FIG 6.

Transcriptional and translational requirements for establishment of superinfection resistance. (A) Effects of transcription inhibitors. Cells were left untreated (UT) or were treated with α-amanitin (10 μg/ml), cordycepin (40 μg/ml), or actinomycin D (act. D; 4 μg/ml) for the duration of the experiment. Following 30 min of inhibitor pretreatment, cells were uninfected (uninf.) or infected with 10 PFU/cell of VACV WR or IHD-J for 150 min at 37°C. Cells then were superinfected with 3 PFU of DiD-loaded secondary VACV WR at 37°C for 90 min. Cells were analyzed by flow cytometry to determine DiD mean fluorescence (DiD fluor.) intensities (MFI), which were normalized to the values obtained for the untreated and uninfected control (cntrl) cells infected with DiD-loaded virus. (B) Effects of translation inhibitors. Protocols were the same as those in panel A, except that emetine (2 μM), cycloheximide (CHX; 66 μM), and anisomycin (1 μM) were used. (C) Inability of UV-irradiated VACV to establish superinfection resistance. Equivalent PFU of IHD-J were irradiated with UV light for the indicated number of seconds (sec UV irr.). Cells were uninfected or infected with irradiated virus particles (preirradiation equivalent of 10 PFU/cell) at 37°C for 180 min. Cells then were superinfected with 3 PFU/cell of LUC-expressing secondary VACV for 150 min. Cells then were lysed and LUC levels measured. (D) Inability of transcription-deficient VACV to establish resistance to superinfection determined by LUC expression. Cells were left uninfected or were infected with 10 PFU/cell of primary VACV containing (+) or lacking (−) L3 protein for various lengths of time at 37°C. Cells then were superinfected with 3 PFU/cell of WRvFire for 150 min at 37°C. LUC activity was determined and plotted as a percentage of the uninfected cell control value. (E) Inability of transcription-deficient VACV to establish resistance to superinfection determined by hemifusion assay. Cells were untreated (UT) or were treated with the indicated inhibitors for the duration of the experiment as described for panels A and B. Following 30 min of inhibitor pretreatment, cells were left uninfected or were infected with 10 PFU/cell of vL3⁺ or vL3⁻ VACV for 150 min at 37°C. Cells then were superinfected with 3 PFU/cell of DiD-loaded VACV WR for 90 min at 37°C and analyzed by flow cytometry to determine the MFI. Data for DiD MFI were normalized to the values obtained for the untreated and uninfected control cells infected with DiD-loaded virus.