{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,9,12]],"date-time":"2026-09-12T02:08:22Z","timestamp":1789178902998,"version":"build-2803163510"},"posted":{"date-parts":[[2026,9,9]]},"group-title":"Microbiology","reference-count":59,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,9,9]],"date-time":"2026-09-09T00:00:00Z","timestamp":1788912000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["R01 GM124378 (IJM, JL)"],"award-info":[{"award-number":["R01 GM124378 (IJM, JL)"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["R01 GM110243 (JL, IJM)"],"award-info":[{"award-number":["R01 GM110243 (JL, IJM)"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["R01 GM150905 (MC)"],"award-info":[{"award-number":["R01 GM150905 (MC)"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,9,9]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Bacteriophage P22 ejects four internal proteins into infected cells to assemble a channel for genome translocation across the cell envelope. However, how these proteins cooperate to build the trans-envelope channel remains enigmatic. Using\n                  <jats:italic>in situ<\/jats:italic>\n                  single-particle cryogenic electron microscopy, we determined near-atomic\n                  <jats:italic>in vivo<\/jats:italic>\n                  structures of P22 infection intermediates, revealing how the tail hub protein gp10 templates the sequential assembly of three internal proteins gp7*, gp20 and gp16 into a \u223c50-nm trans-envelope channel that extends from the virion into the host cytoplasm. Functional analyses explain why phage DNA can be detected in supernatant fluids during infection by channel-defective mutants. Our structures also provide a mechanistic basis for the long-standing observation that a P22 virion lacking gp16 can be complemented, extra-cytoplasmically, by a co-infecting virion lacking gp20, thereby allowing the genome in the gp16-deficient particle to enter the host cytoplasm.\n                <\/jats:p>","DOI":"10.64898\/2026.09.03.749273","type":"posted-content","created":{"date-parts":[[2026,9,9]],"date-time":"2026-09-09T12:20:18Z","timestamp":1788956418000},"source":"Crossref","is-referenced-by-count":0,"title":["Sequential assembly\n                  <i>in vivo<\/i>\n                  of the bacteriophage P22 genome translocation channel"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9375-1916","authenticated-orcid":false,"given":"Huaxin","family":"Yu","sequence":"first","affiliation":[{"name":"Department of Microbial Pathogenesis, Yale School of Medicine; New Haven, CT 06536, USA"},{"name":"Microbial Sciences Institute, Yale University; West Haven, CT 06516, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5190-1840","authenticated-orcid":false,"given":"Jian","family":"Yue","sequence":"additional","affiliation":[{"name":"Department of Microbial Pathogenesis, Yale School of Medicine; New Haven, CT 06536, USA"},{"name":"Microbial Sciences Institute, Yale University; West Haven, CT 06516, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Taehyun","family":"Park","sequence":"additional","affiliation":[{"name":"LaMontagne Center for Infectious Disease, Department of Molecular Biosciences, University of Texas at Austin; Austin, TX 78712, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chunyan","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Microbial Pathogenesis, Yale School of Medicine; New Haven, CT 06536, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1311-7868","authenticated-orcid":false,"given":"Muyuan","family":"Chen","sequence":"additional","affiliation":[{"name":"Division of CryoEM and Bioimaging, SSRL, SLAC National Accelerator Laboratory, Stanford University; Menlo Park, CA 94025, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3108-6735","authenticated-orcid":false,"given":"Jun","family":"Liu","sequence":"additional","affiliation":[{"name":"Department of Microbial Pathogenesis, Yale School of Medicine; New Haven, CT 06536, USA"},{"name":"Microbial Sciences Institute, Yale University; West Haven, CT 06516, USA"},{"name":"Department of Cell Biology, Yale School of Medicine, New Haven, CT 06510, USA"},{"name":"Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0184-7652","authenticated-orcid":false,"given":"Ian J.","family":"Molineux","sequence":"additional","affiliation":[{"name":"LaMontagne Center for Infectious Disease, Department of Molecular Biosciences, University of Texas at Austin; Austin, TX 78712, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026091118401564000_2026.09.03.749273v1.1","doi-asserted-by":"publisher","DOI":"10.1038\/nsmb.1823"},{"key":"2026091118401564000_2026.09.03.749273v1.2","doi-asserted-by":"publisher","DOI":"10.1038\/srep44176"},{"key":"2026091118401564000_2026.09.03.749273v1.3","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1012388108"},{"key":"2026091118401564000_2026.09.03.749273v1.4","doi-asserted-by":"publisher","DOI":"10.1016\/j.virol.2011.06.005"},{"key":"2026091118401564000_2026.09.03.749273v1.5","doi-asserted-by":"crossref","first-page":"eadw7914","DOI":"10.1126\/sciadv.adw7914","article-title":"Structural basis of bacteriophage Ur-lambda infection initiation","volume":"11","year":"2025","journal-title":"Science Advances"},{"key":"2026091118401564000_2026.09.03.749273v1.6","doi-asserted-by":"publisher","DOI":"10.1126\/science.1231887"},{"key":"2026091118401564000_2026.09.03.749273v1.7","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1501064112"},{"key":"2026091118401564000_2026.09.03.749273v1.8","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1605883113"},{"key":"2026091118401564000_2026.09.03.749273v1.9","doi-asserted-by":"crossref","first-page":"163","DOI":"10.1016\/j.jsb.2016.07.019","article-title":"Ultrastructural analysis of bacteriophage Phi29 during infection of Bacillus subtilis","volume":"197","year":"2017","journal-title":"J Struct Biol"},{"key":"2026091118401564000_2026.09.03.749273v1.10","doi-asserted-by":"publisher","DOI":"10.1016\/j.virol.2017.04.017"},{"key":"2026091118401564000_2026.09.03.749273v1.11","doi-asserted-by":"crossref","first-page":"1049","DOI":"10.1038\/s41564-019-0403-z","article-title":"Structural dynamics of bacteriophage P22 infection initiation revealed by cryo-electron tomography","volume":"4","year":"2019","journal-title":"Nat Microbiol"},{"key":"2026091118401564000_2026.09.03.749273v1.12","doi-asserted-by":"crossref","unstructured":"L. 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