{
  "abstract": "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 0.5–80 PFU/cell. This direct entry assay was dependent on a functional virus entry–fusion 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–fusion 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.\n\nIMPORTANCE: 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.",
  "abstract_shown": true,
  "affiliations": [
    "Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, USA"
  ],
  "article_type": "research article",
  "arxiv": null,
  "author_total": 5,
  "authors": [
    {
      "affs": [
        0
      ],
      "name": "Yunliang Zhang"
    },
    {
      "affs": [
        0
      ],
      "name": "Jaroslav Holly"
    },
    {
      "affs": [
        0
      ],
      "name": "James Gibbs"
    },
    {
      "affs": [
        0
      ],
      "name": "Jonathan W. Yewdell",
      "orcid": "0000-0002-3826-1906"
    },
    {
      "affs": [
        0
      ],
      "name": "Bernard Moss",
      "orcid": "0000-0002-2154-8564"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/22fhxssmf8vhn.bib",
  "cited_by": 0,
  "doi": "10.1128/jvi.01115-26",
  "fulltext_url": "https://pmc.ncbi.nlm.nih.gov/articles/PMC13712968/",
  "id": "22fhxssmf8vhn",
  "issns": [
    "0022-538X",
    "1098-5514"
  ],
  "landing_url": "https://doi.org/10.1128/jvi.01115-26",
  "language": "en",
  "licence": "CC-BY-4.0",
  "licence_label": "CC BY 4.0",
  "licence_source": "article",
  "licence_url": "https://creativecommons.org/licenses/by/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "heading",
      "Virus-Cell Interactions"
    ],
    [
      "openalex",
      "2406|Poxvirus research and outbreaks"
    ]
  ],
  "openalex": "W7214727528",
  "pdf_url": null,
  "pmcid": "PMC13712968",
  "pmid": "42808726",
  "published": "2026-09-29",
  "publisher": "American Society for Microbiology (ASM)",
  "ris": "https://research.draconitic.com/paper/22fhxssmf8vhn.ris",
  "sources": [
    {
      "first_seen": "2026-10-31T12:23:27Z",
      "native_id": "PMC13712968",
      "source": "pmc",
      "url": "https://doi.org/10.1128/jvi.01115-26"
    }
  ],
  "subjects": [
    {
      "field": "biology",
      "origin": "openalex:2406|Poxvirus research and outbreaks",
      "subfield": "microbiology"
    }
  ],
  "title": "Real-time tracking of vaccinia virus entry using a split luciferase complementation assay",
  "title_text": "Real-time tracking of vaccinia virus entry using a split luciferase complementation assay",
  "type": "peer-reviewed",
  "updated_at": "2026-10-31T12:23:27Z",
  "url": "https://research.draconitic.com/paper/22fhxssmf8vhn",
  "venue": "Journal of Virology",
  "versions": []
}