Real-time tracking of vaccinia virus entry using a split luciferase complementation assay.

UNLABELLED: 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.