Comparison of VZV promoter activities. MeWo (7 × 10⁴ cells/well) (A) and BSC40 (2.4 × 10⁴ cells/well) cells (B) in 96-well plates were transfected with 125 ng of a reporter plasmid containing the promoter for the indicated open reading frame (ORF) along with 4 ng of a Renilla luciferase-expressing plasmid (pRL-CMV; Promega) as an internal control, by using FuGENE 6 (Roche). pGL3-Basic, a vector plasmid used to clone the promoters, was used as a negative control (−). At 24 h after transfection, the cells were infected with VZV at an multiplicity of infection of ∼0.02, and 1 day later, both firefly and Renilla luciferase activities were determined by a chemiluminescent assay reaction (Dual-Glo luciferase assay system; Promega) followed by measurement of relative light units (RLU) with a luminometer (JNR AB2300; ATTO, Japan). Means and standard deviations (SDs) of RLU from triplicate wells are shown. (C) Correlation of IE62- and infection-dependent promoter activation. MeWo cells were transfected with each reporter plasmid along with an IE62-expressing plasmid, and 2 days later their luciferase activities were measured and compared with those represented in panel A. Each circle represents one promoter-reporter construct. An arrow indicates ORF9.

Characterization of VZV reporter cell line MV9G. (A) Time-dependent activation of the reporter cell line after VZV infection. MV9G cells in 96-well plates were infected with 60 and 600 PFU of VZV and incubated for 2 h. After removal of the inoculums, cells were cultured for the indicated period (in hours), rinsed with phosphate-buffered saline, and stored at −80°C until luciferase activity was measured. Means and SDs of luciferase activities from triplicate wells are shown. (B) MV9G cells were infected with the indicated PFU of VZV and incubated for 2 h. After removal of the inoculums, the cells were cultured for an additional 46 h. Means and SDs of luciferase activities from triplicate wells are shown. (C) MV9G cells were infected with cell-associated virus, and their luciferase activities were measured 2 days later. MeWo cells were infected with the same cell-associated virus, and the numbers of the infected cells used for the inoculation were obtained by immunostaining using anti-VZV IE62 monoclonal antibody (MAb8616; Chemicon International). The luciferase activities were compared with the numbers of the infected cells.

Minimum ORF9 promoter region for responses to VZV infection and to IE62. (A) Reporter plasmids containing truncated ORF9 promoter sequences, 9C, 9D, 9F, 9G, and 9I were constructed. A duplicated form of the minimum essential 93-bp sequence (9G) was cloned into BamHI site of pGL3-Basic, resulting in pGL-T9G. (B) The sequence of the minimum essential region of the ORF9 promoter (9G) and the USF binding motif in the region are shown. A 2-bp alteration (CACGTG to CAtaTG) of the USF binding motif (9U) was introduced into the sequence. (C and D) Activities of the modified promoters represented in panels A and B were analyzed in transient transfection experiments. Means and SDs of luciferase activities from triplicate wells are shown. pGL3-Basic (vector) used for cloning of the promoters was used as a negative control.

Applications of the reporter cell assay. (A) MeWo cells (7 × 10⁴ cells/well) in 96-well plates were inoculated with cell-associated VZV (∼1 × 10³ MeWo cells infected with VZV; cytopathic effect > 70%) (○, “cell-associated”) or with 250 PFU of cell-free VZV (•, “cell-free”) and cultured in the presence or absence of the indicated concentrations of ACV. Two days later, MV9G cells (7 × 10⁴ cells/well) were added to each well and cocultured in the presence of ACV for 1 day. The cells were harvested, and their luciferase activities were measured. Means and SDs of luciferase activities from triplicate wells are shown. (B) MeWo cells (7 × 10⁴ cells/well) in 96-well plates were inoculated with approximately 200 of MeWo cells infected with TK-positive (pOka [•] and CaQu [⧫]) or -negative (rOkaYSR [○] and Kanno [Δ]) strains (22, 23, 28), and cultured in the presence of ACV for 2 days. Then, MV9G cells were overlaid and cocultured for 1 day. The luciferase activities in the ACV-treated cultures were expressed as percent RLU, with the activities obtained in the culture without inhibitor treatment used as a standard. Means and SDs of luciferase activities from triplicate wells are shown. (C) MeWo cells (7 × 10⁴ cells/well) were infected with cell-associated VZV, cultured for 2 days, and then cocultured with MV9G for 1 day (•, “coculture”). MV9G cells (7 × 10⁴ cells/well) were directly infected with 400 PFU of cell-free VZV, and cultured for 2 days (○, “direct infection”). Roscovitine was added at the indicated concentrations throughout the processes. Means and SDs of luciferase activities from triplicate wells are shown.