Development of Robust Varicella Zoster Virus Luciferase Reporter Viruses for In Vivo Monitoring of Virus Growth and Its Antiviral Inhibition in Culture, Skin, and Humanized Mice

Abstract

There is a continued need to understand varicella-zoster virus (VZV) pathogenesis and to develop more effective antivirals, as it causes chickenpox and zoster. As a human-restricted alphaherpesvirus, the use of human skin in culture and mice is critical in order to reveal the important VZV genes that are required for pathogenesis but that are not necessarily observed in the cell culture. We previously used VZV-expressing firefly luciferase (fLuc), under the control of the constitutively active SV40 promoter (VZV-BAC-Luc), to measure the VZV spread in the same sample. However, the fLuc expression was independent of viral gene expression and viral DNA replication programs. Here, we developed robust reporter VZV viruses by using bacterial artificial chromosome (BAC) technology, expressing luciferase from VZV-specific promoters. We also identified two spurious mutations in VZV-BAC that were corrected for maximum pathogenesis. VZV with fLuc driven by ORF57 showed superior growth in cells, human skin explants, and skin xenografts in mice. The ORF57-driven luciferase activity had a short half-life in the presence of foscarnet. This background was then used to investigate the roles for ORF36 (thymidine kinase (TK)) and ORF13 (thymidylate synthase (TS)) in skin. The studies reveal that VZV-∆TS had increased sensitivity to brivudine and was highly impaired for skin replication. This is the first report of a phenotype that is associated with the loss of TS.

Prodrug Strategies for the Development of β-l-5-((E)-2-Bromovinyl)-1-((2S,4S)-2-(hydroxymethyl)-1,3-(dioxolane-4-yl))uracil (l-BHDU) against Varicella Zoster Virus (VZV)

“…This phenomenon was also observed for CDV and ACV. We previously noted this phenomenon with other antivirals against VZV-ORF57-ΔTS, including the thymidine analogue brivudine (BVDU) . We suspect that the loss of TS reduces the dTMP pools in the cell, resulting in less competition with l -BHDU or its prodrugs for incorporation during viral DNA synthesis.…”

“…ARPE-19 cells were seeded in 96-well tissue culture plates 3 d prior to infection and grown to confluence. Cells were infected with cell-free VZV-ORF57-Luc at an MOI of 0.01, as previously described. ,− At 2 h post-infection, the virus inoculum was removed, and the medium was replaced with controls or test compounds in 6 replicate wells. CDV or ACV (positive controls), or l -BHDU and its prodrugs, were incubated with infected cells for 72 h. Virus yield was determined by dividing the average total flux at each concentration by the average total flux of the untreated wells.…”

“…Cells were grown in Dulbecco’s Modified Eagle Medium with 4.5 g/ l -glucose, l -glutamine, and sodium pyruvate (DMEM, 1X, Corning, Manassas, VA) supplemented with 10% heat-inactivated fetal bovine serum (Benchmark FBS; Gemini Bio Products, West Sacramento, CA), penicillin–streptomycin (5000 IU/mL), and amphotericin B (250 μg/mL). The VZV strains used for the cell-based assays included VZV-ORF57-Luc, along with the mutant strains VZV-ORF57-ΔTK, VZV-ORF57-ΔTS, and VZV-ORF57-ΔTKTS . All viruses were passaged no more than 10 times in ARPE-19 cells.…”

Humanized mouse models: A valuable platform for preclinical evaluation of human cancer

“…However, it was not found to accurately reflect the replication rate of VZV in tissues, as its expression was independent of viral gene expression. Recently, Lloyd et al optimized a BAC to be driven by the open reading frame 57 gene, which replicated better and responded to antiviral therapy, with a relatively short half‐life (Lloyd et al, 2022).…”

Direct Relationship between Protein Expression and Progeny Yield of Herpes Simplex Virus 1 Unveils a Rate-limiting Step for Virus Production

“…Therefore, the direct relationship between viral protein expression and progeny virus production, analyzed by considering their heterogeneities at single cell or subpopulation levels, remains to be elucidated. These observations raised a fundamental question as to whether the well-established correlation between viral protein expression and progeny virus production detected at the entire population level by classical time-course experiments 4,6 does reflect a direct relationship between them.…”