primary cell entry receptor","volume":"5","author":"AT Baker","year":"2019","journal-title":"Sci Adv"},{"key":"ppat.1014028.ref026","article-title":"Adenovirus endocytosis","author":"O Meier","year":"2004","journal-title":"J Gene Med"},{"issue":"10","key":"ppat.1014028.ref027","doi-asserted-by":"crossref","DOI":"10.1128\/jvi.00770-23","article-title":"Efficient clathrin-mediated entry of enteric adenoviruses in human duodenal cells","volume":"97","author":"M Becker","year":"2023","journal-title":"J Virol"},{"issue":"3","key":"ppat.1014028.ref028","doi-asserted-by":"crossref","first-page":"477","DOI":"10.1016\/0092-8674(93)90382-Z","article-title":"Stepwise dismantling of adenovirus 2 during entry into cells","volume":"75","author":"UF Greber","year":"1993","journal-title":"Cell"},{"issue":"7","key":"ppat.1014028.ref029","doi-asserted-by":"crossref","first-page":"6056","DOI":"10.1128\/JVI.73.7.6056-6065.1999","article-title":"Fiber swap between adenovirus subgroups B and C alters intracellular trafficking of adenovirus gene transfer vectors","volume":"73","author":"N Miyazawa","year":"1999","journal-title":"J Virol"},{"issue":"6","key":"ppat.1014028.ref030","doi-asserted-by":"crossref","first-page":"2999","DOI":"10.1091\/mbc.e05-02-0121","article-title":"Nuclear targeting of adenovirus type 2 requires CRM1-mediated nuclear export","volume":"16","author":"S Strunze","year":"2005","journal-title":"Mol Biol Cell"},{"issue":"10","key":"ppat.1014028.ref031","doi-asserted-by":"crossref","DOI":"10.1128\/JVI.00164-20","article-title":"Nup358 and transportin 1 cooperate in adenoviral genome import","volume":"94","author":"I Carlon-Andres","year":"2020","journal-title":"J Virol"},{"issue":"4","key":"ppat.1014028.ref032","doi-asserted-by":"crossref","first-page":"2307","DOI":"10.1128\/JVI.02014-12","article-title":"Host cell autophagy modulates early stages of adenovirus infections in airway epithelial cells","volume":"87","author":"X Zeng","year":"2013","journal-title":"J Virol"},{"key":"ppat.1014028.ref033","doi-asserted-by":"crossref","first-page":"229","DOI":"10.1111\/j.0105-2896.2005.00310.x","article-title":"The lysosomal cysteine proteases in MHC class II antigen presentation","volume":"207","author":"LC Hsing","year":"2005","journal-title":"Immunol Rev"},{"issue":"4","key":"ppat.1014028.ref034","doi-asserted-by":"crossref","first-page":"772","DOI":"10.1016\/j.immuni.2024.03.015","article-title":"Vaccine adjuvants: tailoring innate recognition to send the right message","volume":"57","author":"EC Lavelle","year":"2024","journal-title":"Immunity"},{"issue":"11","key":"ppat.1014028.ref035","doi-asserted-by":"crossref","DOI":"10.1101\/cshperspect.a022616","article-title":"Rab proteins and the compartmentalization of the endosomal system","volume":"6","author":"A Wandinger-Ness","year":"2014","journal-title":"Cold Spring Harb Perspect Biol"},{"issue":"4","key":"ppat.1014028.ref036","doi-asserted-by":"crossref","DOI":"10.1080\/19420889.2016.1204498","article-title":"The multiple roles of Rab9 in the endolysosomal system","volume":"9","author":"A Kucera","year":"2016","journal-title":"Commun Integr Biol"},{"issue":"12","key":"ppat.1014028.ref037","doi-asserted-by":"crossref","first-page":"5420","DOI":"10.1091\/mbc.e04-08-0747","article-title":"Rab9 GTPase regulates late endosome size and requires effector interaction for its stability","volume":"15","author":"IG Ganley","year":"2004","journal-title":"Mol Biol Cell"},{"issue":"1","key":"ppat.1014028.ref038","doi-asserted-by":"crossref","first-page":"8440","DOI":"10.1038\/s41467-023-44201-2","article-title":"NDP52 mediates an antiviral response to hepatitis B virus infection through Rab9-dependent lysosomal degradation pathway","volume":"14","author":"S Cui","year":"2023","journal-title":"Nat Commun"},{"issue":"9","key":"ppat.1014028.ref039","doi-asserted-by":"crossref","DOI":"10.1371\/journal.ppat.1011648","article-title":"Noncanonical Rab9a action supports retromer-mediated endosomal exit of human papillomavirus during virus entry","volume":"19","author":"J Choi","year":"2023","journal-title":"PLoS Pathog"},{"key":"ppat.1014028.ref040","doi-asserted-by":"crossref","first-page":"1815","DOI":"10.2147\/OTT.S183748","article-title":"The overexpression of Rab9 promotes tumor progression regulated by XBP1 in breast cancer","volume":"12","author":"Y Liu","year":"2019","journal-title":"Onco Targets Ther"},{"issue":"6","key":"ppat.1014028.ref041","doi-asserted-by":"crossref","first-page":"569","DOI":"10.1097\/CMR.0000000000000595","article-title":"Long noncoding RNA ZFAS1 promotes tumorigenesis through regulation of miR-150-5p\/RAB9A in melanoma","volume":"29","author":"L Liang","year":"2019","journal-title":"Melanoma Res"},{"key":"ppat.1014028.ref042","doi-asserted-by":"crossref","first-page":"1533033820915958","DOI":"10.1177\/1533033820915958","article-title":"Knockdown of Rab9 suppresses the progression of gastric cancer through regulation of Akt signaling pathway","volume":"19","author":"Y Zhu","year":"2020","journal-title":"Technol Cancer Res Treat"},{"issue":"9","key":"ppat.1014028.ref043","doi-asserted-by":"crossref","first-page":"4654","DOI":"10.1128\/JVI.02696-06","article-title":"Comparative seroprevalence and immunogenicity of six rare serotype recombinant adenovirus vaccine vectors from subgroups B and D","volume":"81","author":"P Abbink","year":"2007","journal-title":"J Virol"},{"key":"ppat.1014028.ref044","doi-asserted-by":"crossref","first-page":"174","DOI":"10.1186\/1743-422X-6-174","article-title":"Genetic reconstitution of the human adenovirus type 2 temperature-sensitive 1 mutant defective in endosomal escape","volume":"6","author":"N Imelli","year":"2009","journal-title":"Virol J"},{"issue":"5","key":"ppat.1014028.ref045","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pone.0019564","article-title":"Altering the Ad5 packaging domain affects the maturation of the Ad particles","volume":"6","author":"R Alba","year":"2011","journal-title":"PLoS One"},{"key":"ppat.1014028.ref046","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1007\/978-1-62703-679-5_1","article-title":"Biophysical methods to monitor structural aspects of the adenovirus infectious cycle","volume":"1089","author":"R Men\u00e9ndez-Conejero","year":"2014","journal-title":"Methods Mol Biol"}],"updated-by":[{"DOI":"10.1371\/journal.ppat.1014028","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T00:00:00Z","timestamp":1786924800000}}],"container-title":["PLOS Pathogens"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.ppat.1014028","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T17:41:10Z","timestamp":1786988470000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.ppat.1014028"}},"subtitle":[],"editor":[{"given":"Walter J.","family":"Atwood","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2026,8,10]]},"references-count":46,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2026,8,10]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.ppat.1014028","relation":{},"ISSN":["1553-7374"],"issn-type":[{"value":"1553-7374","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,10]]}}}