Real-time tracking of vaccinia virus entry using a split luciferase complementation assay
- 1Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, USA
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.
IMPORTANCE: 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.
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BibTeX
@article{zhang2026realtime,
title = {{Real-time tracking of vaccinia virus entry using a split luciferase complementation assay}},
author = {Yunliang Zhang and Jaroslav Holly and James Gibbs and Jonathan W. Yewdell and Bernard Moss},
journal = {Journal of Virology},
year = {2026},
month = sep,
doi = {10.1128/jvi.01115-26},
url = {https://doi.org/10.1128/jvi.01115-26},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Real-time tracking of vaccinia virus entry using a split luciferase complementation assay
AU - Yunliang Zhang
AU - Jaroslav Holly
AU - James Gibbs
AU - Jonathan W. Yewdell
AU - Bernard Moss
PY - 2026
DA - 2026/09/29
JO - Journal of Virology
DO - 10.1128/jvi.01115-26
UR - https://doi.org/10.1128/jvi.01115-26
AB - 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. IMPORTANCE: 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.
SN - 0022-538X
SN - 1098-5514
ER -