{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T13:55:28Z","timestamp":1790690128640,"version":"4.1.0"},"reference-count":51,"publisher":"American Society for Microbiology","license":[{"start":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T00:00:00Z","timestamp":1790640000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T00:00:00Z","timestamp":1790640000000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/100017540","name":"Division of Intramural Research","doi-asserted-by":"publisher","award":["1ZIAI000307-44"],"award-info":[{"award-number":["1ZIAI000307-44"]}],"id":[{"id":"10.13039\/100017540","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["J Virol"],"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>A recombinant vaccinia virus (vA4-HiBiT) was constructed with a short, split luciferase HiBiT tag at the C-terminus of the A4 core protein open reading frame. When HeLa or A549 cells stably expressing LgBiT were infected with vA4-HiBiT, the interaction of the split luciferase fragments reconstituted NanoLuc enzyme activity. Upon addition of a membrane-permeable luciferase substrate, a strong luminescence signal indicated core entry into the cytoplasm within 5 min after infection and was proportional to virus multiplicity within the range of 0.5\u201380 PFU\/cell. This direct entry assay was dependent on a functional virus entry\u2013fusion complex, was inhibited by a neutralizing antibody, and was unaffected by an inhibitor of protein synthesis. Entry was enhanced by brief pH 5 treatment of cell-bound virus relative to pH 7 and prevented by pH 3 treatment. Bafilomycin A1, an inhibitor of endosomal acidification, exhibited greater inhibition of entry in A549-LgBiT cells compared to HeLa-LgBiT cells, as did treatment with ammonium chloride, suggesting variations in entry pathways or cell metabolism. Inhibition of entry by latrunculin A and cytochalasin D indicated a role for actin polymerization in both cell lines. Despite the discovery of a conserved furin cleavage consensus sequence in the A16 subunit of the entry\u2013fusion complex, a furin inhibitor did not reduce virus entry unlike results obtained with a human papillomavirus control. These data support the use of this NanoBiT assay for studies of vaccinia virus entry, with application to basic research, antivirals, and neutralizing antibodies.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>Assays to measure virus entry into cells mostly rely on post-entry events such as gene expression, cytopathic effects, or virus yields. Although fluorescence and electron microscopy can provide direct analyses, such methods are labor intensive and semiquantitative. Here we describe a rapid and quantitative real-time direct entry assay for vaccinia virus with application to basic research, antivirals, and neutralizing antibodies.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/jvi.01115-26","type":"journal-article","created":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T13:02:03Z","timestamp":1790686923000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Real-time tracking of vaccinia virus entry using a split luciferase complementation assay"],"prefix":"10.1128","author":[{"given":"Yunliang","family":"Zhang","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/043z4tv69","id-type":"ROR","asserted-by":"publisher"}],"name":"Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases","place":["Bethesda, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jaroslav","family":"Holly","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/043z4tv69","id-type":"ROR","asserted-by":"publisher"}],"name":"Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases","place":["Bethesda, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"James","family":"Gibbs","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/043z4tv69","id-type":"ROR","asserted-by":"publisher"}],"name":"Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases","place":["Bethesda, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3826-1906","authenticated-orcid":true,"given":"Jonathan W.","family":"Yewdell","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/043z4tv69","id-type":"ROR","asserted-by":"publisher"}],"name":"Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases","place":["Bethesda, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2154-8564","authenticated-orcid":true,"given":"Bernard","family":"Moss","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/043z4tv69","id-type":"ROR","asserted-by":"publisher"}],"name":"Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases","place":["Bethesda, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","published-online":{"date-parts":[[2026,9,29]]},"reference":[{"key":"e_1_3_4_2_2","doi-asserted-by":"publisher","DOI":"10.1021\/acschembio.7b00549"},{"key":"e_1_3_4_3_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.virusres.2017.10.015"},{"key":"e_1_3_4_4_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbrc.2025.152236"},{"key":"e_1_3_4_5_2","doi-asserted-by":"publisher","DOI":"10.1128\/jvi.00991-25"},{"key":"e_1_3_4_6_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.heliyon.2024.e31905"},{"key":"e_1_3_4_7_2","doi-asserted-by":"publisher","DOI":"10.1128\/mBio.01590-21"},{"key":"e_1_3_4_8_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.antiviral.2021.105231"},{"key":"e_1_3_4_9_2","doi-asserted-by":"publisher","DOI":"10.1021\/bi00110a002"},{"key":"e_1_3_4_10_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.RA118.004579"},{"key":"e_1_3_4_11_2","doi-asserted-by":"publisher","DOI":"10.3390\/v16071002"},{"key":"e_1_3_4_12_2","first-page":"573","volume-title":"Fields virology","author":"Moss B","year":"2021","unstructured":"Moss B, Smith GL. 2021. 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